ABSTRACT
The side chain of proline is made of an aliphatic five-membered pyrrolidine ring. The side chain is unique among protein-constituting amino acids in that its terminal Cδ is covalently bonded to the preceding pep tide bond nitrogen. The polypeptide backbone at this point has no amide hydrogen for use as a donor in hydrogen bonding. The pyrrolidine ring makes rigid constraints on the rotation about the NCα bond of the peptide backbone and drastically fixes the angle of rotation to approximately −58° (1). The conformational energy of a proline residue depends largely on the angle ψ of rotation about the CαC bond of the peptide backbone. The proline residue, when isolated from other proline residues in a polypeptide chain, has two energy minima at ψ=−55° and ψ=l45° (1). The bulky pyrrolidine ring restricts the available conformational space of the preceding residue in the polypeptide chain if its side chain extends at least to Cβ (1, 2). The peptide bond (Xaai−1Proi) preceding a proline residue, unlike those preceding other amino acid residues, might not be expected to have double bond character due to the lack of an imide hydrogen (3). The Xaai-1Proi peptide bond is more likely to adopt the cis rather than the trans configuration, compared with other peptide bonds (probability: 0.10.3 vs. <10-3) (2, 3). The difference in the standard free energy ∆G° between cis and trans Xaai−1Proi peptide bonds is on the order of +8 to −8 kJ/mol (1 J=0.239 cal) (4), and the activation energy for the cis-trans isomerization is about 54 kJ/mol, which is far less than 84 kJ/ mol for other peptide bonds (2, 3). However, the rate of the cis-trans isomerization is very slow for the Xaai−1 Proi peptide bond, with a half-time of 20 min at 0°C, compared with 1012 s−1 for other peptide bonds (3). The above-mentioned exceptional properties of proline impose significant constraints on the polypeptide conformation of proteins, their folding processes, and their functions (2-9). Thus, proline tends to be a conserved residue (2, 10). In 1988, the frequency of proline was determined to be 5.1 mol% among 1021 unrelated proteins of known sequence (11). This frequency is comparable with that (4.8 mol% among 393 unrelated protein sequences) reported by Chakrabarti and Pal (9). In 1982, we found a big difference in the proline content between oligo-1,6-glucosidases from Bacillus cereus ATCC7064 and B. thermoglucosidasius KP1006 (12). At that time, there was no report about the relationship between proline frequency and thermostability, except one made by Crabb et
al. (13) on D-glyceraldehyde-3-phosphate dehydrogenases from the facultative thermophile B. coagulans and the obligate thermophile B. stearothermophilus. On sequence comparison, they proposed a proline residue in a solvent-exposed variable loop of the B. stearothermophilus enzyme to contribute to its higher thermal stability. In 1987, we found a strong correlation between the number of proline residues and thermostability (expressed as tm*) for five Bacillus oligo-1,6-glucosidases (2, 14-17). Based on this finding, we proposed a general principle for increasing protein thermostability, known as the Proline Rule (16, 18). The rule states that globular proteins can be thermostabilized by increasing the frequency of proline in the second position (i+1) of β-turns.† A direct proof for the proposal was given by Matthews et al. (19). These authors increased the thermostability of bacteriophage T4 lysozyme by replacing an alanine residue with a proline residue in one of the β-turns to decrease the backbone entropy of the unfolded molecule. From 1994 to 1998, we cumulatively thermostabilized B. cereus oligo-1,4glucosidase through stepwise addition of proline residues in 12 separate positions, namely, four i+1 positions in β-turns, four N1 positions in the first turns of α-helices, and four positions in coils (21). The validity of the Proline Rule has been confirmed by sitedirected mutagenesis on various proteins (21-44). Many findings correlating proline residues to thermostability have been reported in numerous proteins from microorganisms including hyperthermophiles (45-63). Directed evolution for protein thermal stabilization has been found to include proline substitutions (64-67). It is furthermore interesting that many psychrophilic proteins have a low number of proline residues (68-72). In 1999, Suzuki (38) proposed the Proline Rule in refined form. The Proline Rule suggests an evolutionary rule for protein thermal adaptation (38, 73-75) and a paradigm for engineering protein thermostability (19, 21-23, 38). This paper describes three steps that we have taken to examine and refine this rule (38).
