SOLEIL shining on the solution-state structure of biomacromolecules by synchrotron X-ray footprinting at the Metrology beamline.

Synchrotron X-ray footprinting complements the techniques commonly used to define the structure of molecules such as crystallography, small-angle X-ray scattering and nuclear magnetic resonance. It is remarkably useful in probing the structure and interactions of proteins with lipids, nucleic acids or with other proteins in solution, often better reflecting the in vivo state dynamics. To date, most X-ray footprinting studies have been carried out at the National Synchrotron Light Source, USA, and at the European Synchrotron Radiation Facility in Grenoble, France. This work presents X-ray footprinting of biomolecules performed for the first time at the X-ray Metrology beamline at the SOLEIL synchrotron radiation source. The installation at this beamline of a stopped-flow apparatus for sample delivery, an irradiation capillary and an automatic sample collector enabled the X-ray footprinting study of the structure of the soluble protein factor H (FH) from the human complement system as well as of the lipid-associated hydrophobic protein S3 oleosin from plant seed. Mass spectrometry analysis showed that the structural integrity of both proteins was not affected by the short exposition to the oxygen radicals produced during the irradiation. Irradiated molecules were subsequently analysed using high-resolution mass spectrometry to identify and locate oxidized amino acids. Moreover, the analyses of FH in its free state and in complex with complement C3b protein have allowed us to create a map of reactive solvent-exposed residues on the surface of FH and to observe the changes in oxidation of FH residues upon C3b binding. Studies of the solvent accessibility of the S3 oleosin show that X-ray footprinting offers also a unique approach to studying the structure of proteins embedded within membranes or lipid bodies. All the biomolecular applications reported herein demonstrate that the Metrology beamline at SOLEIL can be successfully used for synchrotron X-ray footprinting of biomolecules.

Topological characterization of the DnaA-oriC complex using single-molecule nanomanipuation.

In most bacteria, the timing and synchrony of initiation of chromosomal replication are determined by the binding of the AAA(+) protein DnaA to a set of high- and low-affinity sites found within the origin of chromosomal replication (oriC). Despite the large amount of information on the role and regulation of DnaA, the actual structure of the DnaA-oriC complex and the mechanism by which it primes the origin for the initiation of replication remain unclear. In this study, we have performed magnetic tweezers experiments to investigate the structural properties of the DnaA-oriC complex. We show that the DnaA-ATP-oriC complex adopts a right-handed helical conformation involving a variable amount of DNA and protein whose features fit qualitatively as well as quantitatively with an existing model based on the crystal structure of a truncated DnaA tetramer obtained in the absence of DNA. We also investigate the topological effect of oriC's DNA unwinding element.

Cooperative effects on the compaction of DNA fragments by the nucleoid protein H-NS and the crowding agent PEG probed by Magnetic Tweezers.

BACKGROUND: DNA bridging promoted by the H-NS protein, combined with the compaction induced by cellular crowding, plays a major role in the structuring of the E. coli genome. However, only few studies consider the effects of the physical interplay of these two factors in a controlled environment. METHODS: We apply a single molecule technique (Magnetic Tweezers) to study the nanomechanics of compaction and folding kinetics of a 6 kb DNA fragment, induced by H-NS bridging and/or PEG crowding. RESULTS: In the presence of H-NS alone, the DNA shows a step-wise collapse driven by the formation of multiple bridges, and little variations in the H-NS concentration-dependent unfolding force. Conversely, the DNA collapse force observed with PEG was highly dependent on the volume fraction of the crowding agent. The two limit cases were interpreted considering the models of loop formation in a pulled chain and pulling of an equilibrium globule respectively. CONCLUSIONS: We observed an evident cooperative effect between H-NS activity and the depletion of forces induced by PEG. GENERAL SIGNIFICANCE: Our data suggest a double role for H-NS in enhancing compaction while forming specific loops, which could be crucial in vivo for defining specific mesoscale domains in chromosomal regions in response to environmental changes.

RNA folding at millisecond intervals by synchrotron hydroxyl radical footprinting.

Radiolysis of water with a synchrotron x-ray beam permits the hydroxyl radical-accessible surface of an RNA to be mapped with nucleotide resolution in 10 milliseconds. Application of this method to folding of the Tetrahymena ribozyme revealed that the most stable domain of the tertiary structure, P4-P6, formed cooperatively within 3 seconds. Exterior helices became protected from hydroxyl radicals in 10 seconds, whereas the catalytic center required minutes to be completely folded. The results show that rapid collapse to a partially disordered state is followed by a slow search for the active structure.

Time-resolved synchrotron X-ray "footprinting", a new approach to the study of nucleic acid structure and function: application to protein-DNA interactions and RNA folding.

Hydroxyl radicals (.OH) can cleave the phosphodiester backbone of nucleic acids and are valuable reagents in the study of nucleic acid structure and protein-nucleic acid interactions. Irradiation of solutions by high flux "white light" X-ray beams based on bending magnet beamlines at the National Synchrotron Light Source (NSLS) yields sufficient concentrations of .OH so that quantitative nuclease protection ("footprinting") studies of DNA and RNA can be conducted with a duration of exposure in the range of 50 to 100 ms. The sensitivity of DNA and RNA to X-ray mediated .OH cleavage is equivalent. Both nucleic acids are completely protected from synchrotron X-ray induced cleavage by the presence of thiourea in the sample solution, demonstrating that cleavage is suppressed by a free radical scavenger. The utility of this time-dependent approach to footprinting is demonstrated with a synchrotron X-ray footprint of a protein-DNA complex and by a time-resolved footprinting analysis of the Mg(2+)-dependent folding of the Tetrahymena thermophilia L-21 ScaI ribozyme RNA. Equilibrium titrations reveal differences among the ribozyme domains in the cooperativity of Mg(2+)-dependent .OH protection. RNA .OH protection progress curves were obtained for several regions of the ribozyme over timescales of 30 seconds to several minutes. Progress curves ranging from > or = 3.5 to 0.4 min-1 were obtained for the P4-P6 and P5 sub-domains and the P3-P7 domain, respectively. The .OH protection progress curves have been correlated with the available biochemical, structural and modeling data to generate a model of the ribozyme folding pathway. Rate differences observed for specific regions within domains provide evidence for steps in the folding pathway not previously observed. Synchrotron X-ray footprinting is a new approach of general applicability for the study of time-resolved structural changes of nucleic acid conformation and protein-nucleic acid complexes.

Following the folding of RNA with time-resolved synchrotron X-ray footprinting.

The rapid mixing synchrotron X-ray footprinting technique described in this article allows nucleic acid folding and ligand binding reactions to be followed on a millisecond time resolution with single nucleotide resolution. In principle, the change in .OH protection of every nucleotide in a nucleic acid hundreds of nucleotides long can be monitored separately. In addition, a wide range of solution conditions are compatible with the radiolytic generation of .OH. These characteristics of synchrotron X-ray footprinting create opportunities for conducting thermodynamic and kinetic studies of nucleic acids that are both comprehensive and detailed. Kinetic footprinting studies of a number of systems have been initiated by the Center for Synchrotron Biosciences using this technique.

Fractal-like patterns in DNA films, B form at 0% relative humidity, and antiheteronomous DNA: an IR study.

This report details the observation of (a) the intact double helix of DNA at 0% relative humidity in poly(dA).poly(dT) and poly(dA-dT).poly(dA-dT) films, (b) the fractal-like growth of DNA crystals in films of poly(dA).poly(dT), and (c) poly(dA).poly(dT) with adenines in the B form and thymines in the A form. Observation (a) is based on the behavior of the middle ir signature of double-helical base stacking, the 1714 cm-1 peak, at low water activity. This observation is modeled as a trapping of water in the polycrystalline part of these films. We interpret the glycosidic region of the middle ir spectra of the polycrystalline films of poly(dA).poly(dT) at 0% relative humidity to indicate that at least part of the adenine strand in is the B conformation and the thymine strand is in the A form, whereas previous assignments of poly(dA).poly(dT) films at 75% relative humidity indicated the opposite strand conformation assignment [E. Taillandier et al. (1987) Biochemistry, Vol. 26 p. 3361].

The Early Folding Intermediates of the Tetrahymena Ribozyme are Kinetically Trapped.

Abstract The "RNA folding problem" is a fundamental and challenging question in contemporary biophysics. Understanding the mechanism(s) by which RNA molecules fold into compact structures capable of biological activity is important because RNA folding is closely tied to cellular regulation and metabolism and catalytic RNAs are potential reagents for gene therapy. Unlike the "protein folding problem" which has been under study for many decades, the study of RNA tertiary structure stability and folding is a relatively new field of endeavor. Thus, a detailed understanding of both the thermodynamics and kinetics of RNA folding are only now beginning to emerge. Kinetic traps have been observed in the late folding steps of the Tetrahymena ribozyme. In this study we extend our "synchrotron footprinting" analysis of the Tetrahymena ribozyme (Sclavi, et al. Science 279, 1940-1943, 1998) to probe the potential presence of kinetic traps in other steps in the folding mechanism. Examination of the folding in 3M urea demonstrates a significant increase in the rates of folding for early folding steps in the formation of the ribozyme tertiary structure. These data support the conclusion of Williamson and co-workers that the rate-limiting step in the folding of the Tetrahymena ribozyme is kinetically trapped by native interactions (Rook et al., J. Mol. Bio., 281, 609-620, 1998). Kinetic trapping also occurs in the formation of intermediates earlier in the folding reaction, and in these cases nonnative interactions may also play a role in the barrier to folding.

Global mapping of structural solutions provided by the extended X-ray absorption fine structure ab initio code FEFF 6.01: structure of the cryogenic photoproduct of the myoglobin-carbon monoxide complex.

X-ray methods based on synchrotron technology have the promise of providing time-resolved structural data based on the high flux and brightness of the X-ray beams. One of the most closely examined problems in this area of time-resolved structure determination has been the examination of intermediates in ligand binding to myoglobin. Recent crystallographic experiments using synchrotron radiation have identified the protein tertiary and heme structural changes that occur upon photolysis of the myoglobin--carbon monoxide complex at cryogenic temperatures [Schlichting, I., Berendzen, J., Phillips, G., & Sweet, R. (1994) Nature 371, 808--812]. However, the precision of protein crystallographic data (approximately 0.2 A) is insufficient to provide precise metrical details of the iron--ligand bond lengths. Since bond length changes on this scale can trigger reactivity changes of several orders of magnitude, such detail is critical to a full understanding of metalloprotein structure--function relationships. Extended X-ray absorption fine structure (EXAFS) spectroscopy has the potential for analyzing bond distances to a precision of 0.02 A but is hampered by its relative insensitivity to the geometry of the backscattering atoms. Thus, it is often unable to provide a unique solution to the structure without ancillary structural information. We have developed a suite of computer programs that incorporate this ancillary structural information and compute the expected experimental spectra for a wide ranging series of Cartesian coordinate sets (global mapping). The programs systematically increment the distance of the metal to various coordinating ligands (along with their associated higher shells). Then, utilizing the ab initio EXAFS code FEFF 6.01, simulated spectra are generated and compared to the actual experimental spectra, and the differences are computed. Finally, the results for hundreds of simulations can be displayed (and compared) in a single plot. The power of this approach is demonstrated in the examination of high signal to noise EXAFS data from a photolyzed solution sample of the myoglobin--carbon monoxide complex at 10 K. Evaluation of these data using our global mapping procedures placed the iron to pyrrole nitrogen average distances close to the value for deoxymyoglobin (2.05 +/- 0.01 A), while the distance from iron to the proximal histidine nitrogen is seen to be 2.20 +/- 0.04 A. It is also shown that one cannot uniquely position the CO ligand on the basis of the EXAFS data alone, as a number of reasonable minima (from the perspective of the EXAFS) are observed. This provides a reasonable explanation for the multiplicity of solutions that have been previously reported. The results presented here are seen to be in complete agreement with the crystallographic results of Schlichting et al. (1994) within the respective errors of the two techniques; however, the extended X-ray absorption fine structure data allow the iron--ligand bond lengths to be precisely defined. An examination of the available spectroscopic data, including EXAFS, shows that the crystallographic results of Schlichting et al. (1994) are highly relevant to the physiological solution state and must be taken into account in any attempt to understand the incomplete relaxation process of the heme iron for the Mb*CO photoproduct at low temperature.