From the pressure induced frequency shift of photochemical holes burnt into mesomorphyrin substituted horseradish peroxidase, we determined the compressibility of the protein and the vacuum frequency of the chromophore. From the compressibility, an estimation of the volume fluctuations of the biomolecule is possible.
We applied hydrostatic pressure to spectral holes burned into a resorufin doped ethanol/methanol glass. We found that the line shift is perfectly linear with pressure and showed a pronounced dependence on the burn frequency as predicted by theory [J. Chem. Phys. 90, 3274 (1989)]. We exploited the burn frequency dependence to determine the solvent shift of the dye probe and the compressibility of the alcohol glass used. On the other hand, the behavior of the hole width under pressure shows features not predicted by theory: The broadening is, like the line shift, dependent on the burn frequency within the inhomogeneous band, yet in a nonlinear fashion. We attribute the color effect in the pressure induced broadening of the hole to a breakdown of the Gaussian approximation.
We investigated spectral holes burnt at 1.5 K into the origins of several tautomeric forms of mesoporphyrin IX-substituted horseradish peroxidase at pH 8 under pressures up to 2 MPa. From the pressure-induced lineshift the compressibility of the apoprotein could be determined. We found that the compressibility changed significantly when measured at different tautomer origins. It was concluded that there must be a correlation between the tautomer configurations of the chromophore and the actual structures of the apoprotein. As a consequence, specific conformational substates of the protein can be selected by optical selection of the associated tautomers.The solid-state physics of proteins is an intriguing field. Unlike crystals, proteins are finite systems, yet they have a smooth density of vibrational states which is Debye-like at sufficiently small energies (1). Like crystals, proteins are highly ordered (2). Yet disorder plays a very important role, too (3, 4). Disorder manifests itself in inhomogeneously broadened spectral lines, in nonexponential kinetics, in nonArrhenius-type activated processes, in a glass-like specific heat, in dielectric damping, etc. (5-8). Even from x-ray scattering experiments, it became obvious that, at a sufficiently high level of resolution, the structure of a protein is not so well defined (2, 3). There seems to be agreement that a certain extent of structural disorder is a prerequisite for the proper functioning of a protein.A possible model for describing the relation between order and disorder in proteins is based on the concept of conformational substates (4,7,9,10): The basic idea of this model is that a protein can exist in a huge set of substates. Most of these substates are assumed to be in fast equilibrium because their separating barriers are sufficiently small compared with kT. Some ofthem, however, are nonequilibrium states. These special substates may be functionally important. It is structural disorder which gives enough freedom to the protein to reorganize its structure for specific requirements.Here we address the problem of how this reorganization takes place and how this is related to the prosthetic group. Can a slight structural change of the prosthetic group, as induced by a weak chemical interaction, be a signal for the protein structure to be rearranged, for example, to help the binding of special molecules? That is, we address the problem of correlation between the configurations of the prosthetic group and the conformational substates of the protein.The experimental technique we employ is persistent spectral hole burning (11)(12)(13)(14)(15). Hole burning is a special type of saturation spectroscopy. Its specific feature, as compared with similar techniques in NMR and ESR spectroscopy, is the persistence of the hole. This persistence is the reason why the technique can be used to study the ground state by optical means. At liquid-helium temperatures, the width of a spectral hole is close to the natural width of the optical transition involved. For m...
For the first time, conformational relaxation processes have been measured in a small protein, mesoporphyrin-horseradish peroxidase via their influence on spectral diffusion broadening of holes burnt in the fluorescence excitation spectrum of free base mesoporphyrin. Holes were burnt in three 0----0 bands of different tautomeric forms of the chromophore at 1.5 and 4 K, and the spectral diffusion broadening was measured in temperature cycling experiments between 4 and 30 K. The inhomogeneous linewidth for the tautomeric 0----0 bands was estimated to be 60-70 cm-1; the hole width was found narrow, being in the order of 350 MHz (10(-2) cm-1) at 1.5 K what allowed for an extremely sensitive detection of the conformational changes. Though proteins have many features in common with glasses, the spectral diffusion broadening of photochemical holes under temperature cycling conditions in mesoporphyrin horseradish peroxidase has a very different pattern as a function of temperature. Up to 12 K, the linewidth did not significantly change, then around 14 K; a steplike broadening was observed for all three tautomers, although to a different extent. The total magnitude of line broadening up to 30 K was large and also different for the tautomers. We argue that the difference between the behavior of this protein and that of glassy matrices originate from finite size effects; the protein may be characterized by a small number of TLS, and their distribution may bear discrete features.
We use pressure tuning of spectral holes to estimate the compressibility of protein molecules by optical means. We found that the compressibility of mesoporphyrin-substituted horseradish peroxidase increases by a factor of three when it incorporates small aromatic H-donor molecules that bind in the vicinity of its heme pocket. Such a dramatic softening of its packing density corresponds to a jump from a compressibility range characteristic for the solid state into that characteristic for liquids.
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