Conformational Dynamics in Loop Swap Mutants of Homologous Fibronectin Type III Domains

Abstract

Fibronectin type III (FN-III) domains are autonomously folded modules found in a variety of multidomain proteins. The 10th FN-III domain from fibronectin (fnFN10) and the 3rd FN-III domain from tenascin-C (tnFN3) have 27% sequence identity and the same overall fold; however, the CC' loop has a different pattern of backbone hydrogen bonds and the FG loop is longer in fnFN10 compared to tnFN3. To examine the influence of length, sequence, and context in determining dynamical properties of loops, CC' and FG loops were swapped between fnFN10 and tnFN3 to generate four mutant proteins and backbone conformational dynamics on ps-ns and mus-ms timescales were characterized by solution (15)N-NMR spin relaxation spectroscopy. The grafted loops do not strongly perturb the properties of the protein scaffold; however, specific effects of the mutations are observed for amino acids that are proximal in space to the sites of mutation. The amino acid sequence primarily dictates conformational dynamics when the wild-type and grafted loop have the same length, but both sequence and context contribute to conformational dynamics when the loop lengths differ. The results suggest that changes in conformational dynamics of mutant proteins must be considered in both theoretical studies and protein design efforts.

Chimeric fibronectin matrix mimetic as a functional growth- and migration-promoting adhesive substrate

“…The six loops formed at the poles between β strands (termed AB, BC, etc.) are highly variable and can withstand extensive modification without loss of stability [53, 54]. This inherent stability likely facilitated the insertion of the RGDS sequence into the FG loop of FNIII8.…”

Protein conformational flexibility prediction using machine learning

“…Backbone 15 N S 2 have been reported for wild-type and two loop swap mutants of fnfn10 and of tnfn3 [41]. The experimental and predicted S 2 for the wild-type and mutant proteins are shown in figures 5 and 6.…”

“…Generalized order parameters were predicted using the structural coordinates from PDB files 1IGD, 1QQV, 1OR5, and 2RN2, respectively. Experimental generalized order parameters have been reported for wild-type and loop-swap mutants of the tenth fibronectin type III domain of the protein fibronectin (fnfn10) and of the third fibronectin type III domain of the protein tenascin (tnfn3) [41]. The mutants were constructed by interchange of the CC′ and FG loops between fnfn10 and tnfn3.…”

“…The mutants were constructed by interchange of the CC′ and FG loops between fnfn10 and tnfn3. Generalized order parameters were predicted using structural models described elsewhere [41]. GB3, HP67, fren ACP, RNaseH, fnfn10, and tnfn3 are not similar to any of the proteins used to develop the prediction method.…”

Exposing the Moving Parts of Proteins with NMR Spectroscopy

“…The NH studies have become a regular assay in dynamics-function investigations involving mutation and/or ligand binding. Examples are those by Palmer and co-workers 54 , and Peng and co-workers 55 . Using a Lipari-Szabo analysis of NH relaxation rates, the investigators compared the NH S 2 versus sequence for various loop mutants to investigate sequence-dynamics-binding correlations, and thus, the extent to which sequence encodes recognition-specific fluctuations.…”