We report on a study that combines advanced fluorescence methods with molecular dynamics simulations to cover timescales from nanoseconds to milliseconds for a large protein, the chaperone Hsp90.
The process of protein crystallization from aqueous protein
solutions
is still insufficiently understood. During macroscopic crystal formation,
occurring often on time scales from a few hours to several days, protein
dynamics evolves on the molecular level. Here, we present a proof
of concept and a framework to observe this evolving diffusive dynamics
on the pico- to nanosecond time scale, associated with cluster or
precursor formation that ultimately results in emerging crystals.
We investigated the model system of the protein β-lactoglobulin
in D2O in the presence of ZnCl2, which induces
crystallization by electrostatic bridges. First, the structural changes
occurring during crystallization were followed by small-angle neutron
scattering. Furthermore, we employed neutron backscattering and spin–echo
spectroscopy to measure the ensemble-averaged self- and collective
diffusion on nanosecond time scales of protein solutions with a kinetic
time resolution on the order of 15 min. The experiments provide information
on the increasing number fraction of immobilized proteins as well
as on the diffusive motion of unbound proteins in an increasingly
depleted phase. Simultaneously, information on the internal dynamics
of the proteins is obtained.
The crowded environment of biological systems such as
the interior
of living cells is occupied by macromolecules with a broad size distribution.
This situation of polydispersity might influence the dependence of
the diffusive dynamics of a given tracer macromolecule in a monodisperse
solution on its hydrodynamic size and on the volume fraction. The
resulting size dependence of diffusive transport crucially influences
the function of a living cell. Here, we investigate a simplified model
system consisting of two constituents in aqueous solution, namely,
of the proteins bovine serum albumin (BSA) and bovine polyclonal gamma-globulin
(Ig), systematically depending on the total volume fraction and ratio
of these constituents. From high-resolution quasi-elastic neutron
spectroscopy, the separate apparent short-time diffusion coefficients
for BSA and Ig in the mixture are extracted, which show substantial
deviations from the diffusion coefficients measured in monodisperse
solutions at the same total volume fraction. These deviations can
be modeled quantitatively using results from the short-time rotational
and translational diffusion in a two-component hard sphere system
with two distinct, effective hydrodynamic radii. Thus, we find that
a simple colloid picture well describes short-time diffusion in binary
mixtures as a function of the mixing ratio and the total volume fraction.
Notably, the self-diffusion of the smaller protein BSA in the mixture
is faster than the diffusion in a pure BSA solution, whereas the self-diffusion
of Ig in the mixture is slower than in the pure Ig solution.
each other when compared to the intrinsic propensities of a mostly unperturbed arginine in the tripeptide GRG. A conformational analysis based on experimentally determined J-coupling constants from heteronuclear NMR spectroscopy and amide I' band profiles from polarized Raman spectroscopy reveals that nearest neighbor interactions stabilize extended b-strand conformations at the expense of polyproline II and turn conformations. The results from MD simulations with an CHARMM36m force field and TIP3P water reproduce our results only to a limited extent. The use of the Ramachandran distribution of the central residue of GRRRG in a calculation of end-to-end distances of polyarginines of different length yielded the expected power law behavior. The scaling coefficient of 0.66 suggests that such peptides would be more extended than predicted by a self-avoiding random walk.
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