Enhanced Hairpin Stability through Loop Design:  The Case of the Protein G B1 Domain Hairpin

Abstract

A mutational study of the peptide corresponding to the second hairpin of the protein G B1 domain (GB1p) provided a series of mutants with significantly increased fold stability. Mutations focused on improvement of the direction-reversing loop and the addition of favorable Coulombic interactions at the sequence termini. The loop optimization was based on a database search for residues that occur with the greatest probability in similar hairpin loops in proteins. This search suggested replacing the native DDATKT sequence with NPATGK, which resulted in a 4.5 kJ/mol stabilization of the hairpin fold. The introduction of positively charged lysines at the N-terminus provided an additional 2.4 kJ/mol of stabilization, affording a GB1p mutant that is 86 +/- 3% folded at 25 degrees C with a melting temperature of 60 +/- 2 degrees C. The trpzip version of this peptide, in which three of the hydrophobic core residues were mutated to tryptophan, yielded a sequence that melted at 85 degrees C. Throughout, fold populations and melting temperatures were derived from the mutation and temperature dependence of proton chemical shifts and were corroborated by circular dichroism (CD) melts. The study also suggests that the wild-type GB1p sequence is significantly less stable than reported in some other studies: only 30% folded in water at 25 degrees C.

Peptide folding and aggregation studied using a simplified atomic model

“…The magnitude of this difference is similar to that between the different experiments. In fact, the N nat hb -based value agrees well with CD and NMR data [33], whereas the E hp -based value agrees well with Trp fluorescence data [34].…”

“…The same fit gives T m = 321 ± 1 K and and T m = 322 ± 2 K and for GB1m3 and GB1m2, respectively. These values lie close to the melting temperature measured by CD and NMR for GB1m2 (320 ± 2 K [33]), and somewhat below the corresponding result for GB1m3 (333 ± 2 K [33]). …”

Effect of pH, urea, peptide length, and neighboring amino acids on alanine α‐proton random coil chemical shifts

“…Nonetheless, the tryptophan double substitution (14) does exhibit the largest upfield shift for the series (0.19 ppm), and tryptophan in either position is very important to note for -proton chemical shifts and CSI. In fact, the large aromatic amino acid tryptophan appears to dominate over the other smaller aromatic amino acids when two dissimilar aromatic amino acids are adjacent to alanine (22)(23)(24)(25). Whether in the N or C-terminal position, the chemical shift effect seen can be approximated by a single tryptophan residue.…”

“…Several studies have been done to measure the -proton random coil chemical shift value for each of the 20 common amino acids, and while there is general agreement among the studies, there are some notable discrepancies. 6,[18][19][20][21][22][23][24][25] For instance, in four separate studies the histidine random coil chemical shift value has been determined to be 4.630 ppm (pH 7, no urea, 358C, GGX aa A), 18 4.73 ppm (pH 5, 1M urea, 258C, GGX aa AGG, termini protected), 19 4.77 ppm (pH 5, no urea, 48C, GGX aa GG), 20 and 4.79 ppm (pH 2.3, 8M urea, 208C, GGX aa GG, termini protected). 22 Variations in the model systems (peptide length, free vs. capped termini) and the solution environment (pH, urea and temperature) are possible explanations for the different values observed.…”

“…To further complicate matters, studies have also shown that neighboring amino acids can affect the -proton random coil chemical shift value of an amino acid. 19,[23][24][25] Groups studying -hairpin structure have resorted to synthesizing at least two peptides for each hairpin studied, one with -hairpin structure and a control peptide of similar sequence with no hairpin structure, to account for the shift effects of neighboring amino acids. [9][10][11][12][13][14] In the current study it is our goal to add to the collective knowledge of chemical shift effects and correction factors by systematically studying the effect of solution environments (pH, urea) and molecular environments (peptide length, free vs. capped termini, and neighboring amino acids) on -proton chemical shifts.…”