Recursively enriched dynamic combinatorial libraries for the self-selection of optimally stable proteins.

Even at very low template (guest) concentrations, the optimal template-assembled host from a dynamic combinatorial library (DCL) of host fragments may be unobtainable because hetero-oligomers will always be present at higher concentrations than isoenergetic homo-oligomers. Recursively enriched dynamic combinatorial libraries (REDCLs) offer a general solution to this problem that should be applicable to any self-selecting system under thermodynamic control. The utility of the REDCL strategy is demonstrated by determination of the optimal hydrophobic core packing in a template-assembled triple helical protein for which the template is a metal ion and the contributing host fragments are components of a 36-member conformationally restricted peptide library in which each peptide is augmented with a metal-binding moiety. Convergence of the 8436-member DCL to 5 optimal trimers (0.06% of the DCL) is complete after four cycles of enrichment. The core packing of the optimal sequences is shown to be native-like, and to reflect the hydrophobic amino acid preferences found in natural parallel three-stranded coiled coils. The influence of potentially critical amino acids on the outcome of the recursive enrichment is explored in a second REDCL. The same peptide sequences were returned and were shown to populate seven of the 8436 possible trimers, or 0.08% of the DCL.

Protein core packing by dynamic combinatorial chemistry.

We describe the first example of the recursive selection of biologically relevant macromolecules from a dynamic combinatorial library (DCL). A small library of 36 peptides was allowed to undergo self-association in aqueous solution to form 8436 trimers. The stability of each of these trimers was governed by the formation of a well-packed hydrophobic core. The DCL allowed variation of the hydrophobic residues comprising this core over all combinations of glycine, alanine, valine, leucine, isoleucine, and phenylalanine at six positions. The study leads to three important conclusions: (i) fewer than 0.2% of all possible core packing arrangements have high folding stabilities; (ii) these arrangements are stabilized by intimate "jigsaw" packing, not by sequestration of maximum hydrophobic surface area; (iii) a well-defined "rule" for packing of stable cores exists, but this rule is nuanced by the presence of two unexpected amino acid sequences and the absence of one expected amino acid sequence.

Electrostatic determinants of stability in parallel 3-stranded coiled coils.

The optimal positioning of salt-bridging interactions in a parallel alpha-helical homotrimeric coiled coil has been explored in a metal ion-assembled polypeptide trimer of 60 residues; arginine-glutamate pairs are more stabilizing than the corresponding lysine-glutamate pairs, and optimal stabilization is obtained with positively charged arginine residues at the c positions of the alpha-helical heptad and negatively charged glutamate residues at the e positions.