Massive sequence perturbation of a small protein

Abstract

Most protein topologies rarely occur in nature, thus limiting our ability to extract sequence information that could be used to predict structure, function, and evolutionary constraints on protein folds. In principle, the sequence diversity explored by a given protein topology could be expanded by introducing sequence perturbations and selecting variant proteins that fold correctly. However, our capacity to explore sequence space is intrinsically limited by the enormous number of sequences generated from the 20 amino acids and the limited number of variants likely to fold. Here we sought to test whether the sequence space for naturally existing proteins can be explored by simple, sequential degeneration of a complete set of short sequence segments of a model protein, without long-range covariation. Using the Raf ras binding domain as a model of a small protein capable of autonomous folding, we degenerated 72 of 76 positions of the primary structure for the 20 amino acids in segments of four to seven residues defined by secondary structure and selected the folded species for interaction with h-ras by using an in vivo survival-selection assay. The methodology presented allowed for rigorous statistical analysis and comparison of sequence diversity. The ensemble of sequence variants of Raf ras binding domain obtained have recaptured the diversity observed for the ubiquitin-roll topology. A signature sequence for this fold and the implication of this strategy to protein design and structure prediction are discussed.massive mutagenesis ͉ protein-fragment complementation assay ͉ protein structure topology ͉ ubiquitin superfold ͉ Raf ras binding domain

Fold Designability, Distribution and Disease

“…Screening random sequences for ones that fold is one approach [5] to investigate designability. Testing the foldability and functionality of different proteins after mutagenesis in different environments (in vivo or in vitro) is another approach [47][48][49][50]. Such experiments are likely to provide new information important to refining measures of designability and its use to estimate fold fitness.…”

The Relationship Between Relative Solvent Accessibility and Evolutionary Rate in Protein Evolution

“…Mutagenesis experiments have shown that different positions in proteins have widely differing tolerances to amino acid substitutions (Reidhaar-Olson and Sauer 1988;Bowie et al 1990;Lau and Dill 1990;Guo et al 2004;Campbell-Valois et al 2005;Smith and Raines 2006). On average, however, mutations introduced at solvent-exposed sites are less likely to disrupt protein structure and function than mutations introduced at buried sites.…”

The Transition State of the ras Binding Domain of Raf Is Structurally Polarized Based on Φ-Values but Is Energetically Diffuse

“…The main subset of residues was selected on the basis of their low tolerance to mutation in the sequence perturbation experiments. 33 These include all residues of the hydrophobic core, except for W114, which served as the fluorescent probe to follow folding/unfolding reactions (Figure 1(d)), 35 and some residues involved in the topological arrangement of the domain and in the binding interface with ras. Additional mutations were analyzed to cover all regions of the structure, including residues with solvent-exposed sidechains.…”

“…The latter residue might be involved in forming sidechain backbone H-bonds that could stabilize the β-turn at the rate limiting step, 32 as suggested for N14 of protein-L. 15 Q66A and T68A found in a loop, which constitutes part of the ras binding interface, produced insignificant ΔΔG F-U , indicating that they are conserved solely for binding. 33,34 The mutants V70A (0.96) and V72A (0.98) that are located in β2 showed the highest Φ-values at hydrophobic core positions. V69A, which is located on the solvent exposed face of β2, showed Φ-value well above average (0.69).…”

“…Most important for the current study was the observation that our results were consistent with evolutionary conservation of ubiquitin fold sequences and a bi-level organization of the hydrophobic core (Figure 1(c) and (d)). 33 These results constituted the basis for the design of mutants presented below.…”