Aspartic acid-proline sequence

In order to better understand the importance of substrate conformation to enzymatic N-glycosylation, a study was carried out comparing the solution conformation and glycosyl acceptor properties of a series of tripeptides of the general sequence Ac-Asn-Xaa-Thr-NH2 (76). Several amino acids with very distinct conformational properties were substituted in the middle position alanine, leucine, aspartic acid, proline, D-alanine, and a-aminoisobutyric acid. One and two dimensional iH NMR studies were used to determine intramolecular hydrogen bonding patterns, and NOE connectivities. 

Fig. 10. Sequences (see Table 1) of betabeUins. In each case, only one-half of the P-sandwich is shown. The dimer is formed from identical monomeric sets of four P-strands. In the pattern sequence, e is for end, p is for polar residue, n is for nonpolar residue, and t and r are for turn residues. Lower case f is iodophenyialanine lower case a, d, k, and p are the D-amino acid forms of alanine, aspartic acid, lysine, and proline, respectively B is P-alanine (2,53,60,61).

Figure 2.9. Amino acid sequences of human defensins. The conserved positions of six cysteine residues are shown in hatched boxes. Abbreviations A, alanine C, cysteine D, aspartic acid E, glutamic acid F, phenylalanine G, glycine H, histidine I, isoleucine K, lysine L, leucine M, methionine N, asparagine P, proline Q, glutamic acid R, arginine S, serine T, threonine V, valine W, tryptophan Y, tyrosine.

It is noteworthy that there is another limiting factor in the choice of amino acid types at the junction sites which affect the enzymatic process of the intein. For example, in the case of SceVMA (also called PI-Seel) from the IMPACT system, proline, cysteine, asparagine, aspartic acid, and arginine cannot be at the C-terminus of the N-terminal target protein just before the intein sequence. The presence of these residues at this position would either slow down the N-S acyl shift dramatically or lead to immediate hydrolysis of the product from the N-S acyl shift [66]. The compatibility of amino acid types at the proximal sites depends on the specific inteins and needs to be carefully considered during the design of the required expression vectors. The specific amino acid requirements at a particular splicing site depends on the specific intein used and is thus a crucial point in this approach. 

Trypsin cleaves adjacent to arginine and lysine, but has a preference for arginine, particularly at high pH values. In the sequences Arg-X or Lys-X, cleavage is inhibited if there is a proline residue at position X. Cleavage is also inhibited by glutamic acid and aspartic acid, and by the occurrence of additional arginine or lysine residues. 

The cleavage/attachment site consists of 3 amino acids. The first amino acid is the site of attachment and can be glycine, alanine, serine, cysteine, aspartic acid, or asparagine. Only these small amino acids are permissible in chimeric studies [105,108,109] and are the only ones reported in the C-terminal tails of sequenced GPI-anchored proteins [5,104-106]. Serine is most favorable [109] and is present in over half of the known GPI anchor proteins. The second amino acid, on the C-terminal side, can be any amino acid except proline. In contrast, the third amino acid is restricted to only small amino acids, preferably glycine or alanine, although serine and to a lesser extent cysteine, threonine and valine are possible [105,108]. 

The normal mRNA sequence, AUG-CCC-GAC-UUU, would encode the peptide sequence methionine-proline-aspartate-phenylalanine. The mutant mRNA sequence, AUG-CGC-GAC-UUU, would encode the mutant peptide sequence methionine-arginine-aspartate-phenylalanine. This would not be a silent mutation because a hydrophobic amino acid (proline) has been replaced by a positively charged amino acid (arginine). 

Table 15.2. Structural families in the basic loop structures. CONFORMATION is the conformation of the loop (0, ((/ angles, see caption of Figure 15.5) NO. is number observed in proteins analyzed SEQUENCE is the sequence template a,, T are used to denote residues favoring a helix, P strand and type I P turn structures respectively i = polar, = hydrophobic - is any amino acid capital letters are one letter amino acid code (G Glycine D Aspartic Acid, N Asparagine, S Serine, T threonine, A Alanine, H Histidine, P Proline) / indicates alternative, blank indicates loop limits...

Different amino acids favor the formation of alpha helices, beta pleated sheets, or loops. The primary sequences and secondary structures are known for over 1,000 different proteins. Correlation of these sequences and structures revealed that some amino acids are found more often in alpha helices, beta sheets, or neither. Helix formers include alanine, cysteine, leucine, methionine, glutamic acid, glutamine, histidine, and lysine. Beta formers include valine, isoleucine, phenylalanine, tyrosine, tryptophan, and threonine. Serine, glycine, aspartic acid, asparagine, and proline are found most often in turns. 

On the other hand, the equilibrium constant K indicates the tendency to form helical or nonhelical states. K values in excess of unity denote helix formers K values much less than unity, conversely, indicate coil-forming sequences. With proteins, proline, serine, glycine, and aspartine, for example, are typical helix breakers. Lysine, thyrosine, aspartic acid, threonine, arginine, cysteine, and phenyl alanine act as neither helical breakers or formers, whereas all other a-amino acids are typical helix formers. 

---