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Protein dynamics or protein motions are believed to ultimately govern the biological function and activities of protein. Quasielastic neutron scattering (QENS) technique has been proven to be an exceptional tool to study dynamics of proteins in the time scale of picosecond (ps) to nanosecond (ns) [1, 2]. In this study, we use QENS to investigate how a large oligomeric protein, Inorganic Pyrophosphatase (IPPase) from Thermococcus thioreducens with quaternary structural complexity, have distinguishable dynamic characteristics compared to those of the small simple monomeric model protein, lysozyme. IPPase derived from thermostable microorganisms is of extreme interest for biophysical studies because of their inherent chemical and thermal stability and high temperature activity. Two QENS instruments, a backscattering spectrometer (BASIS) and a disk chopper spectrometer (DCS) are used in probing the protein dynamics in different time ranges from 1 ps to 1 ns at different temperatures. In addition, the DCS experiment was performed under the pressure of 1000 bar, mimicking the natural living conditions of IPPase. Our results reveal that the dynamics of IPPase is slower than that of lysozyme in the time range of 10 ps to 0.5 ns [1] while it is faster in the time range of 1 ps to 30 ps. These results are consistent between two instruments and such dynamic behaviors in proteins are believed to be contributed by the rotational motion of the side methyl groups [3]. Distinguishable dynamical behavior found between two proteins reveals local flexibility and conformational substates unique to oligomeric structures. Our results greatly help understanding the relation between protein dynamics and their biological functions.[1] X.-Q. Chu, et al, JPCB. 116, 9917 (2012).[2] X.-Q. Chu, et al, Soft Matter 6, 2623 (2010).[3] X.-Q. Chu, et al, JPCL. 4, 936 (2013).

G-protein-coupled receptors (GPCRs) represent the largest family of proteins in the human genome and comprise about 50% of current molecular drug targets. Rhodopsin is the GPCR involved in visual light perception and occurs naturally in a membrane lipid environment. Rhodopsin photoactivation yields cis-trans isomerization of retinal giving an equilibrium between inactive Meta-I and active Meta-II states. Here we address the question: does photoactivation lead to a single Meta-II conformation or is there an ensemble of substates described by an ensemble-activation mechanism (EAM) [1]? In this context small-angle neutron scattering (SANS) probes rhodopsin-detergent and rhodopsin-lipid complexes through measurement of the intensity of the neutrons scattered as a function of scattering vector I(q). Contrast variation enables us to highlight individual components of a multi-component system without isotopic labeling of the sample. Upon photoactivation, the Meta-I state was stabilized in CHAPS-solubilized rhodopsin, while Meta-II was trapped in DDM-solubilized rhodopsin. The ligand-free apoprotein opsin was obtained by photobleaching rhodopsin in the presence of hydroxylamine. The SANS spectra for the above rhodopsin substates were acquired from 80% D2O solutions and at contrast-matching points for both DDM and CHAPS samples. The data collected in 80% D2O samples provide structural information for both protein and detergent, while the data collected at contrast-matching points give information for the protein structure exclusively. Our experiments demonstrate that for detergent-solubilized rhodopsin, SANS with contrast variation can detect structural differences between the rhodopsin dark-state, Meta-I, Meta-II, and ligand-free opsin states. Dark-state rhodopsin is more conformationally flexible (less-compact) in DDM micelles compared to the CHAPS, which is consistent with an ensemble of activated Meta-II states. Furthermore, the time-dependent structural transitions between Meta-I and Meta-II as observed by time-resolved SANS will be crucial to understanding the ensemble-based activation. [1]A.V. Struts et al. (2011) NSMB18, 392-394.

Life is traditionally seen as being driven by energy from the sun, however deep sea organisms have no access to sunlight, so they depend on nutrients found in the dusty chemical deposits and hydrothermal fluids around the hydrothermal vent zones, where they live. In this study, we use neutron spin-echo spectroscopy (NSE) to measure the inter-domain motions of the inorganic pyrophosphatase (IPPase) enzyme derived from thermostable microorganisms. IPPase is of extreme interest for biophysical studies because of their inherent chemical and thermal stability and high temperature activity. It has a hexameric quaternary structure with a molecular mass of approximately 120kDa (each subunit is about 20kDa molecular weight), which is a large oligomeric molecular structure. Study of the slow inter-domain motions that occur in the protein is the key to understand why IPPase can perform catalytic activity at much higher temperature than normal enzymes, thus can adapt to the extreme environment present at the seabed [1-3]. NSE spectroscopy is able to probe these slow inter-domain motions directly in the time-domain, as has already been established in other studies[4,5]. The length and timescale of NSE are right in the ranges from sub-Angstrom and picoseconds to nanometers and several tens of nanoseconds and beyond. Distinguishable dynamical behavior found between two proteins reveals local flexibility and conformational substates unique to oligomeric structures. Our results greatly help understanding the relation between protein dynamics and their biological functions.[1] X.-Q. Chu, et. al, JPCB 116, 9917 (2012).[2] X.-Q. Chu, et. al, Soft Matter 6, 2623(2010).[3] X.-Q. Chu, et. al, JPCL 4, 936 (2013).[4] R. Biehl, et. al, Soft Matter 7, 1299 (2011).[5] N. Smolin, et. al, Biophys. J. 102, 1108 (2012).