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protein mechanics from neutron diffraction

from the atomic-age-MNT??? dept.
Neutron scattering has been used to quantify thermal positional disorder in proteins. In myoglobin, there is a transition at ~200K from a "harmonic" regime where all the atoms are trapped in single potential wells to an anharmonic regime where jumps between wells become important. The anharmonic regime is important for the biological function of myoglobin (O2 binding), but may cause problems for use of proteins as mechanical elements in nanotechnology. Giuseppe Zaccai wrote a short review paper in [Science 288:1604-1607 2Jun2000] describing the use of neutron scattering to probe protein dynamics. The thermal fluctuations in positions of the atoms in a sample can alter the directions and energies of scattered neutrons. Their mean square scatter from their equilibrium positions "are calculated from the angular dependence of the scattered elastic incoherent intensity." A major advantage of incoherent scattering is that "samples need not be either crystalline or [even] monodisperse". Incoherent neutron scattering is also insensitive to static disorder in samples, which in coherent x-ray diffraction becomes combined with the thermal movement. Neutron scattering is also complementary to x-ray diffraction because the former is very sensitive to hydrogen atoms, which are nearly invisible to x-rays. On the other hand, since neutron scattering cross-sections are considerably smaller than those for x-rays, "~200 mg of material [is] required for a neutron-scattering experiment."

These studies, amongst other techniques, have found "dynamical transitions from harmonic to anharmonic regimes … at ~200K (-73 oC) in various proteins" In the harmonic regime the protein acts like it has a consistent potential well. In experiments on hydrated myoglobin powder, for instance, the scatter is consistent with a 2 N/m spring constant. The mean square scatter in position increases linearly with temperature in this regime.

In the anharmonic regime the atoms start to jump between potential wells, so the scatter starts to increase faster than in the harmonic regime. The effective spring constant drops quite dramatically, to 0.3 N/m in myoglobin. There is considerable evidence that "conformational flexibility is essential for enzyme catalysis and for biological molecular activity in general." In the case of myoglobin, for instance, the static x-ray diffraction structure provides no path for an O2 molecule to diffuse into the molecule and reach the heme binding site. Only fluctuations in the structure permits its biological function of binding O2. Note that these fluctuations start to appear at a temperature where myoglobin is far from denaturing. Evidently typical native, well-folded proteins have 3 well separated states as temperature increases:

  1. frozen into a single potential well
  2. potential well jumping at physiological temperatures
  3. denatured

While inter-well jumping is necessary for biological function, it implies that even a well-folded protein is not a eutactic structure from the viewpoint of molecular nanotechnology. The covalent bonds aren't breaking, but it seems likely that hydrogen bonds which help hold together the 3D structure do break transiently. In other words, the drop in effective stiffness that happens on entering the anharmonic regime makes proteins significantly worse at maintaining positional control in the presence of thermal noise. Ideally, we would prefer structures that are as stiff as possible, except along one or two carefully chosen mechanical degrees of freedom.

Fortunately, this paper also describes experiments with trehalose, a disaccharide that some organisms use to survive dehydration. It "appears to help proteins avoid denaturation by surrounding them with a continuous vitreous layer." When trehalose was added to myoglobin, the protein was held in the harmonic regime to at least 310K. The trehalose also raised the effective spring constant of the myoglobin to 3 N/m. Since trehalose prevents denaturation, presumably the secondary and tertiary structure of myoglobin is left intact. That suggests that the hydrophobic core of the protein is not directly held in place, but rather that edge effects from the trehalose are sufficient to stabilize it.

Overall, the impact on nanotechnology appears to be that designs incorporating protein components must either operate at moderately low temperatures, glaze their proteins with a high-melting hydrophilic shell, or tolerate transient scission of hydrogen bonds in their protein components.

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