Structural and functional investigation of flavin binding center of the NqrC subunit of sodium-translocating NADH:Quinone oxidoreductase from Vibrio harveyi

Na+-translocating NADH:quinone oxidoreductase (NQR) is a redox-driven sodium pump operating in the respiratory chain of various bacteria, including pathogenic species. The enzyme has a unique set of redox active prosthetic groups, which includes two covalently bound flavin mononucleotide (FMN) residues attached to threonine residues in subunits NqrB and NqrC. The reason of FMN covalent bonding in the subunits has not been established yet. In the current work, binding of free FMN to the apo-form of NqrC from Vibrio harveyi was studied showing very low affinity of NqrC to FMN in the absence of its covalent bonding. To study structural aspects of flavin binding in NqrC, its holo-form was crystallized and its 3D structure was solved at 1.56 Å resolution. It was found that the isoalloxazine moiety of the FMN residue is buried in a hydrophobic cavity and that its pyrimidine ring is squeezed between hydrophobic amino acid residues while its benzene ring is extended from the protein surroundings. This structure of the flavin-binding pocket appears to provide flexibility of the benzene ring, which can help the FMN residue to take the bended conformation and thus to stabilize the one-electron reduced form of the prosthetic group. These properties may also lead to relatively weak noncovalent binding of the flavin. This fact along with periplasmic location of the FMN-binding domains in the vast majority of NqrC-like proteins may explain the necessity of the covalent bonding of this prosthetic group to prevent its loss to the external medium

Ultrasound cavitation and exfoliation dynamics of 2D materials re-vealed in operando by X-ray free electron laser megahertz imaging

Ultrasonic liquid phase exfoliation is a promising method for the production of two-dimensional (2D) layered materials. A large number of studies have been made in investigating the underlying ultrasound exfoliation mechanisms. However, due to the experimental challenges for capturing the highly transient and dynamic phenomena in real-time at sub-microsecond time and micrometer length scales simultaneously, most theories reported to date still remain elusive. Here, using the ultra-short X-ray Free Electron Laser pulses (~25ps) with a unique pulse train structure, we applied MHz X-ray Microscopy and machine-learning technique to reveal unambiguously the full cycles of the ultrasound cavitation and graphite layer exfoliation dynamics with sub-microsecond and micrometer resolution. Cyclic fatigue shock wave impacts produced by ultrasound cloud implosion were identified as the dominant mechanism to deflect and exfoliate graphite layers mechanically. For the graphite flakes, exfoliation rate as high as ~5 angstroms per shock wave impact was observed. For the HOPG graphite, the highest exfoliation rate was ~0.15 angstroms per impact. These new findings are scientifically and technologically important for developing industrial upscaling strategies for ultrasonic exfoliation of 2D materials

Active State of Sensory Rhodopsin II: Structural Determinants for Signal Transfer and Proton Pumping

International audienceThe molecular mechanism of transmembrane signal transduction is still a pertinent question in cellular biology. Generally, a receptor can transfer an external signal via its cytoplasmic surface as found for GPCRs like rhodopsin or via the membrane domain like it is utilized by sensory rhodopsin II (SRII) in complex with its transducer HtrII. In the absence of HtrII SRII functions as a proton pump. Here, we report on the crystal structure of the active state of SRII from Natronomonas pharaonis (NpSRII). The problem of a dramatic loss of the diffraction quality upon loading of the active state was overcome by growing better crystals and reducing the occupancy of the state. The determined conformational changes at the region comprising helices F and G are similar to those observed for the NpSRII-transducer complex but they are much more pronounced. Meaning of these differences for proton pumping ability and understanding of the signal transduction by NpSRII is discussed

An Approach to Heterologous Expression of Membrane Proteins. The Case of Bacteriorhodopsin.

International audienceHeterologous overexpression of functional membrane proteins is a major bottleneck of structural biology. Bacteriorhodopsin from Halobium salinarum (bR) is a striking example of the difficulties in membrane protein overexpression. We suggest a general approach with a finite number of steps which allows one to localize the underlying problem of poor expression of a membrane protein using bR as an example. Our approach is based on constructing chimeric proteins comprising parts of a protein of interest and complementary parts of a homologous protein demonstrating advantageous expression. This complementary protein approach allowed us to increase bR expression by two orders of magnitude through the introduction of two silent mutations into bR coding DNA. For the first time the high quality crystals of bR expressed in E. Coli were obtained using the produced protein. The crystals obtained with in meso nanovolume crystallization diffracted to 1.67 Å

Structure of the C. Elegans ZYG-1 Cryptic Polo Box Suggests a Conserved Mechanism for Centriolar Docking of Plk4 Kinases

Plk4 family kinases control centriole assembly. Plk4s target mother centrioles through an interaction between their cryptic polo box (CPB) and acidic regions in the centriolar receptors SPD-2/Cep192 and/or Asterless/Cep152. Here, we report a crystal structure for the CPB of C. elegans ZYG-1, which forms a Z-shaped dimer containing an intermolecular β sheet with an extended basic surface patch. Biochemical and in vivo analysis revealed that electrostatic interactions dock the ZYG-1 CPB basic patch onto the SPD-2-derived acidic region to promote ZYG-1 targeting and new centriole assembly. Analysis of a different crystal form of the Drosophila Plk4 (DmPlk4) CPB suggests that it also forms a Z-shaped dimer. Comparison of the ZYG-1 and DmPlk4 CPBs revealed structural changes in the ZYG-1 CPB that confer selectivity for binding SPD-2 over Asterless-derived acidic regions. Overall, our findings suggest a conserved mechanism for centriolar docking of Plk4 homologs that initiate daughter centriole assembly

Nucleation and Growth of Membrane Protein Crystals In Meso —A Fluorescence Microscopy Study

Since the introduction of in meso crystallization of membrane proteins in lipidic cubic phase (LCP) by Landau and Rosenbusch in 1996, numerous studies attempted to elucidate the mechanism of in meso crystal nucleation and growth. Here we present a fluorescence microscopy study of the crystallization process of the light-driven proton pump bacteriorhodopsin. The crystallization starts with formation of microcrystals, followed by growth of a dominating crystal at the expense of smaller ones and formation of a depletion zone around it. These observations suggest an Ostwald ripening mechanism of the in meso crystal growth. Analysis of the microcrystal spatial distribution suggests that microcrystal nucleation occurs predominately at the LCP domain boundaries

Crystal structure of Escherichia coli-expressed Haloarcula marismortui bacteriorhodopsin I in the trimeric form.

International audienceBacteriorhodopsins are a large family of seven-helical transmembrane proteins that function as light-driven proton pumps. Here, we present the crystal structure of a new member of the family, Haloarcula marismortui bacteriorhodopsin I (HmBRI) D94N mutant, at the resolution of 2.5 Å. While the HmBRI retinal-binding pocket and proton donor site are similar to those of other archaeal proton pumps, its proton release region is extended and contains additional water molecules. The protein's fold is reinforced by three novel inter-helical hydrogen bonds, two of which result from double substitutions relative to Halobacterium salinarum bacteriorhodopsin and other similar proteins. Despite the expression in Escherichia coli and consequent absence of native lipids, the protein assembles as a trimer in crystals. The unique extended loop between the helices D and E of HmBRI makes contacts with the adjacent protomer and appears to stabilize the interface. Many lipidic hydrophobic tail groups are discernible in the membrane region, and their positions are similar to those of archaeal isoprenoid lipids in the crystals of other proton pumps, isolated from native or native-like sources. All these features might explain the HmBRI properties and establish the protein as a novel model for the microbial rhodopsin proton pumping studies

Low-dose X-ray radiation induces structural alterations in proteins.

International audienceX-ray-radiation-induced alterations to protein structures are still a severe problem in macromolecular crystallography. One way to avoid the influence of radiation damage is to reduce the X-ray dose absorbed by the crystal during data collection. However, here it is demonstrated using the example of the membrane protein bacteriorhodopsin (bR) that even a low dose of less than 0.06 MGy may induce structural alterations in proteins. This dose is about 500 times smaller than the experimental dose limit which should ideally not be exceeded per data set (i.e. 30 MGy) and 20 times smaller than previously detected specific radiation damage at the bR active site. To date, it is the lowest dose at which radiation modification of a protein structure has been described. Complementary use was made of high-resolution X-ray crystallography and online microspectrophotometry to quantitatively study low-dose X-ray-induced changes. It is shown that structural changes of the protein correlate with the spectroscopically observed formation of the so-called bR orange species. Evidence is provided for structural modifications taking place at the protein active site that should be taken into account in crystallographic studies which aim to elucidate the molecular mechanisms of bR function

Highly Sensitive Coherent Anti-Stokes Raman Scattering Imaging of Protein Crystals

Serial crystallography at last generation X-ray synchrotron sources and free electron lasers enabled data collection with micrometer and even submicrometer size crystals, which have resulted in amazing progress in structural biology. However, imaging of small crystals, which although is highly demanded, remains a challenge, especially in the case of membrane protein crystals. Here we describe a new extremely sensitive method of the imaging of protein crystals that is based on coherent anti-Stokes Raman scattering