Week Three Lecture Biochemistry of Nutrition, 560B Dr.

Charles Saladino Introduction I think it is safe to say that in terms of functionality, proteins represent the most diverse group of biomolecules encountered in living systems. Let us consider this point: 1. Although not the main source of nutrition in most American diets (whatever that is), proteins can act as a major fuel source. 2. Almost all enzymes are proteins. (Notice I said almost, because there are some ribosomalassociated RNA species that have enzymatic activity.) Simply put, without this class of proteins called enzymes, metabolism would proceed too slowly to be compatible with life. 3. There are numerous groups of proteins that are functionally designed for structural purposes. These would include the collagens (seen in bones, ligaments and tendons, for example), elastins of the skin and arterial walls, and reticular connective tissue components. However, let us not neglect to remind ourselves that integral proteins of a structural nature are also found within membranes, the cytosketeton, microtubules, etc. 4. Antibodies are proteins. 5. There are a large number of coagulation cascade proteins. 6. Storage proteins play an important role in sequestering important ions. For example, ferritin is involved in iron storage, whereas calmodulin is a calcium-binding protein that is important in metabolic regulation. 7. Genetic regulatory proteins are critical to controlling the appropriate expression of gene activity or inactivity. 8. Transport proteins, often integrated into membranes, are important in carrying various substances in and out of cells in a regulated manner. A good example is the glucose transporter. On a grander scale, however, there is albumin, designed to bind to a variety of plasma substances. 9. Proteins, such as myosin and actin, are key components of cardiac and skeletal myocytes and are in large part responsible for the contractile properties such cells. 10. Heat shock proteins can be subdivided into groups of proteins that not only escort proteins from one area of a cell to another site within that same cell (thus acting as transporters), but they also function to protect the three-dimensional structure of enzymes and other proteins in times of metabolic stress. Such stress conditions include aberrant changes in temperature, pH, and oxygen tension. 11. There are a variety of hormones which are protein in nature. These include the well known insulin molecule, glucagon, and a variety of neuropeptides. Other proteins that serve as an example of regulatory proteins at the cellular/tissue level would be angiotensin, involved directly in

vasoconstriction and indirectly in cardiohypertrophy. 12. Finally, many proteins are conjugated to other biomolecules. These would include lipids to form lipoproteins and various carbohydrate moieties to form glycoproteins (as seen in cell membrane receptors) and various mucopolysaccharides, for example. Also, there are metalloproteins, obviously named, such as the Fe-S cluster proteins of the detoxification system and located in the inner mitochondrial membranes in the hepatocytes. In addition, there are hene proteins, such as the welldescribed myoglobin and hemoglobin molecules. From this list of functions, each but briefly described, it is quite evident that as a class of biomolecules, proteins constitute a large spectrum of functions. In addition, and of real importance, is the fact that only proteins can effectively carry out many of these functional duties, due to the absolute requirement for the great specificity that lies within their respective highly-specific, three-dimensional configurations. For example, the effectiveness of antibodies to neutralize precise foreign molecules and agents requires great specificity against its antigen.. The importance of this is seen in autoimmunity, wherein this specificity is lost. The same need for specificity applies to the action of enzymes upon specific substrates and peptide hormones acting as ligands to their respective receptors. In order to appreciate this specificity of action of various proteins, it is critical that we not only understand their respective three-dimensional structures, but we must also appreciate those factors that determine precisely what geometric configurations a particular protein will assume. Besides a host of other factors, such as hydration, energy levels, etc., what we learned last week about pKa will now assume a major role in what the final 3-D array of the protein will in fact be. Protein Structure I understand that making the statement that proteins are comprised of amino acids is doctrinaire to you. So let us start to discuss the various levels of protein structure, which will ultimately determine the functionality of a given protein. The primary structure of a protein is the specific amino acid sequence that comprises that protein; and this primary structure will ultimately be the major determinant of all higher order structure or folding of that protein. In order for a primary structure to be realized, each amino acid must be bonded to an adjacent amino acid through the formation of a strong peptide bond, which in organic chemistry, we would call an amide bond. I refer you to Figure 3.24 in Devlin. Please notice that the amine group of one amino acid is covalently bonded to the carbonyl carbon (the one with the double-bonded oxygen) of the adjacent amino acid. This peptide bond again an amide bond results from the reaction process known as a dehydration synthesis pretty self explanatory. The oxygen from one amino acids carboxyl group and two hydrogen atoms from the amine group (ammonium ion) of the other amino acid are removed enzymatically (in total constituting a dehydration reaction), allowing the formation of a bond between the nitrogen of one amino acid and the carbonyl carbon. of another amino acid. Notice carefully that this peptide bond formation does not involve any part of the R side chain group. Such interactions are for later consideration. If you see the term, dipeptide, this refers to two amino acids joined together by one peptide bond. Thus, a tripeptide is three amino acids (joined by two peptide bonds). Further, if you should see an amino acid sequence, such as Tyr-Phe-Gly-Gly-Leu, the tyrosine is the N-terminal amino acid (i.e. starting with an amine), whereas the leucine is the C-terminal amino acid (i,e. ending in a carboxyl).

Now folks, if we look at the same Figure 3.24 in Devlin, we note that the peptide bond itself is rigid. That is, it does not rotate, any more than the double or triple bond between two carbons can rotate. However, if you look carefully at the figure, there are respective and bonds on either side of the peptide bond. These can and in fact do rotate. So why am I making a point about this? Well, these rotations allow configuration (3-D) changes in the way a polypeptide chain can in fact configure. It also causes some 3-D or conformational constraints, known as steric hindrance, when, for example, two hydrogens are rotated too close to one another. Their respective electrons will repel. What does all this boil down to? Well, it provides for many permutations/combinations of where the R groups of each amino acid will be positioned with respect to one another. This, in turn, will affect possible interactions between R groups, which in turn exert a tremendous affect upon higher order protein structure and thus its function. (Remember lecture one, structure/function relationships????) Before we investigate how interactions between amino acids (known also as amino acid residues) of a polypeptide determine the proteins three-dimensional structure, (as well as its functional role and relationship to other proteins), let us define polypeptides that have similar amino acid sequences and functions as homologous. This is significant, because sequence comparisons among homologous polypeptides have been used to evaluate genetic relationships of different species. For example, a comparison of the mitochondrial oxidation/reduction protein, cytochrome C, among different species reveals a significant amount of sequence conservation. Further, those amino acids within a given protein which do not differ from species to species are referred to as invariant and are believed to be essential for the function of that protein. This is why, for example, there are invariant similarities and some variant differences in the structure of human vs. that of porcine vs. that bovine insulin. It is these invariant amino acids that allow porcine insulin to be substituted for that of human insulin in some cases. I wouldnt be surprised if the variant differences, however, could contribute to immunological reactions by a human against say porcine insulin. Thus, the use of genetically-engineered human insulin produced from bacteria. There are many other examples. Later, when we study energy transformation in the mitochondria, we shall see that the invariant amino acid residues in cytochrome C are essential for interaction with the important heme group. One other point: Some amino acid substitutions do not affect the function of the protein. They are referred to as conservative substitutions, whereas those amino acid substitutions which result in a defective protein are known as non-conservative substitutions. Now we can state the bottom line of primary structure. It is whatever amino acids are bonded together via peptide bonds in whatever order they appear. This primary amino acid sequence forms the basis for all higher order protein structure various types of folding through bonding interactions. When we consider the secondary structure of a protein, we need to realize that a polypeptide chain consists of a regularly repeating structure, called the backbone, and a variable component comprising distinct R side chains of their respective amino acids. The polypeptide is very rich in potential hydrogen bonds, because each amino acid residue contains a carbonyl group (a good hydrogen bond acceptor except for proline) and an amine group (a good hydrogen bond donor). This is the basis of secondary structure, which assumes one of two likely configurations. The first is the alpha () (righthanded) helix, and second is the beta-pleated sheet. Your text shows the -helix in Figures 3.25 and

3.26 and the beta-pleated sheet in Figures 3.28 and 3.29. Note that both of these secondary structures are stabilized through hydrogen bonding within the backbone, not by the R side chains. In both configurations, the R side chains protrude out from the backbone structure and do not participate in stabilizing either structure type. Obviously, in the -helix, for this hydrogen bonding to occur, the carbonyl double-bonded oxygen must point toward the amine hydrogen, which it obviously does. In the pleated sheet, several backbones are joined by hydrogen bonding. Thus, both the -helix and the -pleated sheet represent secondary structure of polypeptides. These major types of secondary structural elements occur in varying combinations and proportions in globular proteins (a term defined below under tertiary structure). Some proteins consist of only helices spanned by short connecting links (such as in some cytochrome proteins), whereas others (such as are found in immunoglobins) contain a great preponderance of -sheets and are without the -helix. Most proteins, however, such as many enzymes, contain significant amounts of both secondary structures. Supersecondary Structures (Motifs) You text hardly mentions these and does not define them as supersecondary structures. However, they are extremely important. In the figure below are shown five of the many types. In biochemistry, a beta sheet is always shown as a flat arrow, whereas a helix is an obvious spiral. The most common supersecondary is the first one in the diagram (). Motifs have significant roles both structurally and functionally. Functionally, many motifs comprise larger domains where many ligands (you know that word?) bind that regulate the activity of the protein. For example, when we discuss phosphorylation as controlling the activity of the enzymes involved in forming and degrading glycogen (so as to store or make glucose available, respectively,) the phosphate groups will be binding to the enzyme at specific domain composed of motifs. So folks, when you get right down to it, these motifs are involved in the regulation of whole metabolic pathways that profoundly affect our metabolism! So here is a perfect example of what I said in my introduction about understanding the how biochemistry happens, not just the fact that it does. Is your appreciation for the complexity of biochemical issues starting to grow? And we havent even gotten to the higher order structure yet, let alone function!

Tertiary Structure It is tempting, as some books almost imply, or just entirely avoid being really correct, to think of tertiary protein structure as just further folding of secondary structures. That idea in and of itself is simply incorrect. Without being too much of a biochemist, I suggest that you can just imagine the near-infinitely possible arrangements of secondary or supersecondary structures. You can investigate this further, if you wish, and I am always happy to give further explanations. The protein structures in your text are relatively simple. Even hemoglobin is not complex (molecular weight 68,000 Daltons), when compared to the millions of Daltons of weight of some protein complexes (even some enzyme complexes). Why not Google image this subject and see what you come up with? For our purposes, I will define tertiary structure as one in which 1) there are secondary structural elements (like the helices and pleated sheets), often in great quantity; 2) a globular protein is formed with efficient packing as the polypeptide chain components fold; 3) a hydrophobic center or interior

occurs (obviously from which water is excluded) in what might otherwise be a significantly hydrophilic protein; 4) and in proteins with approximately >200 amino acids, there will be specific areas called domains. These domains are polypeptide segments with specialized functions for example, where an ion or small molecule might bind. I already mentioned phosphorylating an enzyme by binding at what would probably be a domain. Now your text does not show the types of bonding involved in forming tertiary structure per se. However, if we think about the various chemical groups that for some of the R groups, we would expect ionic, hydrogen, hydrophobic interactions, and last but certainly not least, a disulfide linkage formed between two cysteine molecules, with the sulfhydryl group of the R group of cysteine being located at the end of the R goup. So what am I saying is -helix to -helix (or to itself folded over), -helix to -sheet, and -sheet to sheet bonding to form tertiary structure will occur between various amino acid R groups where appropriate, as well as between R groups and the carbonyl oxygen or hydrogen of the nitrogen attached to the alpha carbon (central carbon) of a given amino acid. These are common and often critical to proper folding structure. Now for those with a little chemistry background, you might say that leucine, isoleucine, alanine, and valine have no R groups that can be charged are and are hydrophobic. So how can they participate in bonding? The answer is hydrophobic interaction. One observes this in the structure of insulin. Look on page 91 of Devlin. Notice that there are two sets of disulfide linkages involving the R group of the cysteine residues that link the alpha and the beta chain of insulin together, and another internal disulfide linkage on the alpha chain. Trivia? Well do you think insulin would function properly if one of those disulfide linkages were to be destabilized?????? Importantly, the tertiary structure now represents the first time we observed not only polypeptide backbone bonding (as in maintaining the helical structure), but the R group side chains of the amino acids also bonding to stabilize this higher level of structure. This raises the permeations and combinations for folding possibilities to an incredible number. However, there is a very important thermodynamic however here. You could only imagine with a large protein, such as albumin with about 600 amino acids and molecular weight of 66,000 Daltons (look up the definition of a Dalton for the first exam, if you are not sure of that term) how many possible ways the protein might fold the number can literally in the tens of thousands! However, the fact is that proteins usually fold only one or two ways. Why is that? There are complexities, such as optimal bond distances and angels, mutual repulsions and attractions, etc. However, the bottom line for our purposes is a thermodynamic one. Which ever folding combination produces the most stable configuration in the aqueous environment of the cell or tissue, then that is the structure that is produced. What do I mean by the most stable configuration? Simply put, the most stable structure is the one with the lowest energy state. At a higher-than-necessary energy state, there would have to be a continual energy input to maintain that condition. If you think about the Second Law of Thermodynamics we discussed in the first lecture, then this concept governing protein folding should make sense to you. (Remember efficiency?) OK? If not, let me know. Remembering that I said that Thermodynamics and the Second Law never go away from us, let us consider a folded protein to be a march away from entropy (which is disorder). Thus, protein folding

represents higher structural order or a more negative entropy. Remembering also that there is no such thing as the free lunch, then if the protein entropy is decreased as it folds, then what entropy is increased to compensate for this? To answer this, we go back to water. When a protein is folding, I mentioned that the interior is starting to become hydrophobic, in that water wants to get away from the hydrophobic R groups. This pushes water together to for cages called clathrates. However, eventually these cages of water collapse, now going from a more organized state (cages) to a less organized state (more bulk water-like). Thus, the positive entropy change in the water largely compensates for the negative entropy change in the folding of the protein. Believe me, I have simplified this; but this, in fact, is the bottom line. Again, appreciate the complexity. By the way, do you think water balance and the osmotic state are important to you now at perhaps a different level than you previously considered???? Note: Sometimes you will come across the structure of a protein as shown in your text with helices and beta sheets (Figure 3.38). These are called ribbon models. Other times you will be given other images that provide different information. For example, in Figures 3.31c and 3.32 and 3.33 you will see what is called a space-filled model, with each sphere. In Figure 3.31b, we have another type of representation called the ball and stick model. I a sure you can see that different models of the same thing provide a different aspect of proteins structure. Quaternary Structure This is in some ways much easier to picture than tertiary structure. Specifically, quaternary structure can be defined as polypeptide chains assembled into multisubunit structures (two or more tertiary structures). Although the term quaternary refers to four, that has nothing to do with having four subunits present. Rather, it is only the level above tertiary. So then, the simplest quaternary structure is the dimer two subunits. When we study hemoglobin next week, we shall see a protein with four subunits (a tetramer) having quarternary structure. In contrast, we will note that myoglobin has but tertiary structure, because it is only a single globular polypeptide. Quarternary structure defines the spatial arrangement of subunits and their interactions, which are not covalent in nature. This type of bond would be too strong to allow a flexible interaction between the subunits. (When we discuss allosterism, then the importance of such flexibility will be come evident.) We must be sure to recognize that the various subunits of a protein with quarternary structure can be identical, but they certainly can also be different. It depends strictly upon the protein. An interesting illustration of multisubunits is the protein coat of the rhinovirus which causes the common cold. The coat of that virus includes sixty copies each of four different subunits. These subunits come together to form an almost spherical shell that encapsulates the viral genome. So where are we in this lecture? I am confident that you have some understanding of the four possible levels of protein structure. First, it must be stated that not all proteins have all levels of structure. Myoglobin has only tertiary structure, whereas hemoglobin has achieved a quaternary configuration. Some growth hormone-like peptides only have primary structure. Also, remember our structure/function concept! The structure is suited to best accommodate the function. That having been said, can you picture the concept that the primary, one-dimensional structure determines all the elaborate higher order structure of a protein? Why? (Dont read the next sentence until you take a shot at a guess). The answer, of course, lies in the side chains of each amino acids and their order, as determined by their respective amino acids sequence. So a fundamental, central principle of biochemistry is that amino acid sequence specifies conformation. This has been proven over and over again. Lets elaborate a bit.

The side chains determine how secondary structures will interact by their availability for bonding or not bonding. Without being too detailed, for example, the -helix is a default conformation. However, the steric hindrances of the side chains of isoleucine and valine tend to destabilize the helix, but are easily accommodated in the -sheet. Also remember that even non-ionizable groups can bond weakly with other non-ionizable groups through hydrophobic interactions). So how each side chain is available for bonding depends upon its charge state (or lack thereof) and its position in the primary sequence. Add that to thermodynamic (energy) constraints, and we have a functional protein all other things being equal. pKa Ah, were you hoping I would forget about that that term the pH at which there is 50% dissociation? Well, lets make things simple. You should remember from last weeks lecture that the pKa relative to the pH will help determine whether a potentially ionizable side chain is in fact ionized or not. This does not apply to all the amino acids, as some do not have potentially ionizable side chains, as in the case of glycine, for example, whereas lysine does. Can you figure out which other amino acids have a pKa value for their side chain? So the bottom line is, the bonding potential of a side chain is most definitely influenced by whether or not a group in the side chain is ionized or not, which you also know is pH-dependent relative to the pKa value. This, in turn, clearly affects the subsequent configuration of the whole protein. Please keep this fact in mind for next weeks discussion on hemoglobin and sickle cell anemia. Additional Material Remember from the opening paragraph of this lecture that amino acids (whether from the diet or from the turnover of protein in the body) can be anabolized into proteins that have many different uses. On the other hand, when we study gluconeogenesis, we will see that glucose can be synthesized from most amino acids. On pages 1043-1046 is reading related to protein digestion. You are responsible for reading this material so as to understand and learn for the first exam. Although it is basic physiology, learn the location and names and the actions of the enzymes involved in protein digestion. You do not have to memorize the specific activators of the various enzymes nor their specific chemical cleavage points shown in Table 25.7.