Structural Comparison of the Two Alternative Transition States for Folding of TI I27

Abstract

TI I27, a beta-sandwich domain from the human muscle protein titin, has been shown to fold via two alternative pathways, which correspond to a change in the folding mechanism. Under physiological conditions, TI I27 folds by a classical nucleation-condensation mechanism (diffuse transition state), whereas at extreme conditions of temperature and denaturant it switches to having a polarized transition state. We have used experimental Phi-values as restraints in ensemble-averaged molecular dynamics simulations to determine the ensembles of structures representing the two transition states. The comparison of these ensembles indicates that when native interactions are substantially weakened, a protein may still be able to fold if it can access an alternative transition state characterized by a much larger entropic contribution. Analysis of the probability distribution of Phi-values derived from ensemble averaged simulations, enables us to identify residues that form contacts in some members of the ensemble but not in others illustrating that many interactions present in transition states are not strictly required for the successful completion of the folding process.

Structural Analysis of an Equilibrium Folding Intermediate in the Apoflavodoxin Native Ensemble by Small-Angle X-ray Scattering

“…The equilibrium ϕ i sim value of residue i in a particular configuration was defined as the fraction of native contacts N i made by that residue in MD with respect to those found in the crystal structure N i nat (i.e., ϕ i sim = N i /N i nat ). To drive the simulations toward satisfying the experimental data, we used the biased MD method of Paci and Karplus 85 and Geierhaas et al 86 A reaction coordinate ρ was defined as the mean squared difference between the N e experimental ϕ-values (ϕ i exp ) and the simulated ones (ϕ i cal ):…”

A Simple Model of Backbone Flexibility Improves Modeling of Side-chain Conformational Variability

“…The proteins used in this study are: ubiquitin (PDB id: 1UBQ) 43 , the third fibronectin type III domain from human tenascin (TNfn3; PDB id: 1TEN) 44 , the tenth fibronectin type III domain from human fibronectin (FNfn10; PDB id: 1FNA) 45 , the B1 domain from protein G (gb1; PDB id: 1PGB) 46 , the SH3 domain in human Fyn (fyn-sh3; PDB id: 1SHF) 34 , the de novo designed three-helix bundle α3D (a3D; PDB id: 2A3D) 6 , eglin c (PDB id: 1CSE) 7; 47 , an SH2 domain of phospholipase C-gamma1 (plcc-sh2; PDB id: 2PLD) 7 , the B1 domain of protein L (proteinL; PDB id: 1HZ6) 31 , HIV-1 protease homodimer bound to the inhibitor DMP323, (hiv-protease-holo; PDB id: 1QBS), flavodoxin bound to flavin mononucleotide (flavodoxin-holo; PDB id: 1OBO) 48 , cytochrome c2 bound to its heme prosthetic group (cytochrome-c2-holo; PDB id: 1C2R) 49 , the N-terminal domain of chicken skeletal troponin C (NTnC; PDB id: 1AVS) 50 , Cdc42Hs 51 , adipocyte lipid-binding protein (albp; PDB id: 1LIB) 52 , muscle fatty acid-binding protein (mfabp; PDB id: 1HMT) 52 , and Ca 2+ -loaded calmodulin (PDB id: 1AHR) 53 .…”

Determination of the Transition State Ensemble for the Folding of Ubiquitin from a Combination of Φ and Ψ Analyses

“…[19][20][21] It has also been shown that it is possible, with the use of restraints on ensembleaveraged properties, to distinguish between multiple pathways and fractional formation of native-like interactions in the TS. [22][23][24][25] In a recent study, the computational strategy of using Φ values as structural restraints has been validated in the case of the protein barnase through the accurate prediction of the specific interactions present in the TS ensemble obtained from double-mutant cycle experiments 5 that were not used as restraints in the simulations. 18 Alternative strategies, which do not rely on experimental measurements, have also been proposed for the validation of TS ensembles.…”

“…In this work, we provide an assessment of the structural information that can be obtained from Φ and Ψ analyses by performing a series of molecular dynamics simulations with restraints on ensembleaveraged properties, 22,24 with the goal of characterizing the TS for folding of ubiquitin. We carried out three series of simulations: (i) using Φ values as restraints; (ii) using Ψ values as restraints; and (iii) using both Φ and Ψ values simultaneously as restraints.…”