Free energy perturbation study on a Trp-binding mutant (Ser88 ->Cys) of the trp-repressor

The Ser⁸⁸Cys mutant of the trp-repressor showed a lower affinityfor the corepressor than the wild-type repressor [G = 1.7 ±0.3 kcal/mol, Chou and Matthews (1989) J. Biol. Chem., 264,18314–18319].A molecular dynamics/free energy cycle perturbation study wasperformed to understand the origin of the decreased affinity.A value (G = 1.58 ± 0.28 kcal/mol) comparable with theexperimental value was obtained by the simulation. Free energycomponent analysis revealed that destabilization of the vander Waals interaction between Ser⁸⁸ and Trp¹⁰⁹ (corepressor)mainly contributed to the decreased affinity of the mutant.The rotational transition of the hydroxyl (sulfhydryl) groupof Ser⁸⁸ (Cys⁸⁸) during the simulations affected the contributionsof Arg⁸⁴ and water to the free energy change in the aporepressorand those of Arg⁸⁴ and Trp ¹⁰⁹ to that in the holorepressor.However, the contributions from different residues compensatedeach other, and the total free energy changes were almost invariablein the various simulations.     

Ca2+-ATPase Molecules as a Calcium-Sensitive Membrane-Endoskeleton of Sarcoplasmic Reticulum

The Ca²⁺-transport ATPase of sarcoplasmic reticulum (SR) is an integral, transmembrane protein. It sequesters cytoplasmic calcium ions released from SR during muscle contraction, and causes muscle relaxation. Based on negative staining and transmission electron microscopy of SR vesicles isolated from rabbit skeletal muscle, we propose that the ATPase molecules might also be a calcium-sensitive membrane-endoskeleton. Under conditions when the ATPase molecules scarcely transport Ca²⁺, i.e., in the presence of ATP and ≤ 0.9 nM Ca²⁺, some of the ATPase particles on the SR vesicle surface gathered to form tetramers. The tetramers crystallized into a cylindrical helical array in some vesicles and probably resulted in the elongated protrusion that extended from some round SRs. As the Ca²⁺ concentration increased to 0.2 µM, i.e., under conditions when the transporter molecules fully carry out their activities, the ATPase crystal arrays disappeared, but the SR protrusions remained. In the absence of ATP, almost all of the SR vesicles were round and no crystal arrays were evident, independent of the calcium concentration. This suggests that ATP induced crystallization at low Ca²⁺ concentrations. From the observed morphological changes, the role of the proposed ATPase membrane-endoskeleton is discussed in the context of calcium regulation during muscle contraction.     

Molecular dynamics simulation of trp-aporepressor in a solvent

Molecular dynamics simulations of Escherichia coli trp-aporepressorwere carried out in the absence and presence of explicit watermolecules. The vacuum simulations resulted in significant deformationof the initial X-ray structure. A solvated simulation with anonbonded cut-off radius of 9 Å gave a better result,and the most satisfactory result was obtained when electrostaticinteractions within a cut-off radius of 18 Å were consideredby a twin-range method. The trajectory from the last simulationwas used to analyze the dynamical properties of the aporepressor.The root-mean-square fluctuations of the residues showed therigidity of the central core and the flexibility of the DNA-bindingsites, consistent with the X-ray temperature factors. The dynamicalcross-correlation map indicated a significant negative correlationbetewen the central core and the two DNA-binding sites, andthus reproduced the three-domain format (a central core andtwo DNA-binding heads) from a dynamical point of view. The coreregion showed weak, but many, intra- and inter-molecular correlations,while the helix-turn-helix DNA-binding motifs were free fromcorrelations with other regions.     

Threonine 81 of the trp repressor of Escherichia coli plays an auxiliary role in the formation of the corepressor binding pocket

A mutational study was performed on the corepressor (Ltryptophan)binding site of the trp repressor of Escherichia coli. Threonine81, one of the residues forming the hydrophobic pocket of thebinding site, was replaced with Ser, Cys and Met by cassettemutagenesis. Biochemical characterization showed that all thesemutations caused a moderate decrease in tryptophan binding activity(free energy change 1 kcal/mol). The results suggested thatthe binding pocket is rather flexible in the vicinity of Thr81.On the other hand, the mutations produced a discernible decreasein the repressor activity in vivo, apparently by weakening oreliminating the hydrogen bond between Thr81 and the operatorDNA, as well as by introducing possible side-chain rearrangement.     

Glycine 85 of the trp-repressor of E.coli is important in forming the hydrophobic tryptophan binding pocket: experimental and computational approaches

Experimental and computational analyses were performed on thecorepressor (L-tryptophan) binding site of the trp-repressorof Escherichia coli to investigate the ligandprotein interactions.Gly 85, one of the residues forming the hydrophobic pocket ofthe binding site, was systematically replaced with Ala, Val,Leu and Trp by cassette mutagenesis. Biochemical characterizationshowed that all these mutations caused significant decreasesin tryptophan binding activity. Free energy perturbation calculationswere performed for the mutants and were consistent with theexperimental results. The lack of a side chain at position 85was concluded to be essential for binding the corepressor; thestructure of the binding pocket was suggested to be tight inthe vicinity of Gly85.