We demonstrate tracking of protein structural changes with time-resolved wide-angle X-ray scattering (TR-WAXS) with nanosecond time resolution. We investigated the tertiary and quaternary conformational changes of human hemoglobin under nearly physiological conditions triggered by laser-induced ligand photolysis. We also report data on optically induced tertiary relaxations of myoglobin and refolding of cytochrome c to illustrate the wide applicability of the technique. By providing insights into the structural dynamics of proteins functioning in their natural environment, TR-WAXS complements and extends results obtained with time-resolved optical spectroscopy and X-ray crystallography.

The time-resolved diffraction signal from a laser-excited solution has three principal components: the solute-only term, the solute-solvent cross term, and the solvent-only term. The last term is very sensitive to the thermodynamic state of the bulk solvent, which may change during a chemical reaction due to energy transfer from light-absorbing solute molecules to the surrounding solvent molecules and the following relaxation to equilibrium with the environment around the scattering volume. The volume expansion coefficient alpha for a liquid is typically approximately 1 x 10(-3) K(-1), which is about 1000 times greater than for a solid. Hence solvent scattering is a very sensitive on-line thermometer. The decomposition of the scattered x-ray signal has so far been aided by molecular dynamics (MD) simulations, a method capable of simulating the solvent response as well as the solute term and solute/solvent cross terms for the data analysis. Here we present an experimental procedure, applicable to most hydrogen containing solvents, that directly measures the solvent response to a transient temperature rise. The overtone modes of OH stretching and CH3 asymmetric stretching in liquid methanol were excited by near-infrared femtosecond laser pulses at 1.5 and 1.7 microm and the ensuing hydrodynamics, induced by the transfer of heat from a subset of excited CH3OH* to the bulk and the subsequent thermal expansion, were probed by 100 ps x-ray pulses from a synchrotron. The time-resolved data allowed us to extract two key differentials: the change in the solvent diffraction from a temperature change at constant density, seen at a very short time delay approximately 100 ps, and a term from a change in density at constant temperature. The latter term becomes relevant at later times approximately 1 mus when the bulk of liquid expands to accommodate its new temperature at ambient pressure. These two terms are the principal building blocks in the hydrodynamic equation of state, and they are needed in a self-consistent reconstruction of the solvent response during a chemical reaction. We compare the experimental solvent terms with those from MD simulations. The use of experimentally determined solvent differentials greatly improved the quality of global fits when applied to the time-resolved data for C2H4I2 dissolved in methanol.

Light absorption can trigger biologically relevant protein conformational changes. The light-induced structural rearrangement at the level of a photoexcited chromophore is known to occur in the femtosecond timescale and is expected to propagate through the protein as a quake-like intramolecular motion. Here we report direct experimental evidence of such ‘proteinquake’ observed in myoglobin through femtosecond X-ray solution scattering measurements performed at the Linac Coherent Light Source X-ray free-electron laser. An ultrafast increase of myoglobin radius of gyration occurs within 1 picosecond and is followed by a delayed protein expansion. As the system approaches equilibrium it undergoes damped oscillations with a ~3.6-picosecond time period. Our results unambiguously show how initially localized chemical changes can propagate at the level of the global protein conformation in the picosecond timescale.

The dynamic properties of nanoparticles suspended in a supercooled glass forming liquid are studied by x-ray photon correlation spectroscopy. While at high temperatures the particles undergo Brownian motion the measurements closer to the glass transition indicate hyperdiffusive behavior. In this state the dynamics is independent of the local structural arrangement of nanoparticles, suggesting a cooperative behavior governed by the near-vitreous solvent.

Elastic incoherent neutron scattering has been used to study the temperature dependence of the mean-square displacements of nonexchangeable hydrogen atoms in powders of a series of homomeric polypeptides (polyglycine, polyalanine, polyphenylalanine and polyisoleucine) in comparison with myoglobin at the same hydration level (h = 0.2). The aim of the work was to measure the dynamic behavior of different amino acid residues separately and assess the contribution of each type of side chain to the anharmonic dynamics of proteins. The results provide direct experimental evidence that the first anharmonic activation, at approximately 150 K, is largely due to methyl group rotations entering the time window of the spectrometer used; however, contributions on the order of 10-20% from the motions of other groups (e.g., the phenolic ring and the methylene groups) are present. Our data also indicate that the dynamical transition occurring at approximately 230 K can be attributed, at least at the hydration level investigated, mainly to motions involving backbone fluctuations.

In this work we study the temperature dependence of the Soret band lineshape of the carbonmonoxy derivatives of sperm whale myoglobin, human hemoglobin, and its isolated alpha and beta subunits. To fit the observed spectral profile we use an analytic expression derived for a system whereby a single electronic transition is coupled to Franck-Condon active vibrational modes, within the adiabatic and harmonic approximation. The vibronic structure of the spectra arises from the coupling with high frequency modes; these modes contribute to the total line shape through a series of Lorentzians with peak positions at vibrational overtones and half width related to the time constant of the population decay of the excited electronic state (homogeneous broadening); moreover, the coupling with low frequency modes broadens each Lorentzian to a Voigtian. Inhomogeneous broadening is modeled as a gaussian distribution of the 0-0 transition frequencies and is therefore added as a constant term to the previous gaussian width. This spectral deconvolution enables us to investigate the different contributions to line broadening and the parameters that characterize the vibrational coupling, as well as their dependence upon protein and solvent composition. The investigation is carried out as a function of temperature in the range 20-300 K; relevant information is obtained by comparing experimental results with theoretical predictions. This work supports a description of the investigated proteins as heterogeneous systems, whose heterogeneity depends on the particular protein and on the composition of the external matrix. The delocalized pi electron cloud of the porphyrin ring is coupled not only to the high frequency vibrational modes of the active site but also to a "bath" of lower frequency modes that involve the entire protein; moreover at suitable temperatures (approximately 200 K), anharmonic motions, which are an obvious prerequisite for the jumping among different conformational substates, become evident.

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Neutron scattering reveals a complex dynamics in polypeptide chains, with two main onsets of anharmonicity whose physical origin and biological role are still debated. In this study the dynamics of strategically selected homomeric polypeptides is investigated with elastic neutron scattering using different energy resolutions and compared with that of a real protein. Our data spotlight the dependence of anharmonic transition temperatures and fluctuation amplitudes on energy resolution, which we quantitatively explain in terms of a two-site model for the protein-hydration water energy landscape. Experimental data strongly suggest that the protein dynamical transition is not a mere resolution effect but is due to a real physical effect. Activation barriers and free energy values obtained for the protein dynamical transition allow us to make a connection with the two-well interaction potential of supercooledconfined water proposed to explain a low-density ! high-density liquid-liquid transition. DOI: 10.1103/PhysRevLett.109.128102 PACS numbers: 87.14.ef, 83.85.Hf, 87.15.HÀ Neutron scattering is a powerful technique to study protein dynamics and energetics. The structure factor SðE ¼ 0; Q; TÞ of elastic incoherent neutron scattering (EINS) is related to the time-position self correlation function of protein-solvent nuclei that, in turn, is related to the energy landscape of the system whose different tiers can be explored by the temperature dependence of the EINS signal. In particular, EINS on D 2 O-hydrated protein powders, which probes the mean square displacements (MSDs) of protein nonexchangeable H atoms, reveals two deviations from harmonic dynamics, at $100-150 K and at $220 K [1,2]. Molecular origin, physical nature and biological relevance of these ''transitions'' are still matter of discussion. The first one is attributed mainly to thermally activated motions of CH 3 methyl groups [1,[3][4][5][6] (for this reason hereon called methyl groups activation, MGA). The second one, called ''protein dynamical transition'' (PDT), has been first interpreted as a glasslike transition [2] directly correlated to the onset of biological activity [7], but this view has been later challenged. Several interpretations of the PDT have been proposed: (i) a change in the protein structural flexibility in response to the glass transition of hydration water [8]; (ii) a result of the protein structural relaxation reaching the limit of the experimental frequency window [9][10][11]; (iii) the protein response to a fragile-to-strong dynamic crossover in the hydration water at 220 K where water structure makes a low density liquid ðLDLÞ ! high density liquid (HDL) transition [12]; (iv) a change in the thermodynamic resilience of the waterprotein system [13]. These models propose physical pictures partially alternative, demonstrating that the question has not been definitively settled yet: (i) and (ii) ascribe the PDT to a temperature dependent relaxation time crossing the instrumental time-scale; (iii) and (iv) interpret the PDT as ...