Franck Travers

Some recent aspects of the use of high-pressure for protein investigations in solution

Abstract Proteins play a key role in the metabolism of living organisms as, for example, catalysts (enzymes), carriers or receptors of various molecules. These marginally stable biopolymers of amino acids can be perturbed in their activity by pressure, temperature and other environmental variables as organic solvents. Changing environmental conditions can induce metabolism dysfunctions. On isolated systems, i.e. purified proteins, high pressure and other perturbing variables can be used as tools for investigation of protein structure/activity relationships and enzyme mechanisms. The elementary basic physical mechanisms of the action of high pressure upon proteins are stated in the first part of this review. This is followed by a section devoted to technical aspects, including methods for generation of high pressure, and some recent developments, namely stopped-flow spectrometry and electrophoresis under high pressure. Then, the use of pressure as a tool for investigation of enzyme mechanisms and for study...

Time resolved measurements show that phosphate release is the rate limiting step on myofibrillar ATPases

The myofibril is a good model to study the ATPase of the muscle fibre. When myofibrillar ATPase reaction mixtures are quenched in acid, there is a burst of Pi formation, due to AM · ADP · Pi or Pi as shown in the scheme: AM + ATP ↔ A·M·ATP ↔ AM·ADP·Pi ↔ AM·ADP + Pi ↔ AM + ADP. Therefore, in the steady state, either AM·ADP·Pi or AM·ADP or both predominate. To determine which, we studied the reaction using a Pi binding protein (from E. coli) labeled with a fluorophore such that it is specific and sensitive to free Pi [Brune, M. et al. (1994) Biochemistry 33, 8262–8271]. We show that the Pi bursts with myofibrillar ATPases (calcium‐activated or not, or crosslinked) are due entirely to protein bound Pi. Thus, with myofibrillar ATPases the AM·ADP·Pi state predominates.

Thermodynamics of the two step formation of horseradish peroxidase compound I

AbstractThe effect of temperature (20 to-38°C), pressure (normal pressures to 1.2 kbar) and solvent (water, 60% DMSO and 50% methanol) on the reaction of hydrogen peroxide or ethyl peroxide with horseradish peroxidase were studied. The formation of compound I was followed at 403 nm in a stopped flow apparatus adapted for high pressure and low temperature work.As with the alkaline form (Job and Dunford 1978) the neutral form of the peroxidase binds peroxide substrates in two steps. It was the combined use of organic solvents and low temperatures which revealed saturation kinetics:

A jump in an Arrhenius plot can be the consequence of a phase transition: The binding of ATP to myosin subfragment 1

E + SE Scompound I,

ATPase and shortening rates in frog fast skeletal myofibrils by time-resolved measurements of protein-bound and free Pi.

where E=horseradish peroxidase and S peroxide substrate. In water and organic solvents at temperatures above-10°C, K1 was too small and k2 too large to be measured, here, K1·k2 was obtained. k-2 was too small for measurement under all conditions. Whereas K1 was insentitive to the peroxide substrate and solvent composition k2 was very sensitive. The thermodynamic parameters ΔH≠, ΔS≠ and ΔV≠ for K1 and k2 were obtained under different experimental conditions and the data are interpreted within the available thermodynamic theories.

Transient-Phase Studies on the Arginine Kinase Reaction

Summary The dielectric constant of water in presence of increasing concentrations of weakly protic solvents depressing the freezing point has been measured. The same measurements have been carried out for various volume ratios between 20°C and the freezing point, as well as in presence of glycine. The conditions in which these solutions are « isodielectricwith water have been established and are part of a series of determinations in view of biochemical investigations at subzero temperatures.

Activation thermodynamics of the binding of carbon monoxide to horseradish peroxidase: Role of pressure, temperature and solvent

The temperature dependence of the kinetics of the binding of ATP to myosin subfragment−1 was studied by an ATP chase technique in a rapid−flow—quench apparatus: A temperature range of 30°C to −15°C was obtained with ethylene glycol as antifreeze. The Arrhenius plot of k 2 is discontinuous with a jump at 12°C. Above the jump ΔH ≠ = 9.5 kcal/mol, below ΔH ≠ = 28.5 kcal/mol. Few such Arrhenius plots are recorded in the literature but they are predicted from theory. Thus, we explain our results as a phase change of the subfragment 1−ATP system at 12°C. This is in agreement with certain structural studies.

The exo- or endonucleolytic preference of bovine pancreatic ribonuclease A depends on its subsites structure and on the substrate size.

Our objective is to propose an overview of the usefulness of skeletal myofibril as an experimental system for studying mechanochemical coupling of skeletal muscles and myosin ATPase activity. The myofibril is a true functional mini-muscle that is able to contract in the presence of ATP. It also contains the machinery necessary for the calcium sensitivity of the contraction. In the absence of calcium, myofibrillar ATPase activity is basal, no shortening occurs and no active force is developed. In the presence of calcium, myofibrillar ATPase is activated and myofibrils either shorten with no external load (native myofibrils) or contract isometrically (cross-linked myofibrils). With this organised system, both chemical and mechanical studies can be carried out. For a decade, our laboratory has been using rabbit psoas myofibrils for exploring myosin ATPase activity. The first challenge was to successfully apply rapid kinetic approaches, such as rapid-flow-quench, to this organised system. Another challenge was to work with myofibrils in cryoenzymic conditions, i.e. in the presence of organic solvents and at sub-zero temperatures. In this overview, we highlight differences between the myosin ATPase in organised systems (myofibrils or fibres) and that of contractile proteins in solution (S1 or actoS1) that we observed using these approaches. We discuss the importance of these differences in terms of mechanochemical coupling. It is concluded that great care should be taken when extrapolating mechanochemical properties of the contractile proteins in solution to the whole muscle.