Site-Specific Determination of Conformational Flexibility from a Side Chain Perspective: Native State Thiol Exchange of E. Coli Ribonuclease H

Abstract

sulfate proteoglycans) supported dimerization or polymerization of the FGFRs are thought to be required to activate the signaling pathway. The D2 domain is suggested to bind with both HSPGs and FGFs to form a ternary complex. Xray and NMR solution structures of the D2 domain have been analyzed using the CAPTURE cation-pi program. The CAPTURE program indicates cation-pi interactions between residues Y155:R152(Xray), W191:R203 (NMR) and possibly F237:K151 (Xray). Biophysical characterization of the mutants at each cation and pi pair, identified by CAPTURE, shows a significant destabilization resulting from the Y155A, W191A and R203E mutations. Results from differential scanning calorimetry show a reduction in melting temperature by 10-14 C for Y155A, W191 and R203 mutants of D2. The reduction in the stability of the D2 domain is corroborated by results of ANS binding, thermal denaturation and a limited trypsin digestion experiments. The HSQC of D2 Y155A shows limited chemical shift perturbation of residues in the vicinity of the mutation site. The W191A and R203E mutations show significant 1H-15N chemical shift perturbations in their HSQC spectra. The results obtained in this study show that cation-pi interactions contribute significantly to the thermodynamic stability of proteins. In addition, our results indicate that cation-pi predictions made on the solution NMR structures are more reliable than those predicted based on crystal structures.