RT Journal Article
SR Electronic
T1 Flexibility and design: conformational heterogeneity along the evolutionary trajectory of a redesigned ubiquitin
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 081646
DO 10.1101/081646
A1 Justin T. Biel
A1 Michael C. Thompson
A1 Christian N. Cunningham
A1 Jacob E. Corn
A1 James S. Fraser
YR 2016
UL http://biorxiv.org/content/early/2016/10/17/081646.abstract
AB Summary Although protein design has been used to introduce new functions, designed variants generally only function as well as natural proteins after rounds of laboratory evolution. One possibility for this pattern is that designed mutants frequently sample nonfunctional conformations. To test this idea, we exploited advances in multiconformer modeling of room temperature X-ray data collection on redesigned ubiquitin variants selected for increasing binding affinity to the deubiquitinase USP7. Initial core mutations disrupt natural packing and lead to increased flexibility. Additional, experimentally selected mutations quenched conformational heterogeneity through new stabilizing interactions. Stabilizing interactions, such as cation-pi stacking and ordered waters, which are not included in standard protein design energy functions, can create specific interactions that have long range effects on flexibility across the protein. Our results suggest that increasing flexibility may be a useful strategy to escape local minima during initial directed evolution and protein design steps when creating new functions.