Serial synchrotron and XFEL crystallography for studies of metalloprotein catalysis

An estimated half of all proteins contain a metal, with these being essential for a tremendous variety of biological functions. X-ray crystallography is the major method for obtaining structures at high resolution of these metalloproteins, but there are considerable challenges to obtain intact structures due to the effects of radiation damage. Serial crystallography offers the prospect of determining low-dose synchrotron or effectively damage free XFEL structures at room temperature and enables time-resolved or dose-resolved approaches. Complementary spectroscopic data can validate redox and or ligand states within metalloprotein crystals. In this opinion, we discuss developments in the application of serial crystallographic approaches to metalloproteins and comment on future directions.     

N-terminal region of human chemokine receptor CXCR3: Structural analysis of CXCR3(1–48) by experimental and computational studies

Our study on the highly charged N-terminal peptide of the human chemokine receptor CXCR3 by spectroscopic methods in solution and by means of molecular dynamics simulations showed that the charge content modulates the intrinsic structural preference of its flexible backbone. Collectively, our findings suggest that the structural organization of a protein should be seen as a part of a continuum in which the ratio between electrostatic and hydrophobic interactions and the intrinsic flexibility are important properties used to optimize the folding. When this ratio changes and the structure is intrinsically flexible, the structural organization of the system moves along the continuum of the possible conformational states. By all this combined information, one can describe the structure of CXCR3(1–48) as an ensemble of conformations. In fact, the peptide shows stretches of negative charges embedded in a flexible sequence which can be used to maximize promiscuous interactions relevant to molecular recognition but globally the peptide appears as a poly-structured globule-like ensemble that is dynamically stabilized by H-bonds. We have approached the study of the most populated ensembles with subset selection to explain our experimental data also by evidencing that the changes into the fraction of charged residues discriminate between dynamically poly-structured states, conceivably because of small free energy barriers existing between the different conformations of CXCR3(1–48). Therefore, the overlap of a highly flexible backbone, negatively charged residues and sites which can be modified by post-translational modifications represent the structural organization that controls the molecular mechanisms underlying the biological functions carried out by CXCR3(1–48).     

Time-resolved cryo-electron microscopic study of the dissociation of actomyosin induced by photolysis of photolabile nucleotides

The rapid release of a substrate or other ligand from photolabile precursors in a thin layer suspension of biological specimens followed by rapid freezing provides a method of trapping and visualizing short-lived states in a dynamic system. We demonstrate here the first successful application of this method to study the interaction of actin filaments with myosin subfragment 1 (S1) after release of nucleotides. The results obtained suggest that structural changes in actin filaments occur as a result of interaction with S1.     

Conformational lability of two molecular chaperones Hsc70 and gp96: effects of pH and temperature

Hsc70 and gp96 are two heat shock proteins with molecular chaperone and immune-related activities. The dynamic conformational properties of heat shock proteins appear to play a critical role in their biological activities. In this study, we investigated the effects of pH and temperature on the conformational states of Hsc70 and gp96. The quaternary, tertiary, and secondary structures of both proteins are evaluated by a variety of spectroscopic techniques, including far-UV circular dichroism, Trp fluorescence, ANS fluorescence, and derivative UV absorption spectroscopy. The results are summarized and compared employing an empirical phase diagram approach. Very similar behaviors are seen for both proteins despite their differences in sequence and tertiary structure. Both proteins show substantial conformational lability in responses to the pH and temperature changes of their environment. This study suggests a natural selection for related functional properties through common conformational dynamics rather than immediate structural homology.     

Regulation of integrin function through conformational complexity: not simply a knee-jerk reaction?

Such diverse biological processes as the maintenance of tissue architecture and the regulation of cell migration are controlled through dynamic changes in integrin receptor conformation. Early analyses of the mechanisms of shape change by integrins led to the definition of three inter-convertible conformational states: inactive, primed and ligand-occupied. Recent advances reviewed in this article have now shown that the integrin molecule contains a number of flexible joints and connections, leading to a broad spectrum of possible conformational states. This conformational complexity is likely to permit fine-tuning of integrin function through regulation of ligand-binding affinity and intracellular signalling.     

An Insight into the Role of Glycerol in Chitosan Films

This work was focused on assessing the influence of the glycerol in chitosan matrices, analyzing the changes produced in the molecular mobility, mechanical, thermal, barrier and structural properties. The addition of glycerol in the matrix decreased the stress values, increasing the elasticity and water vapor permeability of the films, with a marked decrease in glass transition temperature; Detailed analyses of Fourier Transform IR Spectroscopy spectra supported the observed changes, especially in the spectral windows 1700–1500 cm^?1 revealing the modifications at molecular level caused by hydrogen bond interactions between chitosan and water in the presence of glycerol. Positron annihilation spectroscopic (PALS) measurements allowed determining the free volume assuming spherical holes as well as monitoring the structural changes in chitosan films caused by the addition of both, glycerol and water molecules. It was possible to infer that for unplasticized matrices, a sustained increase of the radius between 0.06 and 0.2 of X_water was observed, followed by a plateau up to 0.35. In the other case, with the addition of glycerol, there were two plateaus, the first between 0.25 and 0.37 of X_water, and the second from 0.41 to 0.47. For higher glycerol concentrations, the plasticizer would be mainly bounded to the chitosan pack more efficiently and the water present in the system would be predominantly free in the matrix causing its swelling. Findings on molecular mobility contributed to the understanding of the role of water and glycerol in the structural arrangement and its influence on film properties.     

Molecular mass and volume in radiation target theory

Radiation target analysis is based on the action of ionizing radiation directly on macromolecules. Interactions of this radiation with the molecules leads to considerable structural damage and consequent loss of biological activity. The radiation sensitivity is dependent on the size of the macromolecules. There has been confusion and discrepancy as to whether the molecular mass or the molecular volume was the determinant factor in the sensitivity. Some proteins are known to change their hydrodynamic volume at low pH, and this characteristic can be utilized to compare the radiation sensitivities of these proteins in the two states. The results show that the radiation sensitivity of proteins depends on the mass of the molecule and is independent of the molecular volume/shape.     

Effect of structural modifications on the spectroscopic properties and dynamics of the excited states of peridinin

The spectroscopic properties and dynamics of the lowest excited singlet states of peridinin and two derivatives have been studied by steady-state absorption and fast-transient optical spectroscopic techniques. One derivative denoted PerOlEs, possesses a double bond and a methyl ester group instead of the r-ylidenebutenolide of peridinin. Another derivative denoted PerAcEs, is the biosynthetic precursor of peridinin and possesses a triple bond and a methyl ester group corresponding to the r-ylidenbutenolide function. Ultrafast time-resolved spectroscopic experiments in the visible and near-infrared regions were performed on the molecules and reveal the energies and regarding the structural features and interactions responsible for the unusual solvent-induced changes in the steady-state and transient absorption spectra and dynamics of dynamics of the excited electronic states. The data also provide information peridinin.     

Exposition of humans to low doses and low dose rate irradiation: an urgent need for new markers and new models

In human radiation protection, the shape of the dose effects curve for low doses irradiation (LDI) is assumed to be linear, extrapolated from the clinical consequences of Hiroshima and Nagasaki nuclear explosions. This extrapolation probably overestimates the risk below 200 mSv. In many circumstances, the living species and cells can develop some mechanisms of adaptation. Classical epidemiological studies will not be able to answer the question and there is a need to assess more sensitive biological markers of the effects of LDI. The researches should be focused on DNA effects (strand breaks), radioinduced expression of new genes and proteins involved in the response to oxidative stress and DNA repair mechanisms. New experimental biomolecular techniques should be developed in parallel with more conventional ones. Such studies would permit to assess new biological markers of radiosensitivity, which could be of great interest in radiation protection and radio-oncology.     

Glimpses into measurements of the microcirculation in skin

There are instances when chronic disease states manifest as microvascular disturbances of the skin. Objective assessment of these disturbances would permit a better understanding of the causative factors and, in certain circumstances, influence decisions of clinical management. Recent advances in technology that permit such assessment non-invasively are evaluated in this review.     

Dynamic structural disorder of the FeO<Subscript>2</Subscript> moiety in oxymyoglobin studied by nuclear resonant forward scattering of synchrotron radiation

Oxy- as well as deoxymyoglobin exhibit a pronounced temperature dependence of the quadrupole splitting of the heme iron as detected by conventional M?ssbauer spectroscopy. With nuclear resonant forward scattering (NFS) of synchrotron radiation, which can be viewed as M?ssbauer spectroscopy in the time domain, it is shown that this spectroscopic behavior, although it is phenomenologically similar in the two cases, is based on completely different physical mechanisms. It is demonstrated that stochastic fluctuations of the iron electric field gradient in MbO(2), which are due to the dynamic structural disorder of the FeO(2) moiety, are the reason for the temperature-dependent alterations of the coherent quantum beat pattern in the NFS spectra of MbO(2), in contrast to deoxyMb where transitions between orbital states of iron take place. This subtle spectroscopic difference cannot be inferred from conventional M?ssbauer spectroscopy.     

A review of spectroscopic methods for characterizing microbial transformations of minerals

Over the past decade, advances in surface-sensitive spectroscopic techniques have provided the opportunity to identify many new microbiologically mediated biogeochemical processes. Although a number of surface spectroscopic techniques require samples to be dehydrated, which precludes real-time measurement of biotransformations and generate solid phase artifacts, some now offer the opportunity to either isolate a hydrated sample within an ultrahigh vacuum during analysis or utilize sources of radiation that efficiently penetrate hydrated specimens. Other nondestructive surface spectroscopic techniques permit determination of the influence of microbiological processes on the kinetics and thermodynamics of geochemical reactions. The ability to perform surface chemical analyses at micrometer and nanometer scales has led to the realization that bacterial cell surfaces are active sites of mineral nucleation and propagation, resulting in the formation of both stable and transient small-scale surface chemical heterogeneities. Some surface spectroscopic instrumentation is now being modified for use in the field to permit researchers to evaluate mineral biotransformations under in situ conditions. Surface spectroscopic techniques are thus offering a variety of opportunities to yield new information on the way in which microorganisms have influenced geochemical processes on Earth over the last 4 billion years.     

High probability of disrupting a disulphide bridge mediated by an endogenous excited tryptophan residue

It is well known that ultraviolet (UV) radiation may reduce or even abolish the biological activity of proteins and enzymes. UV light, as a component of sunlight, is illuminating all light-exposed parts of living organisms, partly composed of proteins and enzymes. Although a considerable amount of empirical evidence for UV damage has been compiled, no deeper understanding of this important phenomenon has yet emerged. The present paper presents a detailed analysis of a classical example of UV-induced changes in three-dimensional structure and activity of a model enzyme, cutinase from Fusarium solani pisi. The effect of illumination duration and power has been investigated. A photon-induced mechanism responsible for structural and functional changes is proposed. Tryptophan excitation energy disrupts a neighboring disulphide bridge, which in turn leads to altered biological activity and stability. The loss of the disulphide bridge has a pronounced effect on the fluorescence quantum yield, which has been monitored as a function of illumination power. A general theoretical model for slow two-state chemical exchange is formulated, which allows for calculation of both the mean number of photons involved in the process and the ratio between the quantum yields of the two states. It is clear from the present data that the likelihood for UV damage of proteins is directly proportional to the intensity of the UV radiation. Consistent with the loss of the disulphide bridge, a complex pH-dependent change in the fluorescence lifetimes is observed. Earlier studies in this laboratory indicate that proteins are prone to such UV-induced radiation damage because tryptophan residues typically are located as next spatial neighbors to disulphide bridges. We believe that these observations may have far-reaching implications for protein stability and for assessing the true risks involved in increasing UV radiation loads on living organisms.     

Gamma-radiation induced changes in the physical and chemical properties of lignocellulose

gamma-radiation induced effects on the physical and chemical properties of natural lignocellulose (jute) polymer were investigated. Samples were irradiated to required total doses at a particular dose rate. The changes in the parameters such as the tensile strength, elongation at break, and work done at rupture for the lignocellulose samples on irradiation with the gamma-rays from a cobalt-60 source were measured. The mechanical properties were found to have nonlinear relations with the radiation doses. The chemical stability of irradiated fibers was found to degrade progressively with the increase of radiation dose. Additionally, other chemical changes of the samples due to exposure to high-energy radiation were also investigated using fluorescence and infrared spectroscopic analysis. Differential scanning calorimetry and thermogravimetric studies showed a significant reduction in thermal stability. The wide-angle X-ray diffraction study showed that structural changes of cellulose appeared due to the radiation-induced chemical reaction of lignocellulose.     

Single-molecule spectroscopic characterization of light-harvesting 2 complexes reconstituted into model membranes

The spectroscopic properties of the light-harvesting 2 complexes (LH2) from the purple bacterium Rhodopseudomonas acidophila (strain 10050) in detergent micelles and reconstituted into lipid membranes have been studied by single-molecule spectroscopy. When LH2 complexes are solubilized from their host biological membranes by nondenaturing detergents, such as LDAO, there is a small 2-nm spectral shift of the B850 absorption band in the ensemble spectrum. This is reversed when the LH2 complexes are put back into phospholipid vesicles, i.e., into a more native-like environment. The spectroscopic properties on the single-molecule level of the detergent-solubilized LH2 complexes were compared with those reconstituted into the lipid membranes to see if their detailed spectroscopic behavior was influenced by these small changes in the position of the B850 absorption band. A detailed analysis of the low-temperature single-molecule fluorescence-excitation spectra of the LH2 complexes in these two different conditions showed no significant differences. In particular, the distribution of the spectral splitting between the circular k = +/-1 exciton states of the B850 absorption band and the distribution of the mutual angle between the k = +/-1 exciton states are identical in both cases. It can be concluded, therefore, that the LH2 complexes from Rps. acidophila are equally stable when solubilized in detergent micelles as they are when membrane reconstituted. Moreover, when they are solubilized in a suitable detergent and spin coated onto a surface for the single-molecule experiments they do not display any more structural disorder than when in a phospholipid membrane.     

3D micro-scale deformations of wood in bending: synchrotron radiation muCT data analyzed with digital volume correlation

A micro-scale three-point-bending experiment with a wood specimen was carried out and monitored by synchrotron radiation micro-computed tomography. The full three-dimensional wood structure of the 1.57 × 3.42 × 0.75 mm³ specimen was reconstructed at cellular level in different loading states. Furthermore, the full three-dimensional deformation field of the loaded wood specimen was determined by digital volume correlation, applied to the reconstructed data at successive loading states. Results from two selected regions within the wood specimen are presented as continuous displacement and strain fields in both 2D and 3D. The applied combination of synchrotron radiation micro-computed tomography and digital volume correlation for the deformation analysis of wood under bending stress is a novel application in wood material science. The method offers the potential for the simultaneous observation of structural changes and quantified deformations during in situ micro-mechanical experiments. Moreover, the high spatial resolution allows studying the influence of anatomical features on the fracture behaviour of wood. Possible applications of this method range from bio-mechanical observations in fresh plant tissue to fracture mechanics aspects in structural timber.     

Natural microbial UV radiation filters--mycosporine-like amino acids

Ozone depletion by anthropogenic gases has increased the atmospheric transmission of solar ultraviolet-B radiation (UV-B, 280-315 nm). There is a logical link between the natural defenses of terrestrial and marine organisms against UV radiation and the prevention of UV-induced damage to human skin. UV light degrades organic molecules such as proteins and nucleic acids, giving rise to structural changes that directly affect their biological function. These compounds offer the potential for development of novel UV blockers for human use. The biological role of mycosporine-like amino acids (MAAs) and scytonemin as a defense against solar radiation in organisms, together with their structure, synthesis, distribution, regulation and effectiveness, are reviewed in this article. This review points to the role of MAAs as a natural defense against UV radiation.     

Application of a temperature‐dependent fluorescent dye (Rhodamine B) to the measurement of radiofrequency radiation‐induced temperature changes in biological samples

We have applied a non‐contact method for studying the temperature changes produced by radiofrequency (RF) radiation specifically to small biological samples. A temperature‐dependent fluorescent dye, Rhodamine B, as imaged by laser scanning confocal microscopy (LSCM) was used to do this. The results were calibrated against real‐time temperature measurements from fiber optic probes, with a calibration factor of 3.4% intensity change °C^?1 and a reproducibility of ±6%. This non‐contact method provided two‐dimensional and three‐dimensional images of temperature change and distributions in biological samples, at a spatial resolution of a few micrometers and with an estimated absolute precision of around 1.5 °C, with a differential precision of 0.4 °C. Temperature rise within tissue was found to be non‐uniform. Estimates of specific absorption rate (SAR) from absorbed power measurements were greater than those estimated from rate of temperature rise, measured at 1 min intervals, probably because this interval is too long to permit accurate estimation of initial temperature rise following start of RF exposure. Future experiments will aim to explore this. Bioelectromagnetics 30:583–590, 2009. © 2009 Wiley‐Liss, Inc.     

Effect of microwave radiation on the biophysical properties of liposomes

Steadily growing use of electromagnetic fields, especially in conjunction with wireless communication systems, has led to increasing public concern about possible health effects of electromagnetic radiation. However, besides the well-known thermal effect of electromagnetic fields on biological tissue, there is no clear evidence of further athermal interaction mechanisms with biological systems. The present study was designed to determine the changes in bilayer permeability in egg lecithin multilamellar vesicles after exposure to 900 MHz microwave radiation for a period of 5 h. Specific absorption rate (SAR) of the radiation for the investigated liposome sample was found to be 12 +/- 1 W/kg. Liposomal changes in permeability were monitored using a light scattering technique. Optical anisotropy of the liposome sample decreased dramatically upon exposure to microwave radiation, indicating structural changes in acyl chain packing. IR and NMR ((1)H NMR) studies, which have been employed to reveal structural alterations in microwave, exposed vesicles showed an increased damage upon exposure to microwave. The changes observed in the (1)H NMR spectrum of the microwave exposed sample indicated hydrolysis of carboxylic and phosphoric esters. IR study showed conformational changes in the acyl chains of the lipids upon microwave exposure. However, both IR and (31)P NMR did not show any appreciable changes in the head group part of the lipids.     

Structural, spectroscopic and catalytic activity studies on glutathione reductase reconstituted with FAD analogues

FAD-modified human glutathione reductases were reconstituted from apoenzyme using the FAD analogues 6-SH-FAD, 6-SCN-FAD, 6-OH-FAD, 6-NH2-FAD and 8-OH-FAD. The catalytic activities of the modified enzymes were substantially lower than for the native enzyme. All five species could be crystallized, but only those containing 6-SH-FAD, 6-OH-FAD and 6-NH2-FAD yielded crystals that could be analyzed. X-ray analyses and structural refinements were performed at 0.27 nm and 0.30 nm resolution resulting in R factors around 13.5%. The crystal structures showed the additional non-hydrogen atoms and small conformational changes of the polypeptide that were obviously induced by the substituents of the FAD analogues. The observed changes together with spectroscopic and activity data permit some conclusions about the chemical nature of the substituents.