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115:508 Proteins and
Enzymes
Spring 2002 Lecture 1This course is concerned with proteins, polymeric molecules made up, at least initially, of 20 natural l-amino acids, coded for by DNA, which message is transcribed into mRNA and translated into protein on the ribosomes; thus cyclic peptide antibiotics assembled by bacteria in various ways and including d- and other unnatural amino acids are not included. Once incorporated into proteins, the basic amino acids may be changed into other things; this we will discuss later. Proteins may also have attached prosthetic groups, tightly attached non-amino acid groups, for special purposes; these may be either covalently or non-covalently attached - the operative definition is that they not be dialyzable away. Sometimes in enzymatic reactions these have to undergo a second reaction to return to the original chemical form; but this is really metabolism and outside our concern. Many proteins also bind metal ions tightly. And there are a large variety of other natural modifications of proteins whose significance for their function may or may not be clear. These are an area of considerable interest now. We assume that the properties of a protein can be described as a first approximation by the properties of the constituent amino acids, and it is essential to know these properties; but the properties will be modified considerably by their surroundings, by the three dimensional structure of the protein, and it is the continuing challenge of study of proteins and enzymes to learn how the special properties of a protein result from its unique structure. Sometimes the function of a protein involves a prosthetic group or a metal ion, or a modified amino acid, but not all that often. In some cases, as I mentioned, proteins are found to have non-protein modifications, and the problem is to understand their significance to the function of the protein. This is particularly difficult when the chemical function of the protein is itself unclear, as in oncogene proteins. We will be considering some of the major ways of investigating protein properties, primarily chemical ways and those involving measurement of enzyme activity. The course Physical Biochemistry occasionally given by Dr. Kahn concentrates on the problem of the folding of globular proteins, how do they know how to fold up into the active three-dimensional structure, which is regarded as the major problem in proteins these days, perhaps the only one left with a Nobel prize in it. Because of that course, and the Biophysical Chemistry courses, I don't spend time on three-dimensional structure of proteins; but I have asked Dr. Kahn to give a lecture on modeling protein structures in the computer, which is a real show. If you want more about it, I recommend the books by Creighton, Proteins Structures and Molecular Properties, and by Brandén and Tooze, Introduction to Protein Structure, which are mentioned in the syllabus. If your interest is primarily in protein purification, I recommend Dr. Ward's course Biochemical Separations, which provides a much more basic and comprehensive look at ways of purifying proteins than I have time for, though as you see from the syllabus I do quite a bit. However, that isn’t being given this year, as he is on sabbatical. I should also mention a two-credit course on receptors, occasionally given at the Medical School by Drs. Langer and Rashidbaigi. I will talk some about the general topic of binding of ligands by proteins, and how it is measured, but I have no experience with receptors as such. The course schedule might be summed up as follows: after today, nine lectures on the assay and purification of proteins, how to get them to the point where they can be studied in isolation, as they need to be before you work back toward studying them in vivo, or in as complex a system as you can handle. Then five lectures on enzyme kinetics and related topics. I probably won't be able to cover both allosteric enzymes and organized enzyme systems - there were previously listed in separate lectures, and I rarely was able to speak on the latter, because I was behind and sacrificed the topic to get back on schedule. However, there is an article in the latest Annual Review of Biochemistry on channeling of substrates within multifunctional complexes, which I’d like to read and talk about. Then six lectures on protein modification, natural and chemical - down one this year, because Dr. Lawton is on leave, so we won't have his lecture on kinaes; but I expect to briung back Dr. Poretz on glycosylation. Natural post-translational modifications of proteins are a growth area in studying the specifics of protein action, and artificial modification is a powerful tool in the investigation of the relationships between structure and funcion in proteins. This includes site-specific mutation, which is indeed powerful, but is most useful when a lot is already known about the protein. Then two lectures on the general theory of enzyme catalysis, which is Fersht's favorite subject. Then three on mechanisms of specific enzymes, including tyrosyl-tRNA synthetase which comes almost entirely from Fersht's lab and is covered in detail in his text, and one on catalytic antibodies, another hot topic nowadays. We would like eventually to be able to design enzymes to order, to carry out whatever reaction we want, and this is currently the most promising, if still empirical, initiative in this direction. Dr. Kahn will talk about using computers to change known protein structures into the unknown structures of other proteins - what is called homology modeling; this semester he is teaching a whole course on the subject. I would like to give a lecture on the use of fluorescence in studying proteins, but simply have not been able to find room. I should also mention that since I write out the lectures in detail - though I try not to read them - I make copies available for purchase, at a nominal price of 8¢ a page, in the departmental office, Lipman 125. This is particularly important if you miss a lecture. I shall try to put them on the departmental website if you will promise to come to the lectures and use the posted notes only for review or when you have to miss a lecture. I give three 'take-home' examinations, of increasing importance to your grade, as you get used to the idea. These are very different from the type of exams normally given to undergraduates, where you are tested to find out how many right answers you can identify in an hour or so. I give you a journal paper, which you are to read, and a series of questions on the paper, the answers to which you should be able to figure out if you have understood the material in the lectures up to that point. This is much more like what graduate students do this course is by origin a graduate course - in reading and understanding papers from journals - 'the literature' as we call it. I shall try to give you other papers to read, to be discussed briefly in the following lecture, but this depends on how able I am to get an appropriate paper at the right moment in the course - I don't get to LSM very often. Reading and understanding journal papers, at the level of understanding the techniques as well as the conclusions, is one of the aims of this course, even though I have not in the past worked on this as much as I should. The final item is the course paper, an original research proposal (though without the detail of an NIH grant proposal, which is sometimes assigned in courses at this level). You are to describe a protein and some aspect of it, or of its interaction with other molecules, which is not yet understood, and propose one or more experiments designed to understand it better. For graduate students, this should not be your thesis proposal, though it may be related to it and arising out of reading you have done for it - perhaps something that intrigues you, but which your professor isn't interested in having you work on; and it should not be straightforward purification, which is largely empirical, i.e. you try things until they work, though defining an affinity chromatography method is a reasonable proposal. Nor should it be straight sequencing, or isolating the gene, or anything else which is a standard method whose application to this protein doesn't require much thought. In any case, DNA sequencing and gene isolation are problems in molecular biology, not in protein biochemistry. The paper must focus on a protein problem, not a molecular biology problem. In 1996 I had to reject about five papers because they simply didn't use the material of the course. Molecular biology can be used as a tool in approaching a protein problem, as for instance site-directed mutagenesis or the yeast two-hybrid system for finding a protein which binds to another protein, but the question to be answered must be a protein question. Chemical modification, active-site-directed modification, site-specific mutagenesis, enzyme kinetics, use of physical techniques to investigate conformational changes, are some techniques you might use. I suggest you make a tentative selection of a project early and then come to see me, both for me to say whether I think it is an appropriate project and to get any help I can give you about how to approach it. With most journals now available on line through the library, you probably know how to search it better than I; if not, consult a reference librarian. The point of the paper is to select an appropriate problem and an appropriate technique to solve it, understanding the limitations of the technique as well as its powers. I should perhaps comment here that this course, though not a laboratory course, does expect some lab experience - some years ago a junior undergraduate took it without having taken biochem lab, and she often didn't understand what was going on. When I talk about different types of column chromatography, I don't want to have to explain what column chromatography is to someone who has never done any sort of it. I also say that in a course like this it is hardly possible to make you immediately able to do research in a given area, rather only to make you aware that the area exists, so that you may go on to learn more specifics in an area when you need to. The Rosenberg book does give you a number of experimental protocols to do certain things. What I can do is give you an idea how the various areas fit together, and some idea of strategy, how you use them to answer questions; I suppose one could say that practically everything in the course is methods, except for the few lectures at the end about the mechanisms of specific enzymes. But the problems you will be trying to answer in your work are many, and I can give only a selection of current problems in the area of proteins and enzymes; rather, I try to cover ways of approaching their answers. When you read a journal paper, always make sure you understand the techniques used, how they get their answers; you may find the techniques useful for your own research. The course culminates in a research proposal paper, in which you are to propose some question about an enzyme or other protein, and how to answer it. You may need to know more details of a technique in order to write the paper, but at least I should get you to the point where you can learn more by reading. About the reading listed in the syllabus: the specific textbooks are alternate sources of information at the level of the lectures - probably the best thing to do would be to read the text assignment before the lecture, to help you understand it, but others may find reading afterward more useful. They are also useful references for the future, particularly Scopes if you are going to purify proteins. I put books including the course textbooks - on reserve in the Chang Library on this campus, and I’ve been told they are already available. If you are concerned about the cost of the books, you can try reading them in the library; but if you are going to go on in life purifying and characterizing proteins, Scopes and Rosenberg would be worthwhile investments. The references to Annual Reviews of Biochemistry and to review papers generally indicate what I have used in preparing the lecture. They would be good sources for more information, especially for references on original research papers. I will give more references as we go along in the course. I have been teaching this course for thirty years, and sometimes worry whether I am getting out of date, particularly in light of the mushrooming growth of 'molecular biology'. There is no general agreement on distinguishable differences in definition of biochemistry and molecular biology. A biochemist and a molecular biologist will generally each try to define the other's discipline as a subdiscipline of theirs; but it is at least interesting that, whatever the definition, they know what they are. I would define molecular biology perhaps as synonymous with molecular genetics, as having at its core a genetic way of thinking, based on number (as, number of recombinants) which is very different from a chemical way of thinking based on weight (amount of a substance); and as concerned with the central dogma, the flow of information from DNA to RNA to protein synthesis, the control of each step of this, and the controls within this process of cell function up to differentiation. The manipulation of nucleic acids is central to molecular biology. The term molecular biology arose as a contrast to organismic and physiological biology, as describing understanding of biological processes at the molecular level; but biochemistry has always been at that level. Biochemistry has its origin what was called physiological chemistry, identification and measurement of all the chemical compounds of living systems, and in how they are transformed. It came to be realized that these transformations were carried out by enzymes, which are virtually always proteins. Virtually all cell functions are carried out by enzymes and other proteins. Hence there was much interest in how enzymes catalyze reactions, and more generally in how even non-enzyme proteins carry out their function. I want to emphasize, now and especially when you write your course papers, quantitation, measurement of how much of something you have, of a specific protein or of its biological and chemical activity. Molecular biology has its roots in genetics, and often is content simply to know whether a gene or its product is present or absent in an individual or tissue. You can understand more about a biological function if you can measure how much of the function is there, and especially if you can compare amount of function, such as but not limited to enzymatic activity, to the amount of the protein responsible for the function. I shall try to stress this often in the course. We are realizing more and more things that proteins do in addition to catalyzing reactions of other molecules; the problem is that the functions become very difficult to measure outside the complete biological system in which they occur. This is a practical problem, not a philosophical one; once you have a system for measurement - what we call an assay - most of the approaches we have for studying enzymes are also applicable to the study of other proteins, with the principal exception of enzyme kinetics, the study of the rate of enzyme-catalyzed reactions as influenced by conditions such as but not limited to substrate concentrations. I try to give papers to read on assay and characterization of non-enzyme proteins. And one of the strong points of molecular biology is that it can enable you to express a protein, or repress the normal expression, inside a cell, so that you can at least qualitatively look at its role in a function only observable in the intact cell. Molecular biologists frequently discover the actual biochemical or enzyme function of a protein which has been known only as the product of a gene they have been studying. Scopes, in the introduction to the third edition of his book, makes the point that increasingly often proteins are expressed in and purified from a non-natural host cell, the gene having been introduced into the cell by recombinant DNA techniques. This is an example of the use of molecular biology as a tool. One can thus get a great deal more of the protein - in some cases as much as 50% of the host cell protein - and assay by function may be unnecessary, you just purify the most abundant protein present. Or, knowing the DNA sequence and thus that of the protein, you synthesize peptides of that sequence, have a rabbit make antibodies to the peptides, and use them for assay. This is OK for assay during purification, but of course tells you nothing about the protein's function - particularly important when you have changed the structure in some way. Purification may however be important in itself, if the protein is to be used for therapeutic purposes; the FDA then insists that you get it very pure, purer than you would need it for experimental purposes. This is a major concern in the pharmaceutical industry. Several people have remarked to me that proteins are much harder to work with than nucleic acids, primarily because each protein is a separate problem in purification - one must determine in each individual case how to purify it while retaining its biological activity, as well as how to assay that activity. There are many ways to lose that activity, and in general milder conditions must be used - for instance, nucleic acids are typically precipitated at 70% ethanol, and often heated to at least 65°; neither of these procedures are generally applicable to proteins if you want to maintain their biological activity. Beyond that, one should remember that with rare, if Nobel Prize-worthy, exceptions nucleic acids are only informational molecules, while proteins do virtually everything in cells, including catalyzing the vast number of chemical reactions which continually occur in cells and whose sum is the life process. From the point of view of the enzymologist and proteinologist, the techniques of modern molecular biology are just added ways to study proteins, how do they do what they do. You can learn about the molecular biological methods in other courses; in this course we shall cover the kinds of results available from one of them, site-specific mutagenesis. One matter I always promise at this time but rarely get to: enzymes are classified into six groups, with specific numbers which allow you to recognize an enzyme even if the paper is in Serbo-Croatian, and which are assigned by the Enzyme Commission:
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