Solvent dependence of dynamic transitions in protein solutions

A transition as a function of increasing temperature from harmonic to anharmonic dynamics has been observed in globular proteins by using spectroscopic, scattering, and computer simulation techniques. We present here results of a dynamic neutron scattering analysis of the solvent dependence of the picosecond-time scale dynamic transition behavior of solutions of a simple single-subunit enzyme, xylanase. The protein is examined in powder form, in D2O, and in four two-component perdeuterated single-phase cryosolvents in which it is active and stable. The scattering profiles of the mixed solvent systems in the absence of protein are also determined. The general features of the dynamic transition behavior of the protein solutions follow those of the solvents. The dynamic transition in all of the mixed cryosolvent–protein systems is much more gradual than in pure D2O, consistent with a distribution of energy barriers. The differences between the dynamic behaviors of the various cryosolvent protein solutions themselves are remarkably small. The results are consistent with a picture in which the picosecond-time scale atomic dynamics respond strongly to melting of pure water solvent but are relatively invariant in cryosolvents of differing compositions and melting points.

Correct protein folding in glycerol

Water is the natural medium for protein folding, which is also used in all in vitro studies. In the present work, we posed, and answered affirmatively, a question of whether it is possible to fold correctly a typical protein in a nonaqueous solvent. To this end, unfolded and reduced hen egg-white lysozyme was refolded and reoxidized in glycerol containing varying amounts of water. The unfolded/reduced enzyme was found to regain spontaneously substantial catalytic activity even in the nearly anhydrous solvent; for example, the refolding yield in 99% glycerol was still some one-third of that in pure water, and one-half of that was regained even in 99.8% glycerol. The less than full recovery of the enzymatic activity in glycerol is, as in water, because of competing protein aggregation during the refolding. Lysozyme reoxidation in glycerol was successfully mediated by two dissimilar oxidizing systems, and the refolding yield was markedly affected by the pH of the last aqueous solution before the transfer into glycerol. No recovery of the lysozyme activity was observed when the refolding/reoxidation reaction was carried out in the denaturing solvent dimethyl sulfoxide. This study paves the way for a systematic investigation of the solvent effect on protein folding and demonstrates that water is not a unique milieu for this process.

Noninvasive functional optical spectroscopy of human breast tissue

Near infrared diffuse optical spectroscopy and diffuse optical imaging are promising methods that eventually may enhance or replace existing technologies for breast cancer screening and diagnosis. These techniques are based on highly sensitive, quantitative measurements of optical and functional contrast between healthy and diseased tissue. In this study, we examine whether changes in breast physiology caused by exogenous hormones, aging, and fluctuations during the menstrual cycle result in significant alterations in breast tissue optical contrast. A noninvasive quantitative diffuse optical spectroscopy technique, frequency-domain photon migration, was used. Measurements were performed on 14 volunteer subjects by using a hand-held probe. Intrinsic tissue absorption and reduced scattering parameters were calculated from frequency-domain photon migration data. Wavelength-dependent absorption (at 674, 803, 849, and 956 nm) was used to determine tissue concentration of oxyhemoglobin, deoxyhemoglobin, total hemoglobin, tissue hemoglobin oxygen saturation, and bulk water content. Results show significant and dramatic differences in optical properties between menopausal states. Average premenopausal intrinsic tissue absorption and reduced scattering values at each wavelength are 2.5- to 3-fold higher and 16–28% greater, respectively, than absorption and scattering for postmenopausal subjects. Absorption and scattering properties for women using hormone replacement therapy are intermediate between premenopausal and postmenopausal populations. Physiological properties show differences in mean total hemoglobin (7.0 μM, 11.8 μM, and 19.2 μM) and water concentration relative to pure water (10.9%, 15.3%, and 27.3%) for postmenopausal, hormone replacement therapy, and premenopausal subjects, respectively. Because of their unique, quantitative information content, diffuse optical methods may play an important role in breast diagnostics and improving our understanding of breast disease.

Direct evidence for modified solvent structure within the hydration shell of a hydrophobic amino acid.

Neutron scattering experiments are used to determine scattering profiles for aqueous solutions of hydrophobic and hydrophilic amino acid analogs. Solutions of hydrophobic solutes show a shift in the main diffraction peak to smaller angle as compared with pure water, whereas solutions of hydrophilic solutes do not. The same difference for solutions of hydrophobic and hydrophilic side chains is also predicted by molecular dynamics simulations. The neutron scattering curves of aqueous solutions of hydrophobic amino acids at room temperature are qualitatively similar to differences between the liquid molecular structure functions measured for ambient and supercooled water. The nonpolar solute-induced expansion of water structure reported here is also complementary to recent neutron experiments where compression of aqueous solvent structure has been observed at high salt concentration.