- Uncharged Polar side chains [text Figure 3-5 part 3, 2]: These side chains will generally occur on the surfaces of proteins because of their polarity and hydrogen-bonding characteristics. If they occur on the interior they must generally H-bond with other interior functional groups. The definition of "uncharged" is based on a pH of 7. There are four side-chains, ser (serine), thr (threonine), asn (asparagine), and gln (glutamine), which are neutral under all conditions of pH. (Note that asn and gln are simply the amide forms of asp and glu. It is thus often difficult to determine whether a given residue was a asp or asn etc. in chemical analysis of peptides, since the treatment breaking peptide bonds also will generally break the amide bonds of asn and gln.) Tyr (tyrosine) and cys (cysteine) are uncharged at pH 7, but both ionize at higher pH's (respective pKa's = 9.5-10.9 & 8.3-8.6). Finally, his (histidine - imidazolium grp), has a pKa of 6.4-7.0 and is thus partially charged (positive) at pH 7, and will be charged at low pH's.
- Charged Polar side chains [text Figure 3-5 part 4, 5]: These four side-chains will have very strong tendencies to be on the surface - it costs a great deal of energy to bury an ionic charge in a non-polar interior! It turns out that the sum of the acidic groups in a protein, asp (aspartate) + glu (glutamate), is usually equal to the sum of the sum of the basic groups, lys (lysine - amino grp) + arg (arginine - guanidinium grp). This is expected since we want a net neutral particle at its operating pH (usually around pH 7)
- Amino Acid Chemistry: All aa's share two chemically functional groups, the carboxyl group and the amino group. Thus they will share the chemical reactions of these groups familiar from organic chemistry. Many of these reactions are exploited in the laboratory manipulation of amino acids, peptides, and proteins. Note that these reactions are also common to the side chains of asp, glu (-COOH), and lys (-NH2). Another side-chain with important chemistry is cys (-SH). Biologically the most important reactions are those required for protein formation, particularly the peptide bond (text Figure 3.13).
- pKa's: Note that the pKa's for carboxylic acids tend to have values of about 5, while the pKa of the amino acid -COOH is around 2 (text Table 3.1). What's going on? The shift in pKa can be assigned to the nearby protonated amine. Recall that 'naked' charges are very unstable, while nearby counter-charges stabilize them. Also, from organic chemistry you may recall that negative charges can be stabilized by inductive effects of nearby electron withdrawing groups, such as a protonated, positively charged, nitrogen. Because of the extra intervening carbons the side chain -COOH's of asp and glu are not similarly stabilized, and thus have pKa values closer to the expected 5. Of course we would also expect analogous effects of the negative charge on the carboxyl group on the charged amine. Note the titration curve for amino acids with titratable side groups as exemplified by histidine (text Figure 3.12)
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Peptides and Amino Acid Chemistry
- Peptide bond formation: Note that a peptide bond is simply an amide bond between the alpha carboxyl and amino groups of amino acids. If we write the reacting groups in their unionized (acid and amine) forms, then we can see the reaction takes place with the loss of the elements of water, via an attack of the lone-pair electrons of the amine on the carbonyl carbon of the carboxyl group:
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Let's begin by looking at a well known exemplar of protein structure, Insulin (text Figure 3.24). Note that insulin has two peptides linked together with disulfide bonds. Too successfully analyse the structure of this peptide we first need to break the disulfide bonds (text Figure 3.26) and separate the peptides, most commonly by chromatography. We need to do this to prevent the confusion which would result from analysing the raw protein and discovering multiple N-terminii etc.
We can next analyse the separate peptides by first doing an N-terminal analysis to find the N-terminal amino acid and then doing an Edman degradation which sequentially cleaves amino acids off with repeated chemistry. (text Figure 3.25) Unfortunately this process is limited by the lack of 100% yields in the process so it is only effective for short peptides.
As a result most proteins are hydrolyzed into short peptides by enzymatic and specific chemical cleavage reactions. The cleavage reactions are picked to give a high likelyhood of overlapping peptide fragments. These fragments are then analyzed as above. (text Figure 3.27).
Pathway Diagrams
