Daniel Lavalette

Microscopic viscosity and rotational diffusion of proteins in a macromolecular environment.

The Stokes-Einstein-Debye equation is currently used to obtain information on protein size or on local viscosity from the measurement of the rotational correlation time. However, the implicit assumptions of a continuous and homogeneous solvent do not hold either in vivo, because of the high density of macromolecules, or in vitro, where viscosity is adjusted by adding viscous cosolvents of various size. To quantify the consequence of nonhomogeneity, we have measured the rotational Brownian motion of three globular proteins with molecular mass from 66 to 4000 kD in presence of 1.5 to 2000 kD dextrans as viscous cosolvents. Our results indicate that the linear viscosity dependence of the Stokes-Einstein relation must be replaced by a power law to describe the rotational Brownian motion of proteins in a macromolecular environment. The exponent of the power law expresses the fact that the protein experiences only a fraction of the hydrodynamic interactions of macromolecular cosolvents. An explicit expression of the exponent in terms of protein size and cosolvents mass is obtained, permitting definition of a microscopic viscosity. Experimental data suggest that a similar effective microviscosity should be introduced in Kramers equation describing protein reaction rates.

Competition with Xenon Elicits Ligand Migration and Escape Pathways in Myoglobin

Evidence for ligand migration toward the xenon-binding cavities in myoglobin comes from a number of laser photolysis studies of MbO2 including mutants and from cryo- and time-resolved crystallography of MbCO. To explore ligand migration in greater detail, we investigated the rebinding kinetics of both MbO2 and MbCO under a xenon partial pressure ranging from 1 to 16 atm over the temperature range (293-77 K). Below 180 K xenon affects to a significant, but minor, extent the thermodynamic parameters for rebinding from the primary docking site in each Mb taxonomic substate. Above 200 K the ligand migrates to the proximal Xe1 site but when the latter is occupied by xenon a new kinetic process appears. It is attributed to rebinding from transient docking sites located on the path between the primary and the secondary docking site of both ligands. Ligand escape exhibits a more complicated pattern than expected. At room temperature O2 and CO escape appears to take place exclusively from the primary site. In contrast, at T approximately 250 K, roughly 50% of the CO molecules that have escaped from the protein originate from the Xe1 secondary site.

Viscosity dependence of O2 escape from respiratory proteins as a function of cosolvent molecular weight.

Laser photodissociation of respiratory proteins is followed by fast geminate recombination competing with escape of the oxygen molecule into the solvent. The escape rate from myoglobin or hemerythrin has been shown previously to exhibit a reciprocal power-law dependence on viscosity. We have reinvestigated oxygen escape from hemerythrin using a number of viscous cosolvents of varying molecular weight, from glycerol to dextrans up to 500 kDa. In isoviscous solutions, the strong viscosity dependence observed with small cosolvents is progressively reduced upon increasing the cosolvents molecular weight and disappears at molecular weights greater than about 100 kDa. Thus, viscosity is not a suitable independent parameter to describe the data. The power of the viscosity dependence of the rate coefficient is shown here to be a function of the cosolvents molecular weight, suggesting that local protein-solvent interactions rather than bulky viscosity are affecting protein dynamics.

Kinetic Evidence for Three Photolyzable Taxonomic Conformational Substates in Oxymyoglobin

The kinetics of oxygen geminate binding with the taxonomic substates of MbO2 are reported. The maximum entropy method was used to analyze the rebinding kinetics of MbCO and MbO2 monitored in the Soret. The resulting rate distributions were found to consist of a small number of overlapping bands. A global parametric fit of a series of rate distributions recorded at several temperatures was performed using a Gaussian basis set to resolve the individual enthalpy distributions P(H). This approach was first validated by showing that the well-documented taxonomic substates of MbCO could be recovered. The method was then applied to MbO2. Three taxonomic substates were identified at pH 4.8, whereas only two of them contribute to oxygen geminate rebinding at pH 7.0. These findings show that, similarly to MbCO, MbO2 also exists as three photolyzable and kinetically different taxonomic substates and suggest reconsidering the issue of the photolysis quantum yield of MbO2.

Ligand migration and escape pathways in haem proteins

Biophysical techniques developed during the last three decades have provided an increasingly detailed description of the internal processes associated with ligand capture and release by haem proteins. Myoglobin has long been the paradigm for these studies. More recently, cytochrome P450cam (the camphor-metabolizing cytochrome P450 from Pseudomonas putida) has also received considerable interest. In spite of sharing the same prosthetic group, the Fe(II)-haem, these proteins are structurally unrelated and they perform different functions. Recent works show that both proteins exhibit a common feature: a series of permanent or fluctuating, mostly hydrophobic, cavities of the protein matrix are providing transient docking sites as well as migration, escape and possibly entry pathways for the ligand. Remarkably, these systems of cavities connect the distal and the proximal regions of the haem, a disposition that may contribute to ligand capture enhancement.

Rotational diffusion of Escherichia coli ribosomes: II. - Ribosome attachment to polyurydilic acid

Ribosome attachment to poly(U) has been studied by following the rotational diffusion of polyribosomes in solution. On the average, 13-17 and 50 nucleotides are found to be associated with 30S and 70S ribosome respectively. For an equal length of poly(U), the number of particles in a 30S polysome is four times that in a 70S polysome. The results are consistent with a structure of the polysome in which individual ribosomes are in close contact.