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Peptide Synthesis - A Useful Tool in the Study of Protein Structures and Function

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Peptide Synthesis - A Useful Tool in the Study of Protein Structures and Function
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Christian B. Anfinsen is well remembered as a „public scientist in an age of rapid scientific advancement and social change“ [1]. From 1981 until 1989 he was the chairman of the US National Academy of Sciences’ Committee for Human Rights. He was a fervent advocate of basic research. Some weeks before he came to Lindau in 1983, he had publicly critized the Reagan administration’s severe budget cuts to biomedical research programs at the NIH, together with three other Nobel Laureates. At the beginning of this lecture, he draws a connection between excellent research and teaching by expressing his deep gratitude to Kaj Ulrik Linderstrøm-Lang in whose Carlsberg Laboratory in Copenhagen he had spent „the most formative and exciting time in my life professionally“. Linderstrøm-Lang’s Laboratory is regarded to have been „probably the most scientifically exciting environment for a protein chemist“ [2] in the 1950s. Inspired by this teacher, Anfinsen became one of the best protein chemists of his time. In investigations on bovine pancreatic ribonuclease, Anfinsen could demonstrate already in the late 1950s that all information needed to fold up a protein into its three-dimensional structure is encoded within its linear sequence. It was this work that mainly earned him a Nobel Prize in Chemistry, but in this lecture Anfinsen only briefly mentions it. He prefers to talk about a more recent subject of his research, a nuclease of Staphylococcus aureus whose sequence of 149 amino acids he had determined with the help of affinity chromatography in the late 1960s, a success, which played a role in the decision of the Academy, too. The title of his talk is, as he admits, a bit misleading, because peptide synthesis is rather implied than discussed.In affinity chromatography, molecules passing through a column are recognized and caught by molecules attached to that column. This makes it an ideal method to explore, amongst others, immunological interactions, Anfinsen explains, and describes how he applied it to investigate the antigenic properties of small regions (epitopes) of the nuclease molecule (and other proteins). Specifically, he talks under the impression of the „increasing interest in the possibility of making synthetic vaccines“. He also discusses epitope antibodies as a possible diagnostic tool for sickle cell anemia. More generally, he is interested in understanding how smaller pieces of a polypeptide chain gain a native formation in solution and in determining the equilibrium between the native and the random coil structure of a protein. The high hopes that eminent scientists like Anfinsen had in synthetic vaccines 30 years ago have not yet been fulfilled, however. Although more than a thousand of those vaccines have been examined, and many of them have been clinically tested, not a single one has made it to the market yet. [3] Joachim Pietzsch[1] The U.S. National Library of Medicine: Profiles in Science: The Christian B. Anfinsen Papers; [2] Schellman JA, Schellman CG. Kaj Ulrik Linderstrøm-Lang (1896-1959).Protein Sci. 1997 May;6(5):1092-100.[3] Van Regenmortel MHV. Synthetic Peptide Vaccines and the Search for Neutralization B Cell Epitopes. The Open Vaccine Journal, 2009, 2, 33-44.
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Transcript: English(auto-generated)
Thank you, Professor Vienicke.
It's been obviously become the custom this week to take up most of one's allotted time with an autobiography. But I think I'll skip that now. And perhaps I'll just give you a few salient features of my background and history.
I part my hair on the wrong side, that's one thing. I take four grams of vitamin C every morning for Dr. Pauling's sake. I should take 10, but I take four. I think perhaps the one thing I've never done before is publicly express my gratitude to someone.
And that too was done yesterday by several of the speakers to some individual that had a strong influence on the career of the person. In my own case, I actually started out to be a real chemist. At the University of Pennsylvania, I started in organic chemistry.
But at the end of two years, I found that I was very tired of synthesizing local anesthetics and so on. And I had a chance to go to Copenhagen on an American Scandinavian Foundation fellowship. And very fortunately, selected the laboratory of Professor Lindström Lang. Many of you students, unfortunately,
will probably not have heard of him. I hope you will in times to come. But I went there first in 1939 and 40 and had to come home because of the incursion of visitors from the south. And then I went back in 1954. And I would say that the year I spent there in 1954
was probably the most formative and exciting time in my life, professionally. I was there together with Harold Chiraga, Fred Richards, William Harrington, and Walter Kautzman. We were all there at the same time. And over the whole thing was this figure of Lindström Lang, who was a mathematical genius
in the first place, but extremely talented in music and art and intuition and science. And all of those names that I mentioned, my colleagues that were there at the same time, feel the same way, that this man was really responsible for opening the spigot on all of us and teaching us how to go about studying
scientific problems. I won't say any more about my background, perhaps at the end of this description of the current work, which will be fairly short, actually. I might say a few words about what I'm about to begin, which is really more interesting.
As Professor Wienicke pointed out to you, our activities, my laboratory's activities for a number of years were associated with establishing the fact that polypeptide chains, having been synthesized by cells,
could spontaneously fold into a three-dimensional structure that was unique and reproduced each time without error. And that seems to be commonplace and is true of all macromolecules, all proteins that have been looked at carefully.
The entire problem reached a point where it became important to try to decide why this happened. It was obviously a thermodynamic event and always went the same way. Why did this happen? What could one deduce from the structure of the protein, the polypeptide sequence and so on,
that would tell something about the mechanism by which this folding occurred? And that's still an extremely popular problem. I'm no longer in this business. Having done it for a million years, I had enough. But there are many laboratories that are now looking at the problem
with the use of very complex computer programs. And already there is the beginnings of some success in predicting the three-dimensional structure of small proteins or large polypeptides from their covalent chemistry. We became interested in an aspect of this problem
of how proteins fold and what can fold and what can't in connection with something that we began in my laboratory in 1968 called affinity chromatography. Affinity chromatography based actually
on an observation from a PORASS lab in Uppsala that one could activate a column of sepharose with cyanogen bromide in such a way that amino terminal groups would attach to the sepharose and be bound and would then swing loose and could catch molecules going through the column
that recognize this attached chain. One could also attach coenzymes, other smaller molecules, and catch proteins and so on, the various applications. And we were interested in a combination
of this affinity chromatography method with peptide synthesis, as is promised in the program, about which I will say very little actually, and the antibody molecule in general. And we felt we could work out some procedures for looking at the folding propensities of polypeptides
and for the nature of the surface of molecules, of protein molecules as compared with the internal parts by isolating specific antibodies to small regions of proteins and looking at their relative affinities
by standard immunological methods. There had been a considerable amount of thought on this problem before. My friend Michael Sella, for example, together with Ruth Arnone, suggested years ago, 15, 12, 15 years ago, that two types
of antigenic determinants might exist on protein molecules, so-called conformational determinants, which involve three-dimensional arrangements, and sequential determinants, which are simply the nature of the amino acids in a random chain.
And that some antibodies would require a conformation and some might be satisfied with sequence. We had some ideas about the possibility of an intermediate situation in which the determinants that were sequential
might occasionally form conformational determinants by going back to the structure that they remembered in the protein molecule. And I'll show you some examples about that. The whole problem of the equilibrium between a random coil form
and a three-dimensional conformational form in proteins and polypeptides is becoming very important because of the increase in interest in the possibilities of making synthetic vaccines. I have an example of this sort of thing.
I did this during a sabbatical visit to the Weizmann Institute in Israel about 1971 or two. The first slide, please. The first slide, please. Yesterday it always worked after two times.
This is, this bottom circle here is actually a loop that exists in the hen's egg white lysozyme molecule, the protein is about 17,000 molecular weight,
but at one point in its three-dimensional structure is a loop that sticks out from the molecule. It's exactly like this, except for one change. This alanine in the original is actually a cysteine. And there's a disulfide bridge here, and there's a disulfide bridge from the cysteine here
to another part of the molecule. But to simplify the synthesis of this loop, which we knew had antigenic properties, this had been isolated from the native protein molecule and shown to have antigenic properties when injected into rabbits.
This was synthesized by solid phase techniques of Merrifield and his colleagues, and then deprotected and the SS bridge closed. And this molecule, this synthetic loop, was then attached through the amino group
to a carrier molecule. It could either be a multi-chain polyalanine, a synthetic polymer, or even a sepharose suspension, and injected into animals. And it turned out that this loop induced the formation of antibodies in the animals
that could recognize native lysozyme. It could recognize the loop in the lysozyme molecule. So this was an entirely synthetic antigen, which now could form antibodies against the original native structure. This is precisely the sort of approach that a variety of groups now are using
to attempt to prepare synthetic vaccines. I know at the Scripps Institute in California, there's quite a bit of activity, and I know that, of course, Arnon and her group at the Weissman Institute are very active, and both groups have already made such synthetic vaccines.
By synthetic vaccine, I mean the following. If you take a virus that causes a disease, let's say influenza virus, and if you know the structure of the protein coat that covers this virus, you can then synthesize parts of that coat.
No, don't need it. And as parts of that coat, and attach the synthetic pieces to carriers, inject them into animals, make an antibody which will recognize that flu virus,
and inactivate it. And that's going on at a great rate here and there. Well, so much for that problem. That's a long story in itself. We are interested basically, we were interested basically in this problem of how small a piece of polypeptide chain
could form a native conformation in solution, and how often. We went to work on this particular molecule which we had used as a model for a number of years. This is staphylococcal nuclease, a protein of 149 amino acids,
which has no SS bridges, and which is extremely labile, and will open and close quickly. If you completely denature this molecule so it's an extended coil and solution, and then quickly restore it to the conditions
under which it can fold, it returns to the native structure mainly in the order of about 50 milliseconds, a very rapid folding process. The next slide, once. This is the three-dimensional structure of the molecule.
It has a long amino terminus, then it goes into a anti-parallel pleated sheet, then a loop that sticks out, and then three helices, a nice regular Pauline-Cory helices.
I've marked out the segment from 99 to 126, and 99 to 149. Next slide, please. Next. Now, one thing we need for isolating specific antibodies
is a collection of specific peptides so that we can attach these peptides to a column, pass antibody through it, and catch the antibody that's specific for that piece. And here is the entire molecule with its pleated sheet and its three helical sections.
Here you can get a piece with trypsin cleavage. You can get this one and this one. This is also a trypsin fragment that can be prepared in a special way. It's also possible to make five fragments from this molecule by treatment with cyanogen bromide, which cleaves the following methionine residues, and there are four methionine residues in this protein,
one of which goes from 99 to 149. So that is an easily prepared segment. Also, when you prepare this piece, you have as a side product 127 to 149, both of which I'll mention again in a minute.
This piece, for example, in the native molecule, you see it contains two helical sections. And yet, if you take this peptide that you've produced and purified and study its structure and solution, it's a completely random structure.
It has no helices are apparent. And one is curious whether this molecule has in its sequence the knowledge that's sufficient to form this structure as it appears in the native molecule. The next slide. So here we have a schematic drawing of this fragment,
99 to 149 in a random form. And we ask the question, how often does this random structure shift back and forth into the structure that it occupied originally
in the native protein? This would be the form that would be recognized by an antibody made against native nuclease, whereas an antibody made against a random structure should not recognize the structure. Next slide.
Now here, affinity chromatography comes into play as a very, very useful tool. To study processes of the sort we were interested in, we had to have antibodies that were not precipitating. Most antibodies react with an antigen and precipitate because they are divalent.
They recognize you have antibody mixtures which recognize more than one determinant and they form networks which fall out of solution. So what you want to do is to prepare an antibody that is able to recognize only one antigenic site. Then it will form a complex with the antigen
and will not precipitate but will be soluble and is a much better reagent because it's soluble and can be used in experiments where turbidity is not desirable. So in these experiments, we injected nuclease into goats
and prepared immune sera. The gamma globulin fraction was isolated in the usual way. And then the total gamma globulin fraction was passed through a sepharose nuclease column simply to catch all the antibody that recognized nuclease one way or another.
This fraction, anti-nuclease, was then passed through a second column to which we had attached peptide 99 to 149. Now remember that this antibody was made against native nuclease.
In spite of the fact that this is a random chain, this 99 to 149 peptide, a fraction of this total anti-nuclease population of antibodies was caught on that affinity column. The rest of the antibodies went through
and we had then something which was anti-99 to 149. This antibody with 99 to 149 precipitated. It still had more than one determinant and was able to make a network and fall out of solution. So further fractionation took place. This fraction for the 99 to 149 portion
was passed through another sepharose column to which the peptide 127 to 149 had been attached. That would only recognize the parts of the antibody population that recognized the terminal part of nuclease.
The portion that was not recognized, anti-99 to 126 passed through the column and this other one was eluted subsequently by guanidine hydrochloride. So we have here now an antibody
against a particular region of the nuclease molecule, quite a small region, which is non-percipitating. If you take some antibody which is 7S in the centrifuge and add this to it, you still have 7S. There is no precipitate, no change in molecular weight to speak of.
Now, the next slide please. So we have an antibody that's against one determinant, well-determined section of the molecule and it's non-percipitating. Here we have this antibody. It's the antibody against 99 to 126
and we have a sub N indicating that it's against the native form of that peptide. It was isolated from a antibody mixture against the native structure, not the random. And that will react with native nuclease protein in the native form to form this complex
which will be soluble in solution. We indicate that this is in equilibrium. Now at the same time, we postulate or we ask, does this peptide 99 to 149 exist? Does it enter into an equilibrium with its native form? Is there some small amount of this peptide
which somehow or other knows how to form the three-dimensional structure that this antibody recognizes in the native molecule? And what is its conformational equilibrium? So if we take some antibody, add to it nuclease and varying amounts of this peptide
and then having stirred that around, add some DNA to this solution, DNA being the substrate which is attacked by this nuclease molecule with the cleavage of bonds and shift in the spectrum of the nucleic acid,
you then have a situation in which increasing amounts of the peptide from zero up to as much as you can put in will influence or will compete with the native protein for this antibody in its native format.
Only the native conformation of this peptide is able to compete. Now from the data that one obtains in terms of the rate, the activity of the enzyme as you add increasing amounts of the peptide,
you can calculate what the conformational equilibrium constant is. It turns out that about 0.02%, two hundredths of 1% of a peptide of this kind exists in the native form at any given time. It's a very small amount, but nevertheless, it's quite easy to show
that it does enter into that form and is able to compete with the protein molecule. I should mention that the most critical thing I didn't mention is that the combination of antibody with protein inactivates the enzyme, of course. This is an inactive complex and activity appears
when you add the competing peptide which will displace the protein and allow the enzyme to be active. The next slide please. Now this is a different question. Having found out something about a small piece, we thought we might as well take the big jump.
What about the protein itself? What about the whole structure? Here we have one to 149, the entire nuclease structure and we ask, is it possible that there's an equilibrium for a good, honest, ordinary protein of this kind between the native structure and a random coil structure and if so, what are the properties of this equilibrium?
Next slide. This could be done in a similar way. You take the antibody that we had before against 99 to 126, non-percipitating and we know, no, I'm sorry, this is now 99 to 149.
It's an antibody prepared by injecting 99 to 149 into an animal and that antibody recognizes the random form because essentially all of the molecules in the random state and that random antibody, random form antibody,
now will recognize the peptide 99 to 149 in its random form, make it complex. We ask, is the protein occasionally in a random form and what is the equilibrium constant? You could test that in the following way.
Well, if it can, if it can make a random form, then it will also interact with this antibody in another equilibrium. Now the next slide, in the next slide, we carry out the experiment.
We prepare some carbon-14 labeled peptide with cyanate on the amino group. We have the antibody against the random form of this peptide. We have nuclease presumably or possibly in equilibrium with it and we ask, can this nuclease shift into a random structure
frequently enough so that it can compete with the peptide for antibody? The way to tell this is to take a reagent that's common enough that you can actually buy,
some rabbit, anti-goat immunoglobulin, rabbit made against anti-goat, made against the goat immunoglobulin and add it to this mixture with increasing amounts of nuclease in a series of tests.
By the composition of the, by the radioactivity in the supernatant and in the precipitate, you can estimate these various items and show that increasing amounts of native nuclease
will indeed displace the radioactive peptide from the antibody and that can be seen in these analyses. It turns out once again that the frequency with which native nuclease shifts into the random state
is on the order of three or four tenths of a percent. I have a number for that here. Yeah, 25 degrees. The conformational equilibrium constant was about three times 10 to the three. Now, the next slide shows how you can vary this
in a completely expected way. Here is shown at 25 degrees, the competition by native nuclease for the antibody in the absence and in the presence
of strong inhibitory ligands that are known to tighten up the structure of nuclease. Calcium and thymidine diphosphate are very strong ligands that bind in the active site of nuclease and stabilize, rigidify its structure so that one would expect it to be much less likely to unfold
and be able to compete in this reaction and that's exactly what you see. If you add calcium and thymidine diphosphate, you make the frequency of unfolding much less. At three degrees, I said that at 25 degrees, it was three times 10 to the three. At three degrees, it's 4,000.
At 30 degrees, it was 400. And with thymidine diphosphate and calcium, it was 40,000, where it became very much stabilized. I've shown you these examples to try to convince you
that A, proteins are not completely stable hunks of concrete. They actually do move around a lot and of course, that was known before from experiments involving hydrogen exchange and so on. Nevertheless, here you can actually get a number for the frequency of that event. And also the peptides, in spite of the fact that they have no old friendly neighbors nearby
to help them form the right structures, they do have in their sequence sufficient information to fold up in a solution and to make a relatively unique structure. Of course, it can't be entirely unique, but perhaps you only need halfway to be recognized by the antibody.
It doesn't have to be 100% folded. Now some related experiments I wanted to mention might interest you. These are experiments that were carried out and are still being carried out by Alan Schechter in my laboratory, used to be my laboratory two years ago, on hemoglobin S, sickle cell hemoglobin.
Here we have a drawing of, a kind of drawing of sickle cell hemoglobin showing the portion of the monomers in the beta chains that are modified by the substitution of one amino acid, the allene for glutamic acid, in the polypeptide chain of the beta portion
of hemoglobin S. What Schechter and his colleagues did, next slide, was to synthesize the one to 13 portion of beta S,
which now has this change of valine for glutamic, and attach that to a Scheferose column for antibody fractionation. And he also made some, they also made some smaller peptides. Next slide.
Now with the Scheferose column, with its attached synthetic one to 13 peptide, the total gamma globulin from sheep or goats that had been injected with hemoglobin S, sickle cell hemoglobin, passed through.
The bulk of the material went through the column, but a small fraction was retained on the column, which was specific for the one to 13 sequence. Next slide. Here we have some experiments indicating how nicely this fractionation
really has taken place. Radioactive hemoglobin S was attached to antibiotics. By beta S one to 13, against the antibody, against the small portion. That was attached to radioactive hemoglobin S. And then, increasing amounts of hemoglobin A,
or hemoglobin S, or this peptide, one to one 13, were added to the solutions. And you can see that hemoglobin S displaces the radioactive hemoglobin S from the antibody.
Very nicely, as you would expect. The hemoglobin A has no cross-reactivity at all. It has a different one to 13 than the hemoglobin A. And some release can also be achieved with the peptide itself, with small aliquots of the peptide one to 13.
So that you have then, in this antibody that was isolated against one to 13, you have produced an antibody which is highly specific for hemoglobin S. And indeed, it makes an extremely good diagnostic tool. It's entirely against that form
of the hemoglobin molecule. I think that's my last slide. Lights, please. And I've left a little extra time for the following speakers. I promised you I'd say something about what I'm gonna speak on in three years when I come back.
If I'm invited. And that's some work that we are just beginning, or about to begin. You may have read in a recent issue of Nature, about two or three weeks ago, a short description of some organisms found in the bottom of the Pacific Ocean
in the black chimneys that come out of the tectonic vents. And my colleague, Jody Deming, who's at Hopkins, where I am, and a friend of hers were in the first submarine dive that went down there. And they were able to sample the water coming out of these vents, which is at 350 degrees and 265 atmospheres.
And in this water were eight or 10 species of bacteria happily swimming around. They must have amazing proteins. They must have phenomenal DNA. They must carry out all sorts of reactions. They can take carbonate and make methane or methanol and so on.
So that next time I will come enthusiastically to tell you about these. I hope to tell you about these experiments. Thank you.