Abstract
Protein structures are dynamic, undergoing motions that can play a vital role in function. However, the link between primary sequence and conformational dynamics remains poorly understood. Here, we studied how conformational dynamics can arise in a globular protein by evaluating the impact of individual core-residue substitutions in DANCER-3, a streptococcal protein G domain β1 variant that we previously designed to undergo a specific mode of conformational exchange that has never been observed in the wild-type protein. Using a combination of solution NMR experiments and molecular dynamics simulations, we demonstrate that only two mutations are necessary to create this conformational exchange, and that these mutations work synergistically, with one destabilizing the native structure and the other allowing two new conformational states to be accessed on the energy landscape. Overall, our results show how dynamics can appear in a stable globular fold, a critical step in the molecular evolution of dynamics-linked functions.
Highlights
Protein structures are dynamic, undergoing motions that can play a vital role in function
To investigate how sequence changes can lead to novel modes of conformational exchange in a globular protein, we individually reverted each of the five mutations present in DANCER-3 to the corresponding amino acid found in wild-type G domain β1 (Gβ1) (Fig. 1a, b)
The resulting five DANCER-3 point mutants (D3-F3Y, D3-I7L, D3F34A, D3-L39V, and D3-I54V, each named for the mutation they possess relative to DANCER-3) all adopted the native Gβ1 fold (Supplementary Fig. 1) and were stably folded (Table 1, Supplementary Figs. 2 and 3). 1H-15N heteronuclear single quantum coherence (HSQC) spectra for all variants with the exception of D3-F34A showed a population of additional peaks not observed in the wild-type Gβ1 spectrum (Supplementary Figs. 4 and 5), indicating that these variants adopt more than one distinct structural state, as observed for DANCER-316
Summary
Protein structures are dynamic, undergoing motions that can play a vital role in function. We studied how conformational dynamics can arise in a globular protein by evaluating the impact of individual core-residue substitutions in DANCER-3, a streptococcal protein G domain β1 variant that we previously designed to undergo a specific mode of conformational exchange that has never been observed in the wild-type protein. Despite the demonstrated importance of dynamics for protein function, the link between protein sequence and dynamics remains poorly understood[8], and epistasis resulting from the ruggedness of the protein energy landscape complicates attempts to study how sequence elements contribute to dynamics and thereby function in natural proteins[9] Progress towards this goal has been made by evolution-based studies[10,11,12], with key findings having shown that novel protein functions can arise from new dynamic regimes that reorganize functional sites[13,14]. Our results provide a model for the molecular evolution of conformational dynamics in globular proteins
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