Structural Insights into the Evolution of a Non-Biological Protein: Importance of Surface Residues in Protein Fold Optimization

Abstract

Phylogenetic profiling of amino acid substitution patterns in proteins has led many to conclude that most structural information is carried by interior core residues that are solvent inaccessible. This conclusion is based on the observation that buried residues generally tolerate only conserved sequence changes, while surface residues allow more diverse chemical substitutions. This notion is now changing as it has become apparent that both core and surface residues play important roles in protein folding and stability. Unfortunately, the ability to identify specific mutations that will lead to enhanced stability remains a challenging problem. Here we discuss two mutations that emerged from an in vitro selection experiment designed to improve the folding stability of a non-biological ATP binding protein. These mutations alter two solvent accessible residues, and dramatically enhance the expression, solubility, thermal stability, and ligand binding affinity of the protein. The significance of both mutations was investigated individually and together, and the X-ray crystal structures of the parent sequence and double mutant protein were solved to a resolution limit of 2.8 and 1.65 Å, respectively. Comparative structural analysis of the evolved protein to proteins found in nature reveals that our non-biological protein evolved certain structural features shared by many thermophilic proteins. This experimental result suggests that protein fold optimization by in vitro selection offers a viable approach to generating stable variants of many naturally occurring proteins whose structures and functions are otherwise difficult to study.

The Phylogenomic Roots of Modern Biochemistry: Origins of Proteins, Cofactors and Protein Biosynthesis

“…ANBP shared structural features with extant proteins, including a nucleotide binding pocket similar to those found in ATP-binding proteins and a Znbinding site resembling the typical treble clef Zn binding motif (Krishna and Grishin 2004). Additional in vitro selection experiments designed to improve folding stability of ANBP revealed that surface residues are as important as those buried in the structure (Smith et al 2007). The fine tuned protein (2P09) retained the original structure but folded more stably (Fig.…”

Artificial proteins from combinatorial approaches

“…The initially selected sequences displayed poor biophysical properties (solubility, stability) for structural studies. Further evolutionary optimization of the sequences led first to a truncated version and later to multiple amino acid substitutions [27] that improved the initially selected sequences by surface stabilizing interactions [28]. This man-made progressive amelioration of artificial ATP binding proteins may, in some way, mimic a plausible evolutionary scenario for natural protein evolution.…”

Searching Combinatorial Libraries for Native Proteins with Novel Folds

“…[14] After many iter-A C H T U N G T R E N N U N G ative rounds of in vitro selection and A C H T U N G T R E N N U N G directed evolution, several proteins emerged that bound ATP with high affinity and specificity. [15,16] The three-dimensional structure (Figure 2 B) of one of these proteins has now been solved by solution NMR and X-ray crystallography, and reveals a novel zinc-nucleated a/b fold with a unique topology. [16][17][18] A fundamentally different approach to generating proteins that fold into structures with novel topologies was developed by Baker and co-workers.…”

“…[15,16] The three-dimensional structure (Figure 2 B) of one of these proteins has now been solved by solution NMR and X-ray crystallography, and reveals a novel zinc-nucleated a/b fold with a unique topology. [16][17][18] A fundamentally different approach to generating proteins that fold into structures with novel topologies was developed by Baker and co-workers. [19] Here, a general computational strategy was developed that iterates between protein sequence design and protein structure prediction to create a 93-residue a/b protein with a novel topology.…”

“…[13] B) An ATP binding protein evolved from an unconstrained library of 4 10 12 random sequences (PDB ID: 2P09). [16][17][18] C) An a/b-protein obtained from a library of computational sequences (PDB ID: 1QYS). [19] The two a/b-proteins fold into novel topologies.…”