Handout: Introductory remarks; Schedule; amino acid properties. Reading assignment: Branden-Tooze II, chapter 1 (Introduction); Creighton II, chapter 1.1 to 1.3. (Introduction). [Next lecture: Creighton II, 1.61 (amino acid analysis); 1.62 (sequencing); 1.8 (peptide synthesis)]

II. Secondary Structure. This term refers to the regular local arrangements arising from hydrogen bonds between elements of the polypeptide backbone. There are only a small number of choices: helices, sheets, and turns among which the alpha helix and the beta sheet predominate; these are distinguished by specific positions on the Ramachandran plot. The term supersecondary structure refers to commonly observed motifs or aggregates of secondary structure elements; examples are the alpha-coiled coil and beta barrel)

Before we set out on a more detailed discussion of primary structure, let us take a look at the building blocks themselves, the amino acids. (Amino acid composition could be regarded as the zeroth order structure of proteins.) All amino acids can be described by the general formula (draw) [Could also indicate that P is a special case; the amino acid proline is in fact a cyclic imino acid. This leaves only the carboxyl function as a component that all amino acids have in common. Since the imino nitrogen is as basic as the nitrogen in the amino group, it would be more appropriate to speak of the 20 naturally occurring carboxyl bases]. Note that in all cases where the side chain R is not hydrogen, the central carbon atom represents a center of asymmetry; in other words, amino acids are optically active [= chiral, from the Greek word for hand; therefore the synonymous term handedness for chirality - draw correct form. All amino acids in proteins are of the l-form. [In the SR notation of organic chemistry, it is the S-isomer.] It is not clear why this is so, since chemical mirror images should not differ in their size, shape, charge, or chemical reactivity; most scientist believe that this fact is an accident of evolution and that originally the d-amino acids had had the same chance to become building blocks of biological macromolecules. In fact it is possible to chemically synthesize proteins made up entirely of d-amino acids. Milton et al. (Science 256:1445, 1992) reported the synthesis of the d-enantiomer of HIV protease which, not surprisingly, cleaves substrates which are the mirror images of natural substrates. Others think that decay, which is asymmetric, preferentially destroys d-amino acids (It may interest you that the asymmetry of decay was originally demonstrated by C. N. Yang, who headed the Institute for Theoretical Physics here at Stony Brook). At any rate, it is clear that there is a requirement for uniformity so that the protein synthetic apparatus, i.e. the ribosome, can handle all building blocks in an identical manner. The exception is G in which the side chain equals hydrogen. G accordingly occurs only in one isomeric form. A simple way to remember the absolute configuration is Dickerson's bridge mnemonic (draw). Note that in the RS (Cahn-Ingold-Prelog) notation used by chemists, l-amino acids are S-compounds, with the exception of l-Cys = R-Cys.

Even if we use the proper geometric representation, the molecule is not correctly drawn. After all, amino acids are acids or - more generally speaking - electrolytes, i.e. they contain ionizable groups. At neutral pH, a given amino acid is much more likely to exist in a doubly charged form [here correct the drawing]. This zwitterionic form predominates between pH 2 and 10. Amino acids are weak electrolytes; they are only partly ionized near neutral pH. Their dissociation behavior can be quantitatively described by the Henderson-Hasselbalch equation [derive from law of mass action whose significance for general ligand binding analysis should be stressed ].

So far we have considered ionizable functions that all amino acids share. Side chains impart individuality to an amino acid, and it is to the side chains that we now turn. A list of amino acids and their side chain structures and pK values is given in the handout. These 20 amino acids are coded for by genetic information. Many of them can be modified within the folded protein, e.g. by phosphorylation, without requiring an expansion of the genetic code. However it is now known that a 21^st amino acid is coded for, namely selenocysteine, which occurs in a few enzymes, notably glutathione peroxidase. In this case UGA, one of the normal termination codons, is recruited for the job (Note that a list of 3-letter abbreviations as well as of single-letter designations is also provided; I would strongly recommend committing the single letter code to memory. Although they are not quite as easy to memorize they are widely used). As we shall see side chains and their unique ionizable functions dominate the acid/base or electrolytic properties of polypeptide chains. In addition to carboxyl and amino functions we find guanidinium, imidazol, phenolic hydroxyl groups etc. The pK values given are approximate values; as in the case of -amino and -carboxyl groups, ionization of these functions inside proteins are slightly different. In general, chemical properties of a functional group are determined by context, i.e. the relationship with other functional groups in the folded protein. Here recall that all amino acids have at least 2 ionizable groups - this also holds for proline as imino and amino nitrogens have very similar pKs!!

(i) First let us take a look at the apolar residues which as a rule occur in the interior of folded proteins: G is small and flexible; occasionally, e.g. at helix crossings, it is absolutely necessary because all other amino acids would be too bulky. A is nondescript, a useful brick that is quite abundant in proteins (In E. coli about 10% (13% Schulz and Schirmer; 8.3% acc. to Creighton II, p. 4, rather than the 6.25% expected from the random occurrence of the alanine codons). Branched side chains of V, I, L, F are stiff bricks from which protein interiors are made; the absence of linear alkyl chains is noticeable. The combination of two crosslinked cysteins, C-S-S-C, also referred to as cystine, is very apolar, and so is M. Other special cases are the imino acid P which, as one might expect, has unique steric properties; being a frequent component of so-called reverse turns it is often found on the surface of proteins. Finally there is the aromatic amino acid W which despite its general hydrophobicity can make a hydrogen bond with its indol nitrogen.

(ii) Polar residues are conveniently grouped into nonionizable and ionizable. The former class includes W, already mentioned, and the hydroxylated side chains of S and T and the amides N and Q. They are important because they are rich in H-bonding capability. C and Y become ionized at fairly high pH (in comparison to alcohols, thiols dissociate more readily because of the lower electronegativity of sulfur; phenols because the phenolate anion is stabilized by resonance). Among the ones that are ionized at neutral pH there are 2 acidic, D and E, and 3 basic side chains, R, K, and H. D and E are weaker acids than the alpha carboxyls, because of the absence of a neighboring positive charge that facilitates release of the proton; although they differ only by a methylen group they interact quite differently with the polypeptide backbone (Explain!!). H has a pK value near neutrality and therefore readily participates in proton transfer reaction at physiological pH. It is an important acid-base catalyst that occurs at the active site of many enzymes although it is otherwise a relatively rare protein constituent.

i.e. in a peptide with a single representative each of these elements, W would account for ~77%, Y for ~21%, and cystine for ~2% of the absorbance at 280 nm.