(425c) Simulated Mutagenesis and Structural Analysis for Protein Fragment Engineering

Mutagenesis analyses were performed using Molecular Dynamics Replica Exchange (REM) as a tool for systematically evaluating the structural variability of protein domains of interest in the area of protein biotechnology. A novel REM scheme, termed Multiple Mutant REM (MMREM), was developed to quickly screen diverse mutants based on their relative propensities for attaining favorable structures.

In our initial implementation, the wildtype and mutated hypervariable regions of an anti-hCG llama VHH antibody were simulated via REM on a constrained scaffold. Llama antibody binding domains such as this (which are smaller than those of conventional antibodies) are of interest as minimalistic models for antibody engineering, but are known to have uncommon structures which are not predictable by bioinformatic tools. The loop structures of the wildtype crystal (Spinelli et al., Nat. Struct. Biol. 3:752-757, 1996) were recovered, starting from an ?open? conformation. Furthermore, the structural variability observed for the H1 and H3 loops of the simulated wildtype antibody agrees with that found in the NMR study of this antibody (Renisio et al., Proteins Struct. Funct. Genet. 47:546-555, 2002). In addition to the wildype, seven mutants were simulated via REM with the goal of identifying structural determinants that return the non-canonical H1 loop of the wildtype antibody to the type 1 canonical structure predicted by database methods formulated for conventional antibodies. Two cases with three point mutations yield a stable type 1 H1 structure. Other mutants with fewer mutations were also found to give a significant fraction of such conformations. MMREM was further used to successfully identify additional mutations that stabilize a canonical H1 loop grafted on the llama antibody scaffold. Overall, the mutagenesis results suggest an influence of interloop interactions on the attainment of canonical conformations for this antibody.

In the second implementation of our proposed scheme, wildtype and mutant conformers of the Alzheimer's Aβ42 peptide are studied in order identify ?soluble? - stable mutants that are likely to have reduced proclivity towards aggregation. The results are compared to those obtained experimentally for this system via a recently developed mutant screening method (Fisher et al., Protein Sci. 15:449-458, 2006).

The use of MMREM and REM for screening mutants and assessing structural stability may be useful for elucidating key interactions that lead to the appearance of particular structural features in proteins. The latter can ease the identification of sequences that may promote disease prone conditions and facilitate a rational protein design.