Clive R. Bagshaw

Kinetic analysis of ATPase mechanisms.

At even the simplest level we can expect an ATPase mechanism to comprise the following four steps: the binding of ATP, the reaction of ATP with water on the enzyme, and the release of the products ADP and P 1 . So at the outset techniques are needed to investigate these four processes. The range of techniques needed is soon extended once questions are asked about the role of protons and metal ions, the possibility of a multistep hydrolytic process, multistep substrate and product binding processes, and protein–lipid or protein–protein interactions. Since ATPases and ATP synthases are almost universally involved in some form of energy transduction there is a particular need in an ATPase or ATP synthase reaction to evaluate the equilibrium constants of the steps in the mechanism and to investigate the possibility of alternate reaction pathways. The nature of the coupling process by the protein of the chemical reactions of ATP to the other energetic process, be it muscle contraction, active transport, respiration or photosynthesis, is likewise of profound interest. Finally we would like to know as much as possible about the ATPase or ATP synthase mechanism during the period when the various forms of energy transduction are occurring.

Crystal Structure of the Vinculin Tail Suggests a Pathway for Activation

Vinculin plays a dynamic role in the assembly of the actin cytoskeleton. A strong interaction between its head and tail domains that regulates binding to other cytoskeletal components is disrupted by acidic phospholipids. Here, we present the crystal structure of the vinculin tail, residues 879-1066. Five amphipathic helices form an antiparallel bundle that resembles exchangeable apolipoproteins. A C-terminal arm wraps across the base of the bundle and emerges as a hydrophobic hairpin surrounded by a collar of basic residues, adjacent to the N terminus. We show that the C-terminal arm is required for binding to acidic phospholipids but not to actin, and that binding either ligand induces conformational changes that may represent the first step in activation.

The kinetics of calcium binding to fura-2 and indo-1

The kinetics of Ca2+ dissociation from fura‐2 and indo‐1 were measured using a stopped‐flow spectrofluorimeter. The dissociation rate constants were 84 s−1 and 130 s−1, respectively, in 0.1 M KCl at 20°C. The rate constants were insensitive to pH over the range 7.0 to 8.0. The second order association rate constants were estimated indirectly to be in the region of 5 × 108 M−1·s−1 and thus approach the diffusion‐controlled limit. The results demonstrate that these new generation indicators are well‐suited to measure rapid changes in concentration of intracellular Ca2+.

Myosin cleft movement and its coupling to actomyosin dissociation

It has long been known that binding of actin and binding of nucleotides to myosin are antagonistic, an observation that led to the biochemical basis for the crossbridge cycle of muscle contraction. Thus ATP binding to actomyosin causes actin dissociation, whereas actin binding to the myosin accelerates ADP and phosphate release. Structural studies have indicated that communication between the actin- and nucleotide-binding sites involves the opening and closing of the cleft between the upper and lower 50K domains of the myosin head. Here we test the proposal that the cleft responds to actin and nucleotide binding in a reciprocal manner and show that cleft movement is coupled to actin binding and dissociation. We monitored cleft movement using pyrene excimer fluorescence from probes engineered across the cleft.

Characterization of homologous divalent metal ion binding sites of vertebrate and molluscan myosins using electron paramagnetic resonance spectroscopy

The binding of Ca2+, Mg2+ and Mn2+ to myosins from rabbit skeletal muscle, scallop striated adductor muscle and clam adductor muscle has been investigated. All three myosins bind two moles of divalent metal ion non-specifically and with high affinity (Mn2+ > Ca2+ > Mg2+). In addition, the molluscan myosins bind about a further two moles of Ca2+ specifically. Although rabbit myosin binds some Ca2+ in the presence of an excess of free Mg2+, this binding occurs at the nonspecific sites and should not be taken as evidence for a myosin-linked regulatory system of the type found in molluscan muscles. If such a system exists in vertebrate skeletal muscle, the homologous Ca2+-specific sites must be lost during the early stages of the myosin preparation. The characteristic electron paramagnetic resonance spectrum of the bound Mn2+ was utilized to confirm the homology of the non-specific sites in vertebrate and molluscan myosins. The sites are located on the “regulatory” class of light chain. Mn2+ bound to scallop myosin has a broad electron paramagnetic resonance spectrum, in contrast to the well-resolved spectra that it gives when bound to many other myosin species. This situation was exploited to identify homologous nonspecific, divalent metal-ion sites on the regulatory light chains from a variety of muscle types, including frog skeletal, rabbit cardiac, chicken gizzard and molluscan adductor muscles. When these light chains are combined with desensitized scallop myofibrils the electron paramagnetic resonance spectra of Mn2+ bound to the resultant hybrids are dominated by the signal from the non-specific site of the foreign regulatory light chain.

The significance of the slow dissociation of divalent metal ions from myosin 'regulatory' light chains.

It is well established that Ca2+ binding to troponin C, located on the actin filament, is involved in the activation of contraction of vertebrate skeletal muscle by relieving the inhibitory effect of the troponintropomyosin system [ 11. More recently the discovery of a Ca2+ binding site on the DTNB light chain of the myosin filament has led to the proposal of this being an additional site engaged in the control of contraction [2,3]. These ideas received impetus from the finding that the vertebrate DTNB light chain can substitute for the regulatory light chain of scallop myosin in conferring Ca2+ sensitivity to the ATPase of scallop myofibrils, although the DTNB light chain does not appear to participate directly [4]. Vertebrate skeletal myosin itself does not exhibit a Ca2+ sensitive, actinactivated ATPase in vitro in the absence of troponin and tropomyosin [5,6]. Nevertheless it remains plausible that Ca2+ binding to the DTNB light chain initiates the movement of the myosin crossbridges towards the actin filaments [2,3,7] and such an effect might not be revealed by the actomyosin ATPase in the steady-state, particularly in preparations which lack

Active site trapping of nucleotide by smooth and non-muscle myosins

The folded 10 S monomer conformation of smooth muscle myosin traps the hydrolysis products ADP and Pi in its active sites. To test the significance of this, we have searched for equivalent trapping in other conformational and assembly states of avian gizzard and brush border myosins, using formycin triphosphate (FTP) as an ATP analogue. When myosin monomers were in the straight-tail 6 S conformation, the hydrolysis products were released at about 0.03 s-1. Adoption of the folded 10 S monomer conformation reduced this rate by more than 100-fold, effectively trapping the products FDP and Pi in the active sites. This profound inhibition of product release occurred only on formation of the looped tail monomer conformation. In vitro-assembled myosin filaments released products at a comparable rate to free straight-tail 6 S monomers, and smooth muscle heavy meromyosin, which lacks the C-terminal two-thirds of the myosin tail, also did not trap the products in this way. Phosphorylation of the myosin regulatory light chain had no effect on the rate of product release from straight-tail 6 S myosin monomers or from myosin filaments. Rather, it allowed actin to accelerate product release. Phosphorylation acted also to destabilize the folded monomer conformation, causing the recruitment of molecules from the pool of folded monomers into the myosin filaments. The two processes of contraction and filament assembly are thus both controlled in vitro by light-chain phosphorylation. A similar linked control in vivo would allow the organization of myosin in the cell to adapt itself continuously to the pattern of contractile activity.

Mechanical unfolding of human telomere G-quadruplex DNA probed by integrated fluorescence and magnetic tweezers spectroscopy

Single-molecule techniques facilitate analysis of mechanical transitions within nucleic acids and proteins. Here, we describe an integrated fluorescence and magnetic tweezers instrument that permits detection of nanometer-scale DNA structural rearrangements together with the application of a wide range of stretching forces to individual DNA molecules. We have analyzed the force-dependent equilibrium and rate constants for telomere DNA G-quadruplex (GQ) folding and unfolding, and have determined the location of the transition state barrier along the well-defined DNA-stretching reaction coordinate. Our results reveal the mechanical unfolding pathway of the telomere DNA GQ is characterized by a short distance (<1 nm) to the transition state for the unfolding reaction. This mechanical unfolding response reflects a critical contribution of long-range interactions to the global stability of the GQ fold, and suggests that telomere-associated proteins need only disrupt a few base pairs to destabilize GQ structures. Comparison of the GQ unfolded state with a single-stranded polyT DNA revealed the unfolded GQ exhibits a compacted non-native conformation reminiscent of the protein molten globule. We expect the capacity to interrogate macromolecular structural transitions with high spatial resolution under conditions of low forces will have broad application in analyses of nucleic acid and protein folding.

Reversible movement of switch 1 loop of myosin determines actin interaction

The conserved switch 1 loop of P‐loop NTPases is implicated as a central element that transmits information between the nucleotide‐binding pocket and the binding site of the partner proteins. Recent structural studies have identified two states of switch 1 in G‐proteins and myosin, but their role in the transduction mechanism has yet to be clarified. Single tryptophan residues were introduced into the switch 1 region of myosin II motor domain and studied by rapid reaction methods. We found that in the presence of MgADP, two states of switch 1 exist in dynamic equilibrium. Actin binding shifts the equilibrium towards one of the MgADP states, whereas ATP strongly favors the other. In the light of electron cryo‐microscopic and X‐ray crystallographic results, these findings lead to a specific structural model in which the equilibrium constant between the two states of switch 1 is coupled to the strength of the actin–myosin interaction. This has implications for the enzymatic mechanism of G‐proteins and possibly P‐loop NTPases in general.

Analysis of nucleotide binding to Dictyostelium myosin II motor domains containing a single tryptophan near the active site.

Dictyostelium myosin II motor domain constructs containing a single tryptophan residue near the active sites were prepared in order to characterize the process of nucleotide binding. Tryptophan was introduced at positions 113 and 131, which correspond to those naturally present in vertebrate skeletal muscle myosin, as well as position 129 that is also close to the adenine binding site. Nucleotide (ATP and ADP) binding was accompanied by a large quench in protein fluorescence in the case of the tryptophans at 129 and 131 but a small enhancement for that at 113. None of these residues was sensitive to the subsequent open-closed transition that is coupled to hydrolysis (i.e. ADP and ATP induced similar fluorescence changes). The kinetics of the fluorescence change with the F129W mutant revealed at least a three-step nucleotide binding mechanism, together with formation of a weakly competitive off-line intermediate that may represent a nonproductive mode of nucleotide binding. Overall, we conclude that the local and global conformational changes in myosin IIs induced by nucleotide binding are similar in myosins from different species, but the sign and magnitude of the tryptophan fluorescence changes reflect nonconserved residues in the immediate vicinity of each tryptophan. The nucleotide binding process is at least three-step, involving conformational changes that are quite distinct from the open-closed transition sensed by the tryptophan Trp501 in the relay loop.