Browsing by Author "Suo, Yang"

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Open Access
Conformational kinetics reveals affinities of protein conformational states.
(Proc Natl Acad Sci U S A, 2015-07-28) Daniels, Kyle G; Suo, Yang; Oas, Terrence G
Most biological reactions rely on interplay between binding and changes in both macromolecular structure and dynamics. Practical understanding of this interplay requires detection of critical intermediates and determination of their binding and conformational characteristics. However, many of these species are only transiently present and they have often been overlooked in mechanistic studies of reactions that couple binding to conformational change. We monitored the kinetics of ligand-induced conformational changes in a small protein using six different ligands. We analyzed the kinetic data to simultaneously determine both binding affinities for the conformational states and the rate constants of conformational change. The approach we used is sufficiently robust to determine the affinities of three conformational states and detect even modest differences in the protein's affinities for relatively similar ligands. Ligand binding favors higher-affinity conformational states by increasing forward conformational rate constants and/or decreasing reverse conformational rate constants. The amounts by which forward rate constants increase and reverse rate constants decrease are proportional to the ratio of affinities of the conformational states. We also show that both the affinity ratio and another parameter, which quantifies the changes in conformational rate constants upon ligand binding, are strong determinants of the mechanism (conformational selection and/or induced fit) of molecular recognition. Our results highlight the utility of analyzing the kinetics of conformational changes to determine affinities that cannot be determined from equilibrium experiments. Most importantly, they demonstrate an inextricable link between conformational dynamics and the binding affinities of conformational states.
Open Access
Structural and Functional Studies on Noxious Stimuli Sensing of the Transient Receptor Potential Ankyrin 1 Channel
(2021) Suo, Yang
Transient receptor potential channel subfamily A member 1 (TRPA1) is a Ca2+-permeable cation channel that serves as the primary sensor of environmental irritants, noxious substances, and temperature. Many TRPA1 agonists are electrophiles that are recognized by TRPA1 via covalent bond modifications of specific cysteine residues located in the cytoplasmic domains. TRPA1 is also a temperature activated channel displaying unique species-specific thermo sensitivity. Preceding this work, however, a mechanistic understanding of electrophile sensing by TRPA1 has been limited by a lack of structural information. Moreover, the mechanism by which TRPA1 sense temperature has been elusive. Using cryo-electron microscopy, we determined the structures of nanodisc-reconstituted human TRPA1 in ligand free state and in complex with the covalent agonists JT010 or BITC at 2.8, 2.9, and 3.1 Å, respectively. Our structural and functional studies provide the molecular basis for electrophile recognition by the extraordinarily reactive Cys621 in TRPA1 and grant mechanistic insights into electrophile-dependent conformational changes in TRPA1. This work illustrates the fundamental principles of irritant sensing in humans at the molecular level and provides a platform for future drug development targeting TRPA1. Moreover, we determined the cryo-EM structure of rattlesnake TRPA1 in nanodisc-reconstituted condition at 3.3 Å. This structural revealed a novel N-terminal ankyrin repeat domain that was not resolved in previous structures. Our structural and functional studies on rattlesnake TRPA1 provides a framework in understanding the principles of thermo sensitivity in TRPA1.