Lecture 05. Non-Covalent Interactions, DNA.
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HalbedelsteinBarytAmalgamSenseChemische StrukturEssenz <Lebensmittel>BewegungMeeresspiegelOrdnungszahlZunderbeständigkeitChemische BindungGletscherzungeChemische BiologieNucleinbasenMolekülTautomerieLagerungDNSApothekerinLactitolSetzen <Verfahrenstechnik>ReaktivitätArzneimittelBasenpaarungAktionspotenzialTopizitätRNS-SyntheseElektronentransferISO-Komplex-HeilweiseElementenhäufigkeitChemischer ProzessChemische ForschungDNS-DoppelhelixIsotretinoinFaserverbundwerkstoffPeriodateChemische EigenschaftZusatzstoffStimulationWasserfallHydrophobe WechselwirkungBiochemie
07:53
Hydroxybuttersäure <gamma->ArzneimittelUltraschallschweißenLewisit <Giftgas>Chemische StrukturCHARGE-AssoziationAktionspotenzialZellkontaktChemische BindungOktanzahlKlinisches ExperimentMinimale HemmkonzentrationWasserstoffDipol <1,3->Amine <primär->WasserbeständigkeitWursthülleLipideOrdnungszahlSonnenschutzmittelExplosionBindungsenergieChemische BiologieAktionspotenzialWasserDispersionSammler <Technik>MolekülNeutralisation <Chemie>Elektron <Legierung>Potenz <Homöopathie>Van-der-Waals-KraftAzokupplungPlasmamembranSeitenketteMagnetisierbarkeitErdrutschRNSEukaryontische ZelleFunktionelle GruppeWirtsspezifitätSetzen <Verfahrenstechnik>FaserplatteProteineAtombindungDiamantCHARGE-AssoziationElektronische ZigaretteZunderbeständigkeitFüllstoffMethanisierungBukett <Wein>ZusatzstoffKonkrement <Innere Medizin>Vorlesung/Konferenz
15:46
Chemische BindungDispersionWasserstoffHydroxybuttersäure <gamma->Dipol <1,3->AtombindungUltraschallschweißenMonsterwelleAgar-AgarAlphaspektroskopieSterblichkeitKalisalzeDarmstadtiumGenUmlagerungWursthülleSonnenschutzmittelChemische BiologieGleichgewichtskonstanteCHARGE-AssoziationKonzentratKochsalzKomplikationErdrutschPeyotlMolekülReaktionsmechanismusWasserstoffbrückenbindungProteinePlasmamembranChemische StrukturBenzolringAromatizitätAlignment <Biochemie>ZusatzstoffReaktionsgleichungMassendichteAcetonProlinCobaltoxideOberflächenchemieSetzen <Verfahrenstechnik>ÜbergangszustandMethylgruppeEliminierungsreaktionElektron <Legierung>SenseSystemische Therapie <Pharmakologie>ScherfestigkeitAlterungBukett <Wein>WasserstoffChemische BindungWasserAktionspotenzialDipol <1,3->KörpergewichtUmlagerungElektronendonatorElektronenakzeptorFunktionelle GruppePipetteEisenRingspannungIdiotypKalkammonsalpeterAlpha-1-RezeptorAromatenMolvolumenLigandEukaryontische ZelleChemische ReaktionChemische ForschungKonvertierungVorlesung/KonferenzComputeranimation
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WasserstoffWasserbeständigkeitAlpha-1-RezeptorLigandWasserOberflächenchemieWasserbeständigkeitProteineWasserstoffbrückenbindungHerzfrequenzvariabilitätSeitenketteProteinogene AminosäurenFunktionelle GruppeChemische StrukturBindungsenergieMeeresspiegelOrdnungszahlChemische BindungAlkoholische LösungEisflächeKomplikationRückstandBenzolringFülle <Speise>MolekülTransdermales therapeutisches SystemScherfestigkeitBildungsentropieSomatotropinVerzerrungEssenz <Lebensmittel>KohlenstofffaserStickstoffatomGuanidinArgininMethanisierungKettenlänge <Makromolekül>ClathrateWasserstoffThermoformenSingle electron transferSetzen <Verfahrenstechnik>ZellwachstumAlpha-1-RezeptorZutatBlauschimmelkäseKorngrenzeEukaryontische ZelleInitiator <Chemie>ScreeningHydrophobe WechselwirkungQuellgebietMultiproteinkomplexKohlenstoff-14ZusatzstoffRingspannungCobaltoxideWursthülleStockfischBloom-SyndromGuaninGesundheitsstörungComputeranimation
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Alpha-1-RezeptorLigandWasserbeständigkeitUltraschallschweißenProteineDNS-DoppelhelixRNSSubstrat <Chemie>BiosyntheseDuplikationEnzymMagmaHydroxyethylcellulosenGasverflüssigungDuktilitätHydroxybuttersäure <gamma->Lennard-Jones-PotenzialEnzymModul <Membranverfahren>DuplikationLipideElektronische ZigaretteThermoformenEukaryontische ZelleCHARGE-AssoziationChemische BindungChemische ReaktionGenerikumRingspannungUntereinheitFunktionelle GruppeSpezies <Chemie>SenseRadioaktiver StoffInselTherapietreueBindegewebeWursthülleInterkristalline KorrosionTiermodellPeriodateFließgrenzeGesundheitsstörungDNSHydrophobe WechselwirkungTriterpeneBiologisches MaterialKörnigkeitZusatzstoffSäurePentapeptideSetzen <Verfahrenstechnik>ProteineProteinogene AminosäurenChemische BiologieBiosyntheseScherfestigkeitPolyketideRNSIsoprenAcetylgruppeEsterOrganische ChemieTubeSingle electron transferSpaltflächeEukaryotenOligosaccharideComputeranimationVorlesung/Konferenz
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WasserstoffChemische BindungChemische ForschungHydroxybuttersäure <gamma->ReplikationReaktionsmechanismusFlüssigkeitsfilmEukaryontische ZelleDNSKonzentratWasserReaktionsmechanismusReplikationHelix <alpha->BasenpaarungEukaryontische ZellePauling, LinusDNS-DoppelhelixZusatzstoffPotenz <Homöopathie>ScherfestigkeitMolekülOberflächenchemieSchmierstoffSingle electron transferNucleinbasenDoppelhelixWasserstoffbrückenbindungFunktionelle GruppeElektronendonatorChemische BindungElektronenakzeptorWursthülleRöntgendiffraktometriePedosphäreTranskriptionsfaktorGenomElektron <Legierung>ReplikationsursprungErdrutschZutatChemische StrukturHelicität <Chemie>ProteineSubstrat <Boden>ZellwachstumOrangensaftAusgangsgesteinLinkerThermoformenChemische ForschungProbiotikumLactitolBukett <Wein>DuktilitätFülle <Speise>OrdnungszahlVan-der-Waals-KraftBlauschimmelkäseAdenomatous-polyposis-coli-ProteinEukaryotenKernproteineMitochondriumComputeranimationVorlesung/Konferenz
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Elektronen-Lokalisierungs-FunktionLaminitArzneimittelBasenpaarungGuaninCytosinThyminAdeninPhysiologieAmineAmine <primär->Omega-3-FettsäurenRNSHydroxyethylcellulosenHydroxybuttersäure <gamma->ProteineLipideKernproteineNucleotideBasenpaarungGärrestPhosphateFunktionelle GruppeHydroxylgruppeWassertropfenLactitolSchubspannungSekundärstrukturDNSMeeresspiegelWursthülleNucleinsäurenLigandPolychlorierte DibenzofuraneChemische StrukturChromosomenaberrationGuaninSäureCytosinKonjugateRNSZusatzstoffReaktionsmechanismusSenseProteineHydrolysatFaserplatteHydroxidePhosphonsäureAlkohole <tertiär->OligonucleotideProteinkinase ASpektroelektrochemieAlkoholateTriethylaminComputeranimationVorlesung/Konferenz
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Molekulare ErkennungWasserstoffChemische BindungVerdickungsmittelDNSTautomerieWursthüllePigmentZusatzstoffOrganische ChemieAromatizitätChemische ForschungChromosomRNS-SyntheseChemische StrukturLinkerScherfestigkeitKrebsforschungLactitolMethylgruppeEukaryontische ZelleTumorChemikerChemische VerbindungenSekundärstrukturPolymorphismusMitochondriale DNSBasenpaarungEnoleQuerprofilThermoformenWasserstoffPyrenGuaninBromRNSWaldmoorDNS-SequenzWasserstoffbrückenbindungPosttranslationale ÄnderungPhosgenDoppelhelixMethyltransferase <S>AbschreckenFunktionelle GruppeThyminMolekülTranskriptionsfaktorHelix <alpha->Chemische BiologieNucleinbasenMonomolekulare ReaktionAlphaspektroskopieProteineTellerseparatorVerdickungsmittelErdrutschMeeresspiegelAzokupplungHomidiumbromidGenElektron <Legierung>ReplikationsursprungDoxorubicinBukett <Wein>MesomerieDoppelbindungMolekularbiologieLeucinHelicität <Chemie>UltraviolettspektrumDNS-DoppelhelixDesoxyriboseReaktionsmechanismusUranOrganspendeDeprotonierungHydroxideVerhungernIsotopenmarkierungHydroxylgruppeKohlenstofffaserStickstoffatomAktivität <Konzentration>CobaltoxidePasteTerminations-CodonSpurenelementCHARGE-AssoziationOktanzahlElektronendonatorPrimärelementAdvanced glycosylation end productsBromideFluoreszenzfarbstoffAktives ZentrumDenaturierenPolymereChemische BindungElektronentransferSchönenGezeitenstromCidreMetallPastisBiochemieWasserfallCytosinCadmiumsulfidKoordinationszahlSenseKonvertierungLäsionComputeranimation
Transkript: Englisch(automatisch erzeugt)
00:08
My name is Javert. Was that at least as good as Russell Crowe? OK. So, we're ready to get started. First, a quick quote, chemistry is to biology,
00:21
what notation is to music. To me, this really grabs at the essence of chemical biology in the sense that the notations on a musical scale allow creativity. They allow other performers to interpret the works in new ways and give the work context.
00:41
Chemistry does that in biology. Chemistry gives us an opportunity for us to be creative about biology and invent new ways of thinking about biology. It's sort of the underlying basis at the level of atoms and bonds as I keep saying for biology. And to me, in some way, this really captures what we're trying to do in this class.
01:04
OK. So, this week, we're, it's already week three, which is amazing. Oh, hang on. OK. So, it's week three. So, we're up to chapter three. And we're going to be talking about DNA.
01:22
Our knowledge of DNA was really set in place by the people in front of you. These are the giants really in the field of structural biology who determined structures of DNA in the 1950s. This includes the great Rosalind Franklin whose very accurate x-ray diffraction structures.
01:41
Her pictures of the x-rays diffracting off of fibers of DNA set in motion the determination of the structure. She was working with Maurice Wilkins. And two physicists, Francis Crick and Jim Watson, went on to solve the structure of DNA. And as we'll see in a moment, really one of their key insights was at the level of atoms and bonds in the sense
02:02
that they discovered an interesting tautomerization of the DNA bases that made it possible to have what we now call Watson-Crick base pairing between the strands of DNA. I'm getting a little ahead of myself, but that's where we're going in the next week or so. So, we're going to be finishing up non-covalent interactions, then talking
02:21
about DNA structure, DNA property, and finally DNA reactivity of small molecules. This is a large chapter. We have a lot to talk about. So, bear with me. Things are going to go not faster. It's going to be the same speed. But we're going to gloss through a few topics that are less important. And when we do, this means then
02:41
that you can focus your reading and your study just on the level of detail that we're covering in the class. OK. Some announcements. In the textbook, read chapter three. Again, skim concepts not presented in lecture. Don't get too worked up about them. Chapter three problems, do all the odd-numbered
03:01
and all the asterisk problems. In addition, I want to encourage you to get involved here at UC Irvine. This is super important. Many of you I know aspire to become physicians or scientists or pharmacists or whatever it is that you aspire to do. All those big plans require preparation.
03:21
They require some evidence that you've gone beyond the ordinary. And I want to encourage you to do this, OK? One way to get involved is to look around for opportunities to volunteer. This is one that's run by my friend who is one of the founders called the Social Assistance Program for Vietnam. If you go to this website, there are opportunities
03:41
to volunteer to spend two weeks in Vietnam in a rural part of Vietnam administering medicine. You know, you'll probably not be of course, you know, drilling people's teeth and, you know, doing open heart surgery. But you will get a unique opportunity to actually see those types of things happening. And that's really important if you aspire
04:01
to that kind of career. It provides evidence that you're qualified, that you're committed and that you're someone who's altruistic. All of those things, professional and graduate schools look for in your application. You need to be doing those things now, OK? And I'm on your side on this, OK? I will help you get and find those opportunities.
04:23
I'll bring them to your attention like this one. And if there's something in particular that I can do to connect you with, let me know and I'll do my very best on your behalf. OK. Along those lines, our laboratory always has openings for talented undergraduates. It's competitive but you have a chance to participate
04:41
at the full level of graduate student. Undergraduates in our laboratory are doing actual science. They're publishing papers with us. They're making discoveries and they're participating as full members of the team. OK. Here's how you apply. Send me a paragraph describing your career goals and how research in our laboratory would advance those
05:01
career and educational goals. In addition, send me a copy of your college level transcripts. This includes any transcripts at community colleges if you're transferring. Many of my best students are transfer students. Send me those transcripts as well. And also send me three names and email addresses of TAs who know you well in lab sections, OK?
05:22
And I'm going to email them and I'm going to ask them, what was this person like in the laboratory? Were they, you know, the first one out of the room or the last one out of the room? Did they, you know, follow you around the laboratory asking you, does this look pink, does this look pink or were they pretty independent? OK. So I'll find out about that sort of thing
05:41
and then that's how I make a decision on who to accept into the laboratory. OK. And then, of course, the resume. This is pretty standard. If you're interested in doing research here at UC Irvine, which I highly, highly encourage you to do, this is a pretty good way to go about it, OK? This is an effective way to get noticed and to get
06:00
that job that you need, OK? Any questions about these opportunities, why I think they're important, things like that? OK. See me in office hours if there's something in particular that you want from me and I'll try to hook you up. OK. Office hours this week, speaking of which. On tomorrow, I'll have my usual office hour, 2.45 to 3.45,
06:21
the usual location. Thursday, I'll have my office hour 11 to 1, usual location. In addition, Miriam will have her office hour Fridays, 1.30 to 2.30. And Krithika, could you raise your hand? So Krithika is our new TA. She'll be joining the team. And Krithika, does this time work for you, Tuesdays, 2.30, 3.30?
06:41
Good. OK. And she'll be having her office hours Tuesday. So notice that we spread out our office hours so that there's one every day of the week except Monday because I know you're very busy on Monday doing all kinds of things. I hope you're having fun yesterday. But, yeah, so every day of the week, there's an office hour. They're staggered so they're at different times
07:01
so you can have your questions answered. And again, Krithika is a graduate student in my laboratory. She knows this material as well as I do. She's really smart. You can go to her office hour and get an answer that's as good as an answer that I will give you, OK? And for that matter, you can also email the TAs with your abundant questions, OK?
07:22
I'm looking at you where I can't find that person. OK. There's like one person in the class. He must send me 10 emails a day. But, you know, I will do my best but you can also email the TAs as well, OK? Oh, along those lines, I sent you an email saying, don't send me book or potential journal articles. And the reason is I must have, I opened my inbox and I had
07:43
like 15 of those. And it got to the point where I was bouncing messages because the inbox was so full. So, if you send me those, I can't do very much with them, OK, it might clog my box. So, what I propose we do is instead of you emailing me them, instead bring them to my office hours, bring them to Krithika's office hours or Miriam's office hours and ask your questions then, OK?
08:04
Now, the standard question I get asked is, is this article appropriate? And my answer to that is if you follow the guidelines, it will be appropriate. Now, in addition, when you're writing your summary, your report on the journal article, focus on the aspects
08:20
of the article that fit the definition of chemical biology. OK, so a paper and cell for example is going to be a very meaty paper. It's going to cover about 10 pages. It's going to have, you know, eight or nine figures. And some of those figures, some of those experiments aren't exactly what we would call chemical biology. Don't focus on those. Focus on the ones that are chemical biology related.
08:42
OK? Otherwise, I don't know that you know the definition of chemical biology. OK, any questions? All right. Guess what? We're heading into midterm season. This is week three. So week four is next week. We will have a midterm next Thursday,
09:01
a week from this Thursday. There will be a review session in advance by the TAs. Time to be announced. Krithika will arrange for this. The seating for the midterm will be assigned. Miriam, you'll do the assignment. It's really essential that you bring your UCI student ID. We will check the IDs to make sure you're seated
09:20
in the right seat. If you're not seated in the right seat, it will be treated as an academic honesty infraction. No notes, no calculators, no electronic devices. You don't need them. You're smart. OK? Any questions about the upcoming midterm? OK, now I know you want to know what will be on the midterm. OK. So let me tell you. It will cover through Tuesday's lecture one week from today.
09:43
And so we will be about halfway through chapter four on Tuesday. OK? So plan to read through about halfway through chapter four. That's the chapter on RNA. And that's where I expect to be for Tuesday's lecture. It's possible I might get behind, but I'm going to really try hard not to do that.
10:02
OK? All right. I will also post a practice midterm to the website. And you can use that along with the discussion worksheets, the assigned problems as a guide for what will be on the midterm. OK? So the midterm will look very much like a compilation of discussion worksheets, of assigned problems,
10:21
and the practice midterm. OK? And it will be about as long as the practice midterm as well. So when the practice midterm comes out, I'll post two versions. One version will be blank, one version will be the key. The blank version you should print out and then give yourself an hour and 20 minutes and make sure that you can handle it. OK? And at the end of that,
10:41
then check your answers against the key. But give yourself a real practice. OK? That's pretty important, I think. OK. So anyway, that's the plan. Any questions about the midterm coming up? I know you will have lots of questions. I look forward to hearing about them in my office hours. All right. I want to go back to finish up our discussion
11:02
of non-covalent interactions. And where we left off last time was with charge-charge interactions. I'm now ready to talk to you about interactions between atoms that are uncharged. OK? Neutral atoms that are interacting with each other.
11:22
These are described by a Leonard Jones potential which is an equation that describes how these neutral atoms would interact with each other. Another way of describing these neutral atoms, another term that's used and probably one that you encountered is a London dispersion force. OK? So when you have two, say, neon atoms that cozy
11:42
up next to each other, then they will interact through a London dispersion potential or force. And that's what I'm describing here. OK? So it's just a couple of different ways of saying the same thing. This happens a lot in biology, not necessarily between neon atoms but certainly between aliphatic side chains, hydrophobic side chains
12:02
in proteins, in interactions with each other, interactions with lipids at the plasma membrane of the cell, and a whole host of other non-covalent hydrophobic, hydrophobic type interactions. This turns out to be a very potent and very strong force in biology.
12:21
OK? So we need to understand it better. So the energy, the potential energy of a van der Waals interaction, yet another word to describe it, is equal to, is proportional to 1 over R to the 12th minus 1 over R to the 6th. These terms, the sigma term deals with the diameter in this epsilon ij, not so important.
12:41
So let's ignore that. Let's focus in on the 1 over R to the 12th term and the 1 over R to the 6th term. First, notice that it's minus 1 over R to the 6th and minus in potential terms means more stable in energy, lower on this y-axis of potential energy over here. OK? So that's going to be our attractive term.
13:03
Hydrophobic, if things attract each other, OK, not just due to repulsion from water, we'll talk about that next, but hydrophobic things want to stick to each other and they're going to do this with an attraction that's proportional to 1 over R to the 6th. The fact that it's 1 over R to the 6th as opposed to R
13:21
to the 2nd in the charge-charge interactions means that this is a much shorter range attraction. This attraction takes place on a very tiny distance scale. OK? Now, eventually, the two atoms in this case as described here are two molecules or two molecules bang
13:44
into each other and go past the point where they're attracted to each other. OK? And at that point, their electrons are trying to overlap with each other. That's really bad news, right? We know by the Pauli exclusion principle that that's not allowed and so in the same way that my fingers are never going to fuse with each other, just going to bang off of each other,
14:02
the atoms push away from each other and they push away from each other with the repulsion force or repulsion potential that's proportional to 1 over R to the 12th. OK? And so this means that this is extremely short-ranged and extremely sharp, right? To the 12th power is a large number.
14:21
So this means that this really dramatically pushes apart the atoms if they happen to get too close to each other. Turns out that there are a whole series of other non-covalent interactions that we find in biology that actually contribute quite a bit of non-covalent binding energy.
14:41
Here, for example, are the dispersion interactions that we've discussed before on the previous slide. And so these include things like aliphatic-aliphatic interactions, but also aliphatic interacting with hydrophilic molecules. So here's water interacting with methane. They're going to interact with each other
15:01
and have some attraction. This number here, minus 0.5 to minus 0.7 kcals per mole is pretty low. OK? This is not a tremendously strong interaction. Where it gets strong is when you have a molecule that has a hard, large number of functional groups, each one with 0.5 kcals per mole here, 0.5 here, 0.5 here.
15:23
And when you sum up across all of those, you're starting to talk about big energy. OK. Now, just to give you an idea, you need to know one fact that I think is really important. And the fact is important enough that I'm going to try to write it on the board over here in the corner.
15:41
The fact is that a factor of 1.4 kcals per mole will be a factor of 10 in equilibrium constants. OK? So, 1.4 kcals per mole is a magic number in chemical biology.
16:02
OK? So look for 1.4 kcals per mole because that tells you that that's favored tenfold over non-binding. In other words, the interaction is going to be 10 times more likely to form than not form. OK? It's a factor of 10 in terms of equilibrium constants. OK? So if we're talking about something over here that's only 0.5, 0.7 kcals per mole,
16:23
you have to start summing up a whole bunch of these to get anywhere in terms of enforcing the interaction. On the other hand, some of these other interactions can be quite strong. And let's take a closer look at those next. OK? So, for example, we've talked a little bit
16:43
about hydrogen bonding. Hydrogen bonding, of course, has a donor and an acceptor. And here's a range of strengths. Hydrogen bonds vary enormously in strength from about 1 kcals per mole all the way to 7 kcals per mole. The strength of the interaction depends enormously
17:02
on the identity of the donor and acceptor. When the donor and or acceptors are charged, if either one is a charged functionality, the strength of this hydrogen bond goes up enormously. And this kind of makes sense, right? Because remember earlier, I described a hydrogen bond
17:21
as a kind of a special case of a charge-charge interaction in which a hydrogen is being shared between two atoms. OK? So, if one of these happens to be charged, that's going to be a much stronger charge-charge interaction. Speaking of charge-charge interaction, salt bridges are the Coulombic potential
17:40
that we saw on Thursday. These are the charge-charge interactions. These vary also enormously depending upon the environment that the salt bridge happens to find itself in, where water can shield this charge. Water or counter ions can shield this charge,
18:02
decreasing it considerably and making the interaction much, much weaker. So, a salt bridging interaction, which is another way of saying charge-charge interaction, found in a hydrophobic environment, say the interior of a plasma membrane is going to be a much stronger interaction
18:22
than one that's found out in water where there's plenty of water and counter ions to shield the charge, OK, where those provide a counter against the charge. Recall that those environmental terms are embodied by the 1 over 4 pi epsilon term in the Coulombic potential
18:43
that I showed you on Thursday. OK. In addition, there's also dipole-dipole interactions which are alignments of densities of charge where we have a little bit more negative charge on the oxygen over here. The dipole is pointing in this way on the, to the right
19:03
on the upper acetone and to the left on the lower acetone. The two of these dipoles want to cancel each other out. By canceling each other out, that will give you a more optimal interaction and that's where some potential energy.
19:20
Finally, there's also a whole series of aromatic or arene interactions. And in general, these include both face-to-face interactions where you have two faces of a benzene ring that are interacting with each other. Notice in this picture over here that the top benzene ring is offset from the bottom one
19:42
and this makes sense. We're going to be looking at regions of electron density, interacting with regions of electron poverty, OK, that that's actually the basis for the interaction. And so, for that reason, we also see very commonly edge-to-face interactions, OK. So, this is the one that we'll see in a moment
20:01
when we start looking at pi stacking in DNA, but in addition, you can have a edge of an aromatic system interacting with a face of another aromatic system down here and that's as strong, right. It has the equivalent strength. Even though you expect, you know, face-to-face to be ideal, that's actually not what we see when we start looking
20:21
at large numbers of aromatic interactions. We see these edge-to-face interactions all the time. OK. And then finally, there's some other ones that are really bizarre and they include charged interacting with the electron-rich aromatic rings. And this kind of makes sense, right. You have something that's positively charged.
20:44
You have something that's very electron-rich in terms of the ring system. So, these cation-pi interactions, which is what this one is called, are found pretty ubiquitously in biology, oftentimes playing a commanding role, playing a really key role in chemical biology, OK.
21:02
So, these are ones that I'd like you to memorize. I'd like you to know something about their strengths, which one is strong, which one is weak. I don't want you to memorize the numbers per se, but I want you to know something and be conversant on relative strengths, OK. Relative strengths matters, OK. And one thing, one last thing to keep in mind if we're going
21:21
for this 1.4 kcal per mole, again, you can have a summation of a large number of interactions to achieve that 1.4 kcals per mole or even more. And I'll show you an example of that very shortly. Now, it turns out that it's actually a little bit tricky to start comparing energetics when you design in, say,
21:42
the perfect cation-pi interaction. What ends up happening is that you get a complication due to water, OK. So, let's imagine that you had designed in the perfect cation-pi interaction. And in doing so, you put this positively charged thing that forces all of the water around it
22:02
to rearrange itself or reorient itself. Turns out that's actually a complicated thing of orientation of water, but it cannot be neglected, OK. So, what we do is we make a very important simplifying assumption and I'll talk more about water on the next slide. But before I do, water, since we just have
22:22
to acknowledge in advance, water can complicate everything, right. It's present at 55 molar concentration in your cells and we can't neglect it, OK. It has its own energetics. It's, as I showed on this slide over here, for example, it's interacting with hydrophobic things. So, its own energetics are really complicated, OK,
22:43
and actually very hard for us to understand and pin down. And so, it's really difficult to estimate the entropy lost or gained in an interaction due to that rearrangement of water when you start making changes. So, what we'd like to do is compare things that are
23:01
as similar to each other as possible, OK. This is the simplifying assumption that I alluded to earlier. Here, for example, is an example of that, OK. So, here's two possible transition states and transitions are two possible mechanisms. Mechanism number one involves an SN2 reaction.
23:22
Mechanism number two involves the same molecule undergoing an E2 elimination reaction. And the key here is that the molecules are identical, OK. That extreme similarity makes the comparison in between these two much easier to make, OK.
23:40
And so, for example, if we're looking at two proteins, we can look at empty protein versus ligand bound protein. But on the other hand, we're not trying to make all kinds of changes to the protein structure over here. Problem is, proteins are rarely, you know, like looking like this when the ligand is unbound.
24:00
So, these simplifying assumptions will start to cause all kinds of problems. Here's one though that works. You can make a single change to the surface of a protein and then compare the altered protein, compare its interaction with a ligand. So, for example, we could change this isopropyl group
24:21
to a methyl group and then compare what's happened, what's different in that receptor ligand interaction, OK. So, all you've done is remove two methyl groups. That's about as simple as it gets, right? So, that type of experiment is an easy one to make comparisons to, OK.
24:42
And again, by doing that, we're trying to minimize how much the water has to rearrange itself at that interface, OK. It turns out actually this assumption works most of the time. So, in short, being good scientist, not changing lots of variables at the same time pays off in biology
25:00
because underlying everything we do is this complicated solvent that we operate in called water. Let's take a closer look at the structure of water, OK. So, here is water in ice and notice how neatly regular it is and how nicely ordered it is. And then here's water in solution as liquid water. And it's just crazy complicated.
25:22
First, notice that there are all these dots or dotted lines are the hydrogen bonds. These hydrogen bonds are pretty much maximized. Water is not passing up any opportunities to hydrogen bond to itself, OK. But, the hydrogen bonds in the liquid solution are non-optimal, OK.
25:41
Water in solution, each water molecule is jam-packed with other water molecules and oftentimes the hydrogen bonds are slightly distorted or they don't have the right distances. Those little distortions and that lack of perfect distances makes the hydrogen bonds in liquid water weaker than they are in solid water.
26:03
Furthermore, a molecule of water in its own, you know, with a lot of other molecules of water is behaving kind of like it's on a crowded dance floor, OK. So, it's bouncing around wildly against this other,
26:21
you know, molecules that are nearby and interacting with lots and lots of different molecules nearby, constantly breaking interactions and forming new ones. Welcome, OK. So, water is actually very complex, weak and distorted hydrogen bonds, OK.
26:41
In addition, when water cozies up to hydrophobic surfaces, it tends to form a very ordered structure that starts to look a lot like the structure found in ice. And this works by water satisfies its propensity to form hydrogen bonds by forming a clathrate-like structure.
27:01
So, for example, here is a molecule of methane encapsulated in one of these clathrates of water where clathrate is just simply a structure of water that satisfies its desire to form hydrogen bonds with itself, OK, or with other molecules of like kind, OK. This really dramatically changes the strengths
27:22
of nearby non-covalent interactions, OK. This does things to strengthen those non-covalent interactions because every time one of those, let's just say hydrophobic-hydrophobic interactions breaks, then water has to slot in between the now broken interaction and form one
27:41
of these clathrates, OK. That formation of the clathrate, the formation of an ordered structure costs energy. It's a loss of entropy. This is a more ordered structure than the structure of disorganized water that I showed you earlier in solution, OK. So, for this reason, hydrophobic molecules are driven
28:05
against each other. They want to find each other in water. And this is sometimes referred to as a, this is actually a water-driven effect.
28:21
I'm forgetting the technical word for this. Miriam? OK. Anyway, so, oh, sorry. It's sometimes referred to as a hydrophobic effect, OK, in water. OK. Now, let's take a closer look at a receptor-ligand interaction now zooming in at the level
28:43
of atoms and bonds. This is a molecule called human growth hormone. And yes, Lance Armstrong admitted to Oprah that he took human growth hormone to win to help him recover basically from different stages of the Tour de France during all seven of his victories. It really annoys me actually.
29:03
I could say a lot more about that, but I'm going to hold myself back. OK. Now, when human growth hormone binds to its receptor on the surface of cells, it's stimulating growth and recovery of those cells. It's stimulating protein production, et cetera. And when it binds to the surface of the cell,
29:22
to the binding partner on the surface of the cell, its receptor, then all of the region that's colored in on the surface is buried, OK? So, in other words, human growth hormone binding protein binds over here and then makes contact with each of these colored atoms.
29:41
OK. Everything that's in white here is still out in water, out in the solvent. It's not interacting with the receptor at all. Now, when I was a postdoc, I repeated a classic experiment that was done by Jim Wells. And Jim Wells and his coworkers found that even though there are 19 residues that are buried
30:02
on the surface, there are 19 amino acids that are buried, only the ones in red are actually contributing binding energy, OK? So, notice that all of this other stuff that is in blue that is buried is not at all contributing any binding interactions. So, although there's interactions
30:21
between these side chains of these two proteins, there is no binding energy that's being exchanged or gained by that interaction, OK? So, just because two molecules find each other, or two functional groups find each other in space does not ensure that there's actually going to be a net gain in binding energy.
30:41
Because again, that net gain in binding energy includes both the strength of the interaction but must also include the water ordering and disordering term which we've been calling entropy earlier, OK? So, in order for this interaction to take place, you're going to be pushing out ordered water and gaining some entropy in some places
31:01
and in other places losing some entropy. OK. Now, when we look even more closely, let's just zoom in on this red patch over here. This red patch has been termed a hotspot of binding energy. That's where the binding energy allowing these two molecules to interact with each other is found, OK? This is the essence of the non-covalent interaction
31:22
between human growth hormone and its binding partner, human growth hormone binding protein. And in green, these are the functional groups that are found in this red patch, OK? So, the red patch is over here and now I'm showing you the functional groups where in green, these are carbon atoms, in blue,
31:43
that's a nitrogen and in red, that's an oxygen. OK. Notice that the hydrophilic functionalities, the guanidine of an arginine over here, a bunch of nitrogens, another nitrogen over here, an oxygen, an oxygen over here. Notice that those are around the periphery
32:00
of this red region. They're around the outside of this hotspot of binding energy. The center of the hotspot is largely hydrophobic, OK? Notice that it has lots and lots of carbons. There's a benzene ring and smack in the center, there's this aliphatic chain that's capped by an amine functionality.
32:21
But nevertheless, this is an aliphatic chain. There's aliphatic functionalities over here and over here and over here, et cetera, OK? So, in other words, the outside hydrophilic, the inside hydrophobic. And so, when molecules, functional molecules find each other, this is a very common way for them to interact
32:41
with each other through a small set of residues that form this hotspot of binding energy, which again, kind of looks like a core sample through a protein. Outside is hydrophilic, inside, hydrophobic, OK? Any questions so far? OK. Let's talk one last, about one last section
33:06
of Chapter 2 before we move on to Chapter 3. There's this concept that the bio-oligomers on Earth are highly modular. We've discussed this before. This also extends to the polyketides and the terpenes which are composed of isoprenes and the polyketides
33:22
which are caused, which are composed of either malonyl or acetyl subunits that are strung together where the red bonds indicate that where the connection between these modules such as the amino acids as individual modules in a protein, OK?
33:40
And furthermore, this is also found in oligosaccharides where you have this glycosidic bond that connects the glycan fragments together. There's also a numerical amplification in biosynthesis. So, if there's only one or two copies of DNA per cell, depending upon whether it's a prokaryotic cell
34:02
or eukaryotic cell, some prokaryotic cells admittedly are more than one, but let's just simplify it. Then, to RNA, each DNA is transcribed 10 to 50 times and then each RNA is translated, say, 10 to 20 times. So, in the end, you end up with this massive amplification
34:23
of signal going through the cell where with one copy of DNA you can end up with millions of products from some enzyme reaction down here. Last thought, form follows function in biology. These, the bonds that join together,
34:42
the oligomeric subunits are, have a strength that follows their function, their functional requirements, OK? And so, for example, when we look at the half-life of lipids, we find that actually the ester bonds
35:01
in a lipid have a half-life on the order of a year or so. OK? So, esters, not so stable. Compare that against DNA down here which has a half-life on the order of 220 million years, OK? That's its half-life for DNA. And in retrospect, this kind of makes sense, right?
35:23
Because DNA has to be a, you know, has to be a bioligomer for the life of the organism, OK? And so, we're now at the point where we're routinely taking advantage of this tremendous stability of DNA to amplify DNA from even extinct organisms like wooly mammas, like species
35:47
of prototypical humans that haven't lived on the planet for tens of thousands of years. That sort of thing is going on right now in laboratories taking advantage of the tremendous stability of DNA. Now, your hair which is a protein has a lifetime
36:03
on the order of, you know, 300 years or so. And you can see that, right? We can find, you know, we give, well, anyway, so I guess it depends on the human that we're talking about. My hair obviously doesn't exist that long. But, you know, so certainly the lifetimes here are
36:22
following their function, right? Proteins don't have to last as long. Question, how does one get a PhD that's going to take you five or six years studying and trying to measure these half-lives of 220 million years? Anyone have any ideas how to do that experiment?
36:41
I can guarantee it to you, it's not like, you know, you set up this test tube and then you check it every 20 years, OK? To see how much gets cleaved. How would you do this?
37:01
Yeah. How would you do it? OK. Small amount of RNA. Now, I would use a large amount because very little is going to get degraded. How would you do this though? Yeah. OK.
37:26
But then you wouldn't know if the decomposing environment is different than in the cell, right? We want to know about the half-lives in the cell, right? Yeah. Model organism. Now, I want to know what it's going to be,
37:43
you know, in this cell or this other one over here. Question over here? OK. You're definitely going to use radioactivity because you need something that's super sensitive. How would you do this?
38:04
OK. You're getting close. What is your name? Brian? OK. Brian is getting close. So, the suggestion was radioactivity. Brian's suggestion is you look for a tiny little quantity and radioactivity gives you that sensitivity. But are you going to do this for 220,000 years or 220 million years?
38:21
OK. So, how are you going to do this experiment? We have the sensitivity. We're going to look for tiny little quantities and extrapolate back. How are you going to model 220,000 years? Yeah. Carl? OK. Look at fossils. Yeah. We do that.
38:41
Yeah. OK. C14? C14. OK. Yes.
39:02
OK. So, but the problem is you want to know all the conditions it's experienced over, you know, say 100,000 years or something, right? So, I mean, how do you, you want to do this in a controlled circumstance. You want to have everything just in a little test tube
39:21
where you know exactly what's been added to the test tube, right, but you don't want to wait around for 220,000 years or 220 million years. What are you going to do? OK. I'd like you to look this one up. This is one that you should be able to design.
39:41
Look it up and then when we come back on Thursday we'll talk about this. But I'd like everyone to have looked this up. OK. This is important. OK. Let's talk, let's summarize what we've been talking about in terms of non-covalent interactions. These are completely ubiquitous in biology. Good news, we only have to learn two equations
40:01
which govern all interactions in chemical biology. Those were the Coulomb's law for the charge-charge interactions and the Lennard-Jones potential for the uncharged interactions. OK. And so if we know those two equations, we're set. What's really important, what's important to us is not
40:21
that we're going to be plugging in, you know, charge of this and then, you know, radius of this. What's important to us are the relationships, the distance dependence, the 1 over R squared versus 1 over R6, that type of distance dependence makes a big difference. And knowing that sort of thing and having sort of an intuitive grasp of that is going to be very important.
40:43
So, and I'll just give you a quick example. For example, we now know if DNA is negatively charged, it's going to attract other charged ions to it from great distances, right? Because it's distance dependent, it's only 1 over R to the second power versus 1 over R to the sixth.
41:01
In addition, we've learned that these non-covalent interactions are very sensitive to the environment, the distance and the geometry. Water is a really slippery molecule to understand, to say the least, as malleable structure and it can dramatically alter the strength
41:20
of non-covalent interactions. This makes it really tough for us to draw any generalities because water is an intermediate lubricant between all of these interactions and it plays a complicated and sometimes hard to us, hard for us to define role. And there's still big arguments that are going
41:42
on in water chemistry to this day. For example, there's an argument going on about how many ions are found on the surface of water or what's the pH at the surface of water. And there's been a set of dueling papers that have appeared that contradict each other. The first paper had a title like the pH of the surface
42:03
of water is more acidic. The next article by the competitor said the pH of water at the surface is more basic. And the two and these groups have been arguing backward and forth and both making very reasonable arguments for years. OK. The truth is what we found is actually it's somewhere
42:22
in between those two and you can actually see evidence for either one and it turns out to be a very minor effect that's not so important in biology. But the point is, is that water itself is such a complicated fluid that we're still using the latest techniques to try to understand it better. It's not fully understood.
42:40
Hydrogen bonds have donors and acceptors and they're also very susceptible to competition with water for those hydrogen bonds. I would like you to know the approximate strength, the relative strengths, not the approximate but the relative strengths and distance dependence of non-covalent interactions, that's important.
43:01
OK. So that's a summary of chapter two. Any questions about chapter two? Yes, Chelsea. Yeah, I really want you to know that.
43:21
OK. That's super important. That's that Henderson-Hasselbalch equation. That hopefully you learned in Chem 1. You definitely need to know that. Other questions? OK. Let's move on. I want to talk to you about the structure of DNA. This is the classic structure of DNA first proposed by Watson and Crick in I believe 1952 or, yeah,
43:43
1952, somewhere in there. The structure of DNA has two strands running in opposite directions to each other. So they're anti-parallel to each other. These strands are held together by phosphodiester bonds which we'll look at more closely.
44:01
So here's a schematic diagram of what the structure of DNA looks like. And here's a space filling view where each one of these spheres is a van der Waals sphere to approximate where the atoms are, where the outermost electrons of the atoms are. One thing to notice is that DNA has two grooves.
44:22
OK. It has the distance here between these two strands is very close versus the distance here between the two strands being much further away. These are going to be called the minor and major grooves respectively. And this is the origin of the fact that DNA is a double helix.
44:41
I think it's commonly thought that DNA is a double helix because it's two relatively rod-shaped molecules that are twisted with each other. But that's actually not the case. It's a double helix because it has a minor groove and a major groove. And I believe the next slide will show us that more closely.
45:00
OK. So, in blue, this is the major groove of DNA and in green, this is the minor groove. In red, this is the phosphodiester backbone of DNA that we've seen before. OK. So again, notice that there are two helices that are running parallel to each other, a major groove
45:21
and a minor groove. OK. The structure of the bases is going to set up this major and minor groove relationship. As we will see shortly, DNA bases, base pairs form a U shape and that U shape ensures that you're going to get a major and a minor groove where the inside
45:40
of the U is going to be this minor groove and the outside will be the major groove. But I'm getting a little bit ahead of myself. The reason why this is important is as we'll see in a moment, proteins like to interact with the major groove of DNA whereas they can't fit in to the much closer interspecies of the minor groove of DNA.
46:00
Rather, small molecules will fit into this minor groove and try to largely avoid the less cozy major groove of DNA. OK. So, almost immediately, we can start to make some predictions about where stuff binds just knowing that DNA is a double helix, double by virtue of the fact that it has two parallel helices,
46:22
minor and a major groove. So, this DNA structure immediately sets up replication. This is the original 1953 paper by Watson and Crick and this is the very last sentence of the paper in which they had this incandescent understatement. It has not escaped our attention,
46:41
has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material. OK. So, if you have two strands of DNA running anti-parallel to each other, you can simply separate out the two strands
47:01
and then get a perfect copy of one strand over here and a perfect copy of the second strand over here. OK. So, here's the parent strand of DNA and again, here are the two new strands in orange and blue. Note too that DNA forms a right-handed helix.
47:20
OK. Does everyone see that you can trace out along the right, with your right hand over here, the structure of DNA? I think it's worth trying that, whereas your left hand kind of slips off. It doesn't trace it out effectively. OK. Does everyone see that? So, it's DNA is always a right-handed helix.
47:44
You know, so this beautiful structure of DNA is one that was solved by X-ray crystal structure. Before then, there were a large number of wrong, incorrect predictions about DNA structure, including by people who I, you know, think the world of. I think are, you know, absolute heroes in science.
48:01
For example, the great Linus Pauling who proposed a triple helix of DNA where the phosphodiester backbones would be in the center of the molecule and the bases would be out on the outside. This kind of, this is somewhat, this is intellectually attractive if you don't think about the fact that you have two parents.
48:22
But furthermore, it's attractive because at least the base pairs would be out here in space where they can interact with transcription factors. We now know, of course, that that's not correct. Instead, we'll take a look in a moment at where the transcription factors interact. Before we do, let's zoom out a little bit. OK. So DNA in the cell is concentrated in two regions,
48:45
a nucleosome in the prokaryotic cell. So it's kind of concentrated in the very center of an E. coli cell. In a eukaryotic cell, of course, DNA is found exclusive, is found in the nucleus and also the mitochondria. But let's just focus on DNA that's
49:00
in the nucleus for today. The bases themselves are connected together to form oligonucleotides through these phosphate, these phosphodiester functionalities. OK. So this is called a phosphodiester functionality.
49:22
The DNA also has a directionality associated with it. OK. So there's, if we look closely at this deoxyribose base, there is a five prime end. There's a five prime hydroxy over here and a three prime hydroxy over here. And so, the convention is to always write DNA
49:43
in the direction from five prime to three prime. In the same way that we read English going left to right, DNA is always read out five prime to three prime. This is a really important convention. OK. Everyone on the planet follows this convention.
50:01
And I'm going to hold you to it as well. OK. Because if you read the DNA in the opposite direction, you get a different word coming out. OK. It spells something else that might not be this. It will almost certainly not be the same thing and it might actually be, you know, it might actually cause a lot of trouble. So we're always going to be reading this five prime
50:22
to three prime directionality. So this sequence here would be read out as A, C, G, and T. OK. Where the structures of A, C, G, and T are shown here. OK. Don't bother memorizing the structures of these.
50:42
I'll simply give them to you on the midterm. OK. So, at a graduate level, you should know these. Miriam will need to know these for her oral exam. But the rest of you are in luck because I'm not going to test you on them at least for this class. OK. And again, the directionality matters a lot.
51:02
If there is a five prime phosphate, this five prime phosphate is indicated by a lowercase P. Finally, last bit of nomenclature, oligonucleotides that are connected together are often referred to as oligos and that's how I'll describe them. OK. Now, I realize oligos is not the most descriptive
51:22
nomenclature because it just simply means an oligomer or something. But that's the convention that we've been operating under for 50 years. OK. So, oligos will refer to oligonucleotides. Typically, DNA oligonucleotides composed of deoxynucleic acid.
51:41
OK. Now, even though DNA is the bases of DNA are called bases, it turns out they're not that basic and few are protonated at physiological pH. It's this is kind of one of those historical anomalies. Here's a bunch of PKAs. For example, starting with triethylamine.
52:01
Here is the pKa of the protonated triethylamine, the conjugate acid of triethylamine, pKa of 10.8. Here is the pKa of cytosine, thiamine, adenine, and guanine. And you could see none of these would be remotely considered bases. Whereas this one over here, triethylamine,
52:23
definitely a base, OK, as evidenced by the fact that it's conjugate acid is, you know, 10.8 pKa. OK. Questions so far?
52:40
All right. Now, DNA, of course, is missing a two prime hydroxyl. OK. So, here's RNA. It has a two prime hydroxyl over here. This two prime hydroxyl makes RNA considerably less stable than DNA. I didn't point this out. Let me go back to it when we talked earlier about half-lives.
53:02
Let me just zoom back to that really fast. The half-life of RNA is considerably lower than the half-life of DNA. OK. So, here's the half-life of RNA, 220,000 years, whereas the half-life of DNA at 220 million years is much, much greater.
53:24
OK. A thousand fold difference in stability for the phosphodiester backbone of the DNA versus the phosphodiester backbone of RNA. This makes sense. OK. The two prime hydroxyl of RNA sets you
53:41
up for hydrolysis using an intramolecular attack. OK. So, here's again the structure of RNA. Here's the two prime hydroxyl. This two prime hydroxyl can act as a nucleophile to attack the phosphodiester backbone
54:03
of the RNA setting up cleavage. Does anyone want to see the mechanism of that? OK. All right. Let's take a quick look. OK. So, in this mechanism, let me just draw
54:27
out the structures and then I'll blank the board. OK. One sec. OK. So, in this mechanism, here's our structure of.
54:55
So, here's our backbone structure of RNA. And I'm just going to draw this as base over here.
55:03
OK. OK. So, if there is any base that's present, let's just say hydroxide. This can deprotonate the two prime hydroxyl giving us
55:26
like alkoxide adjacent to the phosphodiester backbone of the DNA.
55:49
This neighboring alkoxide can now attack the backbone, the phosphodiester backbone giving you a five-membered ring intermediate, OK, which I'll show down here.
56:25
Five-membered ring intermediate and this intermediate collapse leading to cleavage of the RNA. OK. So, here is that collapse.
56:43
OK. So, we're going to be making two strands of RNA that are separated from each other. OK. So, here's one strand over here
57:04
and then here's the second strand down here. OK. I'm going to just differentiate these
57:23
as base one and base two. OK. So, notice that the strand has actually cleaved apart. You can then hydrolyze this phosphodiester backbone, this phosphodiester back to a phosphomonoester using another equivalent of hydroxide.
58:05
And then finally, collapse of this tetrahedral intermediate gives us the product. OK. Questions about this mechanism?
58:20
All right. Now, notice again if DNA lacks this two prime hydroxyl over here and I just want to make this totally explicit, I'm going to label it two prime hydroxyl, three prime, five prime, OK. So, DNA lacks the two prime hydroxyl
58:42
and therefore does not have an opportunity for this intramolecular nucleophilic attack on the phosphodiester backbone. So, for this reason, DNA is a thousand times more stable than RNA, right, lacking this intramolecular nucleophile. Make sense?
59:01
Questions about this? OK. Let's go back. Turns out that when you look at the liability of the bases, we see actually a different trend, OK.
59:23
And actually, I think I'm going to skip that. OK. Moving on. OK. I'd like you to learn what I just told you. Don't worry so much about the base stability. DNA bases are subject to important modifications. These modifications have dramatic roles
59:40
on the phenotype of organisms, OK. So, for example, methyl groups are often transferred to DNA. I showed you structures of DNA bases. Again, they're subject to massive modification by methyltransferases and other modifications. So, for example, here's
01:00:00
5-methylcytosine over here, 4-methylcytosine and then N6-methyl adenine. These modifications can dramatically alter transcription levels. They can set up the organism to transcribe some genes more often, OK? So, for example, I'm lacking pigmentation.
01:00:23
The genes that encode pigmentation are in my skin cells, my epidermal cells, yet they're not transcribed very often. And so, it's likely that my DNA has not been methylated in those regions. However, when I go out and spend a lot of time in the sun, I'm getting additional little spots called freckles which are resulting
01:00:43
from methylation of those DNA sequences which in turn then turns on transcription of the pigmentation and results in freckles, OK? So, the environment, the environment that you're exposed to can alter this, these transcription patterns.
01:01:03
It's one of the ways that organisms like ourselves respond to changes in the environment. It's a very important way in fact. And oftentimes, this goes through methylation of DNA. This DNA methylation is really as important as sequence or genomics.
01:01:20
And this is an area called epigenetics that's really an area of very active research that's taking place in chemical biology. OK. So, we've looked at structures of the bases themselves. We've looked at structures of the phosphodiester backbone. Let's start putting things together to start to understand the structure of DNA. The bases themselves are slightly U-shaped, OK?
01:01:43
So, here's a base between A and T, adenine and thymine. Notice that this base is composed of two hydrogen bonds. Here's a base of G and C which has three hydrogen bonds. But notice more importantly that the bases are U-shaped
01:02:01
or equally importantly, OK, U-shaped here. The inside of this U where the R is going to be the towards the ribose, the deoxyribose ring, the inside of this U is going to form the minor groove which I showed you on an earlier slide. The outside of the curvy part of this U is going
01:02:21
to form the major groove. As you have these U's that are stacked on top of each other and each one is slightly offset with each other, this is outside is going to result in a much bigger helix than the inside over here, OK? And here's what this looks like, OK? So, here's a trace of the phosphodiester backbone
01:02:42
and then I've highlighted just one Watson-Crick base pair, OK? And again, notice that it's U-shaped that there's more section traced out over on this side, that will be the major groove and the inside will be the minor groove. Furthermore, the green arrows define hydrogen bond donation
01:03:02
and acceptance by the base pair. And notice that there is a pattern to this, that there is an acceptor-acceptor donor, OK? So, this is a donor-acceptor donor over here. So, there's actually a little bit of a pattern
01:03:21
to whether this is a G on this side and a C on this side or C and G on the opposite sides. So, in other words, A and T are not the same as T and A because they're going to present a different pattern of hydrogen bonds for molecular recognition where, again,
01:03:43
the proteins are going to be the transcription factors are going to be interacting over here in the major groove and small molecules will be interacting in this minor groove down here. I should mention that there's also some protein DNA interaction in the minor groove. It tends to be more minor, however.
01:04:03
OK. Let's take a close look at one example of a transcription factor and how it works. This is the transcription factor phosgene. It consists of a leucine zipper which is a two helices that interact with the DNA like chopsticks, OK?
01:04:22
So, these are fitting neatly in the major groove. It turns out the major groove has exactly the right size to accommodate an alpha helixal protein, OK? So, this phosgene is absolutely perfect. It fits neatly in the major groove. Now, these hydrogen bond donating functionalities are
01:04:40
going to then read out the sequence of the DNA and look for a specific sequence of DNA to interact with trying to form complementary hydrogen bonds, trying to form complementary van der Waals interactions in this sequence, OK?
01:05:00
Let's take a close look now at the forces holding together the DNA double helix. Earlier, I alluded to the fact that AT base pairs form two hydrogen bonds and GC base pairs form three. Which one is stronger? Just, you know, from a crude approximation. Yeah, three is stronger than two, right?
01:05:22
OK. So, in addition to this, the DNA structure is held together by pi stacking between the bases. Again, this is a face-to-face interaction typically not perfectly face-to-face, rather it's typically offset. And that offset leads the bases to stack not directly
01:05:42
on each other but slightly twisted from each other, setting up this helical structure that we're now familiar with. In order for this base pairing to take place, the base pairing that I showed on the previous slide, you need a particular tautomer of these aromatic rings, OK?
01:06:02
And the first one that should strike you as funny is this one over here. Because you can imagine another resonance structure that would make this C aromatic, right? Notice that the C has its non-aromatic in this tautomer shown here, right?
01:06:21
It only has two pi electrons rather than the requisite six that it would need to be aromatic, OK? That's almost, that's bizarre to begin with, OK? So, what's going on here is that there is a preference for this tautomer versus this one. This one is actually thermodynamically more stable.
01:06:41
And the reason for this is that the carbon-oxygen double bond over here is quite strong. I will tell you that I think any chemist looking at this could not have predicted this in advance. And in fact, actually this tremendously slowed structure determination of the original structure of DNA back in the 1950s.
01:07:01
Watson and Crick were physicists and weren't as familiar with the whole notion of tautomerization as their chemical counterparts who are racing to solve the structure of DNA. And so, for them, this did not look funny whereas to us, I think it does look funny, right? Because it lacks aromaticity whereas the structure
01:07:21
on the left is aromatic. Again, this happens to be just a little bit more stable because of the strength of the carbon-oxygen double bond but I don't think anyone would have predicted that, OK? I think now we, you know, with our 21st century guys we could predict it but going back in time, I don't think we could have predicted it so readily.
01:07:40
Similarly, over here, these amadines are actually going to be more stable in the aromatic structure than in the amadine structure. And in this case, that's due to the much poor overlap between a carbon-nitrogen double bond than a carbon-oxygen double bond, OK? So, all of this leads to the base pairs
01:08:02
with the hydrogen bonding preferences that are shown here, OK, whereas for example, this is a non-aromatic ring that could be aromatic if it's tautomerized but it doesn't prefer to be tautomerized whereas this one over here seems to prefer to have an amadine in this structure
01:08:20
because of the strength of a carbon-nitrogen double bond. OK, here's another example of that over here. This one prefers aromatic because carbon-nitrogen double bonds are relatively weak. OK, pretty interesting, huh? A natural basis, however, could dramatically shift these
01:08:41
preferences for tautomerization. And a good example of this is 5-bromouracil, OK? So, if this compound here is fed to organisms, what happens is an unusual tautomerization preference where the enol form of bromou is actually more preferred
01:09:10
than it would be if there was no bromine over here, OK? So, most of the time, it forms the regular base pair. However, some of the time,
01:09:20
it can actually form the incorrect base pair because it can actually more readily access this enol form of the base, OK? So, that's due to the electron withdrawing functionality of bromine over here, OK? That's changing this tautomerization preference.
01:09:41
The consequence of this are really dramatic because the Watson-Crick base pairing is not followed as closely. What ends up happening is the DNA comes out with all kinds of bizarre breaks and lesions, OK? So, here's chromosomes from a normal organism.
01:10:00
I think it's a hamster in this case. And then here's chromosomes from hamsters that were exposed to bromouracil. And you could see they have all kinds of bizarre shapes to them. Things are incorrect, OK? So, this causes cancer and breakages in DNA which then eventually lead to cancer cells
01:10:22
and tumors in the organism, OK? All right, so furthermore, it turns out that we can test the importance of the strengths of these hydrogen bonds by synthesizing a natural basis.
01:10:40
So, this is one of the great things about chemical biology. If you have this hypothesis that something is important, then you could test that hypothesis by synthesizing compounds which are, say, missing that key functionality. So, from Watson and Crick, we expect to find that hydrogen bonds are holding together the structure of DNA
01:11:00
and chemists went out and synthesized variants of DNA basis that were lacking that ability to hydrogen bond, OK? Structures of these are shown here, OK? So, for example, this compound here is simply a pyrene in place of a base and it actually prefers to base pair
01:11:23
with a missing base over here, OK? So, these guys over here, no hydrogen bonding, no hydrogen bonding over here. And yet, these actually prefer to pair with each other, OK? So, you can actually have completely a natural basis, missing hydrogen bonds that are yet able to form base pairs
01:11:45
with each other preferentially. What this tells us is that there's more going on in DNA structure than simply hydrogen bonding. Hydrogen bonding is a nice simplifying assumption for our biochemical friends, our molecular biology friends.
01:12:01
But in actuality, the pi stacking of DNA is a driving interaction. The edge-to-edge interactions of aromatic functionalities are also driving this interaction between the strands of DNA. And so, while we can do quite a bit with hydrogen bonding, there's quite a bit more that's left to be explored.
01:12:23
OK. Last thought, I've been showing you, or it's not last thought. I've been showing you, oh, before I get to that, here's, here for example, is this illustration here emphasizes the importance of pi stacking in here, OK?
01:12:44
So, one thing is that bigger bases tend to pi stack better. For example, the guanine base tends to pi stack better than say cytosine. In addition, I've been showing Watson-Crick base pairing where it's a canonical base pair, Gs and Cs,
01:13:00
have three hydrogen bonds, As and Ts have only two. Other kinds of hydrogen bonding possibilities are not only possible but have been observed. These were proposed by Carl Hoogstein and we observed these a lot in RNA structure. We don't necessarily see these in DNA but we definitely see these in RNA and they're going to come up later.
01:13:20
So, I'll just show you the structures here. This is an alternative to the usual AT base pair and this is an alternative to the usual CG base pair, this one being driven by a protonation event, protonation of this nitrogen over here. OK. So, this is actually, these are sort of edge-to-edge interactions rather than the sort of neat,
01:13:43
more typical Watson-Crick base pair. OK. Any questions about the structure of DNA? Anything whatsoever? I want to change gears then and start talking about how small molecules interact with DNA. The first mode that small molecules can interact with DNA is to actually slip into this pi stack of DNA.
01:14:04
So, aromatic compounds can slide into the pi stack of the DNA and we're going to see the consequences of this can be quite destructive. Let's take a look at some examples. This is a class of molecules called intercalators meaning that they intercalate into the pi stack of the DNA.
01:14:22
They get integrated into the DNA structure. So, in order to fit into this pi stack, these molecules must be also hydrophobic and also aromatic, right? They will form competing pi-pi stacking interactions
01:14:41
with the DNA and so they must also be aromatic. Note too that in order to force the way into the pi stack, these molecules force the DNA double helix to slightly unwind to accommodate the DNA intercalator. Here are some examples of this.
01:15:00
These are examples of intercalators. Notice that they're all aromatic compounds. They're all flat and aromatic to slide into the pi stack. Many of these molecules also have positive charge. Positive charge is useful, right? Because the DNA with the phosphodiester backbone of the DNA is negatively charged.
01:15:21
This gives the molecule a way to be attracted to the DNA through a long range charge-charge interaction, right? So these molecules are going to seek out DNA like a homing missile. And once they slide into the pi stack, the consequences can be pretty bad or actually fairly useful.
01:15:40
OK? Let me show you an example of a useful intercalation over here on the right. This is actually an agarose gel which is an important way that chemical biology laboratory separate out DNA structures. Different DNA sequences can be separated out on the basis of their size using these agarose gels. I'll show you what that looks like in a couple
01:16:02
of slides from now. To visualize the DNA however, this molecule over here, ethidium bromide, is incorporated into the gel and it gets concentrated into the DNA by an intercalation interaction. So, it slips into the pi stack of the DNA and it's a fluorescent molecule.
01:16:22
Many aromatic compounds are fluorescent. We've talked about fluorescence before. And so you can actually shine UV light on the gel and wherever you see these pinkish bands, that's where the DNA is present. And so you can actually take a razor blade for example and cut out the DNA of a particular size.
01:16:42
Here's a couple of more DNA intercalators. Here's one that's designed to intercalate and then have a little linker and then intercalate down below the compound. Here's what it looks like structurally. So there's intercalator, linker, intercalator up here for example.
01:17:00
I think that this is it right over here. These are also compounds that are used to treat cancer. So, donamycin, adriamycin are used as anti-cancer compounds. They're some of the first rounds of anti-cancer compounds that are used as chemotherapeutics.
01:17:21
And we'll talk more about their mechanism of action later in the class. We're not quite there yet. OK. Let's stop here. When we come back next time, we'll be talking more about the structure of DNA.