A de novo designed protein–protein interface

Abstract

As an approach to both explore the physical/chemical parameters that drive molecular self-assembly and to generate novel protein oligomers, we have developed a procedure to generate protein dimers from monomeric proteins using computational protein docking and amino acid sequence design. A fast Fourier transform-based docking algorithm was used to generate a model for a dimeric version of the 56-amino-acid b1 domain of streptococcal protein G. Computational amino acid sequence design of 24 residues at the dimer interface resulted in a heterodimer comprised of 12-fold and eightfold variants of the wild-type protein. The designed proteins were expressed, purified, and characterized using analytical ultracentrifugation and heteronuclear NMR techniques. Although the measured dissociation constant was modest (;300 mM), 2D-[ 1 H, 15 N]-HSQC NMR spectra of one of the designed proteins in the absence and presence of its binding partner showed clear evidence of specific dimer formation.

Computational Enzyme Design

“…Huang et al achieved micromolar binding affinities by using a design minimization approach in which the best amino acid identities and rotamers were predicted for the protein-protein interface. [132] Improved affinities were obtained when the naturally occurring protein-protein interfaces were used as a guide.…”

Manipulating the substrate specificity of murine dihydrofolate reductase enzyme using an expanded set of amino acids

“…The conformational stabilities of mDHFR variant:ligand complex structures were calculated using the RosettaDesign version 3.4 as described previously with minor changes [35,53,54]. A fixed backbone protein design protocol has been successfully used for computational design of diverse proteins [55][56][57][58][59][60][61]. In particular, a flexible backbone protocol was effective in calculating conformational stability of a protein variant with a conservative single mutation often leading to a minimal backbone displacement upon the mutation.…”

Purification Systems Based on Bacterial Surface Proteins

“…However, one interesting example that relates to engineering of novel binding surfaces is the computational de novo design of a protein-protein heterodimer based on the C1-domain (Huang et al 2007). Through rational design, molecules that spontaneously formed heterodimers could be produced.…”