May 19, 2008

2009 Joint NSLS-CFN Users' Meeting Workshop

Solution Studies of Macromolecules: Global and Local Structure

In recent years, small angle scattering (SAXS) and x-ray footprinting (XF) are two techniques that have grown in popularity among the life science community to study the structure of large biomolecular complexes. SAXS and XF can be used to study an individual functional protein or nucleic acid as well as a large assembly of proteins and protein nucleic acid complex in solution near physiological conditions. SAXS provides information about tertiary structure and folds or global structure whereas XF provides information on the scale of a single nucleotide or amino acid side chain. Both of the techniques can be used to carryout time-resolved studies to analyze structural changes in response to variation in external conditions, like the process of protein-protein and protein-ligand interaction, steps in the assembly/dissociation of large complexes. Most importantly, both SAXS and XF can be used to study supramolecular complexes that control most of the fundamental processes in living organism such as ribosomal protein synthesis, DNA replication machinery, gene expression and regulation, protein degradation pathway, etc. Often, these two methods are complementary to high-resolution techniques like nuclear magnetic resonance and x-ray crystallography, where there are difficulties to structural dynamics in supramolecular complexes. The combination of SAXS and XF has already proven valuable for understanding the relationship between tertiary contact formation and global compaction during the processes of macromolecular folding and formation of protein structural intermediates, and has highlighted the value of simultaneous global and local structural measurements by SAXS and XF.

Participants of the Solution Studies of Macromolecules: Global and Local Structure Workshop

As a part of the 2009 NSLS and CFN Users’ Meeting, the workshop entitled "Solution Studies of Macromolecules: Global and Local Structure" was held on May 20, 2009, at Brookhaven National Laboratory. The workshop was organized by Sayan Gupta of the Center for Proteomics and Bioinformatics, Case Western Reserve University. The objective of the workshop was to discuss the experimental and data analysis methodologies of XF and SAXS with examples of their application to investigate RNA folding, protein-protein and protein-nucleic acid interactions, the structure of large complexes and membrane proteins, and structural studies inside living cells. The workshop provided a venue for XF and SAXS users as well as the structural biology community to share ideas as well as discuss future directions for their research.

This workshop provided an opportunity to discuss the synergy between XF and SAXS technologies and its contribution to the development of world-leading life science user facility for macromolecular structure and function at NSLS-II.

The workshop began with a talk by Sarah Woodson (Johns Hopkins University) on "Probing 30S Ribosome Assembly with X-ray Hydroxyl Radical Footprinting." Woodson discussed how her research group has used x-ray mediated hydroxyl radical footprinting experiments to map the kinetic pathway of 30S ribosome assembly in vitro and applied this method to whole cells, in order to follow the assembly of new ribosomes in their natural environment. The RNA cleavage patterns observed in cells are consistent with in vitro data. Woodson demonstrated that in vivo footprinting with a synchrotron x-ray beam can provide rapid and detailed structural information on RNA-protein complexes in whole cells.

Joanna Krueger (University of North Carolina Charlotte) discussed "Constraints for Building Structural Models of Gelsolin, an Actin Dimer and Their Complex in Solution Using Small-Angle Scattering Data." Krueger described SAXS data on Ca2+-activated gelsolin and the Ca2+-gelsolin:actin dimer complex that provides structural insights into the regulatory activity of gelsolin. Krueger demonstrated that SAS-constrained models provided new insights into understanding the mechanism by which gelsolin nucleates F-actin formation with broad implications as to the mechanism for actin polymerization in general.

Sayan Gupta (Case Western Reserve University), in his talk "Introduction to X-ray Protein Footprinting," described the concept, beamline facility, and application of x-ray footprinting to study membrane protein structure and dynamics in solution. Gupta describes how intense synchbrotron x-rays is employed to produced hydroxyl radicals in solution on a microseconds to milliseconds timescale and used to produce stable covalent modifications at the solvent accessible side chain amino acid residues. Gupta described how to analyze data by mass spectroscopy to get the information about the side chain solvent accessibility or mobility of side chain mobility in various states of proteins that provides detailed structural information to map tertiary contacts of interactions or local conformational changes. He demonstrated how one can study membrane proteins in their functional states of interest and gain insight into the dynamic processes involved in their function.

Janna Kiselar (Case Western Reserve University) discussed "The Role of Mass Spectrometry in Probing the Structure of Macromolecular Complexes." Kiselar described how the x-ray footprinting approach, in combination with mass spectrometry, offers an important solution method for characterizing protein-ligand, protein-protein interactions and conformational changes of macromolecules in solution with resolution at the single side-chain level. Kiselar described implementation of the single reaction monitoring method to probe covalent modification at the side chain residue, which will significantly increase the detection ability and increase the structural resolution of this x-ray protein footprinting technique.

In his talk "Facilities for Static and Time-Resolved X-ray Solution Scattering at the BioCAT Beamline 18ID at the Advanced Photon Source" Thomas C. Irving (Illinois Institute of Technology) discussed the BioCAT-NIH Biotechnology Resource dedicated to, and optimized for, structural studies of partially ordered biological materials exploiting the exceptional source properties of the Advanced Photon Source at Argonne National Laboratory. Irving briefly described the features of the small-angle instrument on the BioCAT beamline 18ID in the context of recent experimental results from static and time-resolved scattering from macromolecules in solution. Irving showed how high source brilliance can enable new classes of experiments and hinted at some directions for the future.

B. Tracy Nixon (Pennsylvania State University) described "SAS Studies of Two Component Signal Transduction and AAA+ ATPase Function." Nixon demonstrated the use of solution scattering (SAXS and SANS) to examine how two-component signal transduction regulates function of bacterial enhancer binding proteins, which are members of the large superfamily of AAA+ ATPases, and to elucidate structural changes in the catalytic cycle of such AAA+ ATPases. Nixon highlighted the use of conventional and newly developed instrumentation and experimental protocols, including attempts at 10-msec resolved SAXS. He explained that these advances are expected to open up new avenues in the field of other large macromolecular assemblies like nucleosome.

In his talk, "Monitoring Conformational Dynamics of Macromolecules and Water with Radiolytic Footprinting" Mark R. Chance (Case Western Reserve University) described the hydroxyl radical labeling from the water embedded in transmembrane domains of G-protein coupled receptor (GPCR)-Rhodopsin and its photo-activation-induced changes in local structural constraints mediated by those in water. Chance demonstrated a possible general mechanism for water-dependent communication in family A GPCRs. He highlighted the utility of radiolytic footprinting for observing membrane proteins dynamics and also mentioned that protein and nucleic acid footprinting has had synergy with SAXS studies for many years as they provide a specific connection between local and global measure of structural change in solution. He then reviewed examples from ribozyme folding and gelsolin activation to illustrate this synergy between the two techniques. Chance highlighted the recent technological improvements in protein footprinting, which promise to make the technology easier to perform and extend its applicability to more complex samples.

The final speaker of the workshop, Lin Yang (Brookhaven National Laboratory), described "Biomolecular Solution Scattering at NSLS: Current Status and Outlook." Yang described the newly constructed, dedicated SAXS beamline, X9, which is currently being commissioned. Yang reviewed the technical development initiated at X21, and the improvements realized in the X9 SAXS instrument. He also presented commissioning results and discussed the outlook of the biomolecular solution scattering program at X9.

Following the presentations, a tour of NSLS beamline X9 and X28C was given by Lin Yang and Sayan Gupta to demonstrate their configurations and experimental strategies.

The workshop, which drew in nearly 50 attendees from various research institutes and universities, was very productive and will bring in users who want to utilize SAXS and XF simultaneously using NSLS beamlines for their research. This workshop has initiated a scientifically enriching and mutually beneficial relationship for all users who are a part of the life science community at the NSLS.

For more information:
Sayan Gupta
Center for Proteomics and Bioinformatics
Case Western Reserve University
Email: sayan@bnl.gov