Y. Michael Peyser

Inorganic phosphate regulates the binding of cofilin to actin filaments

Inorganic phosphate (Pi) and cofilin/actin depolymerizing factor proteins have opposite effects on actin filament structure and dynamics. Pi stabilizes the subdomain 2 in F‐actin and decreases the critical concentration for actin polymerization. Conversely, cofilin enhances disorder in subdomain 2, increases the critical concentration, and accelerates actin treadmilling. Here, we report that Pi inhibits the rate, but not the extent of cofilin binding to actin filaments. This inhibition is also significant at physiological concentrations of Pi, and more pronounced at low pH. Cofilin prevents conformational changes in F‐actin induced by Pi, even at high Pi concentrations, probably because allosteric changes in the nucleotide cleft decrease the affinity of Pi to F‐actin. Cofilin induced allosteric changes in the nucleotide cleft of F‐actin are also indicated by an increase in fluorescence emission and a decrease in the accessibility of etheno‐ADP to collisional quenchers. These changes transform the nucleotide cleft of F‐actin to G‐actin‐like. Pi regulation of cofilin binding and the cofilin regulation of Pi binding to F‐actin can be important aspects of actin based cell motility.

Tryptophan-130 is the most reactive tryptophan residue in rabbit skeletal myosin subfragment-1

Rabbit skeletal muscle myosin subfragment‐1 (S‐1) was reacted with dimethyl(2‐hydroxy‐5‐nitrobenzyl)sulfonium bromide (DHNBS) resulting in modification of 0.8 tryptophan residues per S‐1. In order to assign the most reactive tryptophan of the 5 S‐1 tryptophans, antibodies were raised in rabbits against bovine serum albumin modified with DHNBS. The antibodies reacted with the 27 kDa tryptic fragment of DHNBS‐treated S‐1, indicating that the reactive tryptophan resides on this domain. The 27 kDa fragment was isolated from DHNBS‐treated S‐1 and was further cleaved at a single cysteine residue by 2‐nitro‐5‐thiocyanobenzoic acid. This cleavage resulted in two peptides, each of them containing one tryptophan. The antibodies reacted with the smaller peptide consisting of residues 122–204. The only tryptophan residing on this peptide is Trp130, and this is therefore the most reactive tryptophan of S‐1.

Effect of Ionic Strength on the Conformation of Myosin Subfragment1–Nucleotide Complexes

The effect of ionic strength on the conformation and stability of S1 and S1-nucleotide-phosphate analog complexes in solution was studied. It was found that increasing concentration of KCl enhances the reactivity of Cys(707) (SH1 thiol) and Lys(84) (reactive lysyl residue) and the nucleotide-induced tryptophan fluorescence increment. In contrast, high KCl concentration lowers the structural differences between the intermediate states of ATP hydrolysis in the vicinity of Cys(707), Trp(510) and the active site, possibly by increasing the flexibility of the molecule. High concentrations of neutral salts inhibit both the formation and the dissociation of the M**.ADP.Pi analog S1.ADP.Vi complex. High ionic strength profoundly affects the structure of the stable S1.ADP.BeF(x) complex, by destabilizing the M*.ATP intermediate, which is the predominant form of the complex at low ionic strength, and shifting the equilibrium to favor the M**.ADP.Pi state. The M*.ATP intermediate is destabilized by perturbation of ionic interactions possibly by disruption of salt bridges. Two salt-bridge pairs, Glu(501)-Lys(505) in the Switch II helix and Glu(776)-Lys(84) connecting the catalytic domain to the lever arm, seem most appropriate to consider for participating in the ionic strength-induced transition of the open M*.ATP to the closed M**.ADP.Pi state of S1.

Near UV circular dichroism from biomimetic model compounds define the coordination geometry of vanadate centers in MeVi- and MeADPVi-rabbit myosin subfragment 1 complexes in solution

The circular dichroism (CD) spectrum was measured from vanadate (Vi) cyclic esters of chiral vicinal diols, hydroxycarboxylates, and cyclodextrines as a function of Vi concentration ([Vi]) and at the lowest energy transitions of the vanadium. At low [Vi] and in the presence of excess vicinal diols, hydroxycarboxylates, or cyclodextrines the CD signal intensity scales linearly with [Vi] indicating the predominance of a monomeric cyclic ester. At higher [Vi], the signal intensity in the presence of the vicinal diols and hydroxycarboxylates become nonlinear in [Vi], indicating formation of a dimeric cyclic ester. Vanadium-51 NMR (51V-NMR) indicates the coordination geometry of several of these model Vi centers in solution and identifies the CD signals characteristic to Vi trigonal bipyramidal (tbp) and octahedral (Oh) coordination geometries from monomeric and dimeric species. The CD spectra from monomeric and dimeric forms of the tbp-coordinated model compounds have two apparent transitions with amplitudes of opposite sign at wavelengths > or = 240 nm. Spectra from the monomeric and dimeric Oh coordinated species are distinct from the tbp-type spectra over the same wavelength domain because of the presence of two additional transitions with opposite sign amplitudes. These model spectra were compared to the vanadate CD spectra from Vi bound to rabbit myosin subfragment 1 (S1) in solution, in the presence of divalent metal cations (MeVi-S1) or trapped with MeADP (MeADPVi-S1). Polymeric MeVi binds to the active site of S1 and the vanadate centers in MnVi-S1 or CoVi-S1 produce a CD signal resembling that from the tbp model. The trapped ATPase transition state analog MeADPVi produces a different CD signal resembling that from the Oh model.