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

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

Thermodynamic Additivity of Sequence Variations: An Algorithm for Creating High Affinity Peptides Without Large Libraries or Structural Information

BACKGROUND: There is a significant need for affinity reagents with high target affinity/specificity that can be developed rapidly and inexpensively. Existing affinity reagent development approaches, including protein mutagenesis, directed evolution, and fragment-based design utilize large libraries and/or require structural information thereby adding time and expense. Until now, no systematic approach to affinity reagent development existed that could produce nanomolar affinity from small chemically synthesized peptide libraries without the aid of structural information. METHODOLOGY/PRINCIPAL FINDINGS: Based on the principle of additivity, we have developed an algorithm for generating high affinity peptide ligands. In this algorithm, point-variations in a lead sequence are screened and combined in a systematic manner to achieve additive binding energies. To demonstrate this approach, low-affinity lead peptides for multiple protein targets were identified from sparse random sequence space and optimized to high affinity in just two chemical steps. In one example, a TNF-α binding peptide with K(d) = 90 nM and high target specificity was generated. The changes in binding energy associated with each variation were generally additive upon combining variations, validating the basis of the algorithm. Interestingly, cooperativity between point-variations was not observed, and in a few specific cases, combinations were less than energetically additive. CONCLUSIONS/SIGNIFICANCE: By using this additivity algorithm, peptide ligands with high affinity for protein targets were generated. With this algorithm, one of the highest affinity TNF-α binding peptides reported to date was produced. Most importantly, high affinity was achieved from small, chemically-synthesized libraries without the need for structural information at any time during the process. This is significantly different than protein mutagenesis, directed evolution, or fragment-based design approaches, which rely on large libraries and/or structural guidance. With this algorithm, high affinity/specificity peptide ligands can be developed rapidly, inexpensively, and in an entirely chemical manner

The mobilization and effect of endogenous bone marrow progenitor cells in diabetic wound healing

Diabetic patients suffer from impaired wound healing, characterized by only modest angiogenesis and cell proliferation. Stem cells may stimulate healing, but little is known about the kinetics of mobilization and function of bone marrow progenitor cells (BM-PCs) during diabetic wound repair. The objective of this study was to investigate the kinetics of BM-PC mobilization and their role during early diabetic wound repair in diabetic db/db mice. After wounding, circulating hematopoietic stem cells (Lin 12c-Kit+Sca-1+) stably increased in the periphery and lymphoid tissue of db/db mice compared to unwounded controls. Peripheral endothelial progenitor cells (CD34+VEGFR+) were 2.5- and 3.5-fold increased on days 6 and 10 after wounding, respectively. Targeting the CXCR4\u2013CXCL12 axis induced an increased release and engraftment of endogenous BM-PCs that was paralleled by an increased expression of CXCL12/SDF-1\u3b1 in the wounds. Increased levels of peripheral and engrafted BM-PCs corresponded to stimulated angiogenesis and cell proliferation, while the addition of an agonist (GM-CSF) or an antagonist (ACK2) did not further modulate wound healing. Macroscopic histological correlations showed that increased levels of stem cells corresponded to higher levels of wound reepithelialization. After wounding, a natural release of endogenous BMPCs was shown in diabetic mice, but only low levels of these cells homed in the healing tissue. Higher levels of CXCL12/SDF-1\u3b1 and circulating stem cells were required to enhance their engraftment and biological effects. Despite controversial data about the functional impairment of diabetic BM-PCs, in this model our data showed a residual capacity of these cells to trigger angiogenesis and cell proliferatio

The Role of Romantic Involvement and Social Support in Italian Adolescent Mothers' Live

This study examined the experience of mothering in adolescence in the context of romantic involvement and social support. 30 adolescent mothers completed measures of adolescent self-development and motherhood, romantic relationship experience, availability of and satisfaction with social support, and parenting stress. Findings suggest that young mothers benefit greatly from the support of a partner, as evidenced by a more functional understanding of motherhood and the maternal role, greater satisfaction with social support, and lower levels of psychological distress. Education on social support should be made available to young mothers to address stress associated with adolescent parenting