Imaging via Correlation of X-Ray Fluorescence Photons.

We demonstrate that x-ray fluorescence emission, which cannot maintain a stationary interference pattern, can be used to obtain images of structures by recording photon-photon correlations in the manner of the stellar intensity interferometry of Hanbury Brown and Twiss. This is achieved utilizing femtosecond-duration pulses of a hard x-ray free-electron laser to generate the emission in exposures comparable to the coherence time of the fluorescence. Iterative phasing of the photon correlation map generated a model-free real-space image of the structure of the emitters. Since fluorescence can dominate coherent scattering, this may enable imaging uncrystallised macromolecules.

Ultrafast dynamics and scattering of protic ionic liquids induced by XFEL pulses.

X-rays are routinely used for structural studies through scattering, and femtosecond X-ray lasers can probe ultrafast dynamics. We aim to capture the femtosecond dynamics of liquid samples using simulations and deconstruct the interplay of ionization and atomic motion within the X-ray laser pulse. This deconstruction is resolution dependent, as ionization influences the low momentum transfers through changes in scattering form factors, while atomic motion has a greater effect at high momentum transfers through loss of coherence. Our methodology uses a combination of classical molecular dynamics and plasma simulation on a protic ionic liquid to quantify the contributions to the scattering signal and how these evolve with time during the X-ray laser pulse. Our method is relevant for studies of organic liquids, biomolecules in solution or any low-Z materials at liquid densities that quickly turn into a plasma while probed with X-rays.

Molecular dynamics investigation of a redox switch in the anti-HIV protein SAMHD1.

HIV-1 is restricted in macrophages and certain quiescent myeloid cells due to a "Scorched Earth" dNTP starvation strategy attributed to the sterile alpha motif and HD domain protein-SAMHD1. Active SAMHD1 tetramers are assembled by GTP-Mg+2-dNTP cross bridges and cleave the triphosphate groups of dNTPs at a K m of ~10 µM, which is consistent with dNTP concentrations in cycling cells, but far higher than the equivalent concentration in quiescent cells. Given the substantial disparity between the dNTP concentrations required to activate SAMHD1 tetramers (~10 µM) and the dNTP concentrations in noncycling cells (~10 nM), the possibility of alternate enzymatically active forms of SAMHD1, including monomers remains open. In particular, the possibility of redox regulation of such monomers is also an open question. There have been experimental studies on the regulation of SAMHD1 by Glutathione driven redox reactions recently. Therefore, in this work, we have performed all-atom molecular dynamics simulations to study the dynamics of monomeric SAMHD1 constructs in the context of the three redox-susceptible Cysteine residues and compared them to monomers assembled within a tetramer. Our results indicate that assembly into a tetramer causes ordering of the catalytic core and increased solvent accessibility of the Catalytic Site. We have also found that glutathionylation of surface exposed C522 causes long range allosteric disruptions extending into the protein core. Finally, we see evidence suggesting a transient interaction between C522 and C341. Such a disulfide linkage has been hypothesized by experimental models, but has never been observed in crystal structures before.

Allosteric Signal Transduction in HIV-1 Restriction Factor SAMHD1 Proceeds via Reciprocal Handshake across Monomers.

The sterile alpha motif and histidine-aspartate domain-containing protein 1 (or SAMHD1), a human dNTP-triphosphohydrolase, contributes to HIV-1 restriction in select terminally differentiated cells of the immune system. The catalytically active form of the protein is an allosterically triggered tetramer, whose HIV-1 restriction properties are attributed to its dNTP-triphosphohydrolase activity. The tetramer itself is assembled by a GTP/dNTP combination. This enzyme uses the strategy of deoxynucleotide starvation, which is thought to prevent effective reverse transcription of the retroviral genome-hence, restricting HIV-1 propagation. HIV-2 and SIV have evolved defenses against SAMHD1, underscoring its role in restriction. Previous studies have provided high-resolution structures of GTP/dNTP-bound enzyme complexes but have not been able to provide information on dynamics. In this study, we have used correlation network analysis along with MD techniques to study the flow of allosteric information across the active complex. We have found evidence of a reciprocal allosteric "handshake" occurring across monomeric units. We have also uncovered a short linker region as the nexus for funnelling the regulatory signal from phosphorylation at T592 from the surface to the interior core of the protein.

Uncovering allostery and regulation in SAMHD1 through molecular dynamics simulations.

The human sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a retroviral restriction factor in myeloid cells and non-cycling CD4+ T cells, a feature imputed to its phosphohydrolase activity-the enzyme depletes the cellular dNTP levels inhibiting reverse transcription. The functionally active form of SAMHD1 is an allosterically triggered tetramer which utilizes GTP-Mg+2 -dNTP cross bridges to link and stabilize adjacent monomers. However, very little is known about how it assembles into a tetramer and how long the tetramer stays intact. In this computational study, we provide a molecular dynamics based analysis of the structural stability and allosteric site dynamics in SAMHD1. We have investigated the allosteric links which assemble and hold the tetramer together. We have also extended this analysis to a regulatory mutant of SAMHD1. Experimental studies have indicated that phosphorylation of T592 downregulates HIV-1 restriction. A similar result is also achieved by a phosphomimetic mutation T592E. While a mechanistic understanding of the process is still elusive, the loss of structural integrity of the enzyme is conjectured to be the cause of the impaired dNTPase activity of the T592E mutant. MD simulations show that the T592E mutation causes slightly elevated local motions which remain confined to the short helix (residues 591-595), which contains the phosphorylation site and do not cause long-range destabilization of the SAMHD1 tetramer within the timeframe of the simulations. Thus, the regulatory mechanism of SAMHD1 is a more subtle mechanism than has been previously suspected. Proteins 2017; 85:1266-1275. © 2017 Wiley Periodicals, Inc.