Spasic, A. | Search bioRxiv

bioRxiv2022

Gαs slow conformational transition upon GTP binding and a novel Gαs regulator

G proteins are major signaling partners for G protein-coupled receptors (GPCRs). Although stepwise structural changes during GPCR-G protein complex formation and guanosine diphosphate (GDP) release have been reported, no information is available with regard to guanosine triphosphate (GTP) binding. Here, we used a novel Bayesian integrative modeling framework that combines data from hydrogen-deuterium exchange mass spectrometry, tryptophan-induced fluorescence quenching, and metadynamics simulations to derive a kinetic model and atomic-level characterization of stepwise conformational changes incurred by the {beta}2-adrenergic receptor ({beta}2AR)-Gs complex after GDP release and GTP binding. Our data suggest rapid GTP binding and GTP-induced dissociation of Gs from {beta}2AR and G{beta}{gamma}, as opposed to a slow closing of the Gs -helical domain (AHD). Yeast-two-hybrid screening using Gs AHD as bait identified melanoma-associated antigen D2 (MAGE D2) as a novel AHD-binding protein, which was also shown to accelerate the GTP-induced closing of the Gs AHD.

molecular biology↗

bioRxiv2018

Chemically Accurate Relative Folding Stability of RNA Hairpins from Molecular Simulations

This study describes a comparison between melts and simulated stabilities of the same RNAs that could be used to benchmark RNA force fields, and potentially to determine future melt-ing experiments. Using umbrella sampling molecular simulations of three 12-nucleotide RNA hairpin stem loops, for which there are experimentally determined free energies of unfold-ing, we projected unfolding onto the reaction coordinate of end to end (5' to 3' hydroxyl oxygen) distance. We estimate the free energy change of the transition from the native con-formation to a fully extended conformation--the stretched state--with no hydrogen bonds between non-neighboring bases. Each simulation was performed four times using the AM-BER FF99+bsc0+{chi}OL3 force field and each window, spaced at 1 [A] intervals, was sampled for 1 s, for a total of 552 s of simulation. We compared differences in the simulated free energy changes to analogous differences in free energies from optical melting experiments using ther-modynamic cycles where the free energy change between stretched and random coil sequences is assumed to be sequence independent. The differences between experimental and simulated {Delta}{Delta}G{degrees} are on average 1.00 {+/-} 0.66 kcal/mol, which is chemically accurate and suggests analo-gous simulations could be used predictively. We also report a novel method to identify where replica free energies diverge along the reaction coordinate, thus indicating where additional sampling would most improve convergence. We conclude by discussing methods to more economically perform such simulations.

biophysics↗