Jen Bohon

Synchrotron Protein Footprinting Supports Substrate Translocation by ClpA via ATP-Induced Movements of the D2 Loop

Synchrotron X-ray protein footprinting is used to study structural changes upon formation of the ClpA hexamer. Comparative solvent accessibilities between ClpA monomer and ClpA hexamer samples are in agreement throughout most of the sequence, with calculations based on two previously proposed hexameric models. The data differ substantially from the proposed models in two parts of the structure: the D1 sensor 1 domain and the D2 loop region. The results suggest that these two regions can access alternate conformations in which their solvent protection is greater than that in the structural models based on crystallographic data. In combination with previously reported structural data, the footprinting data provide support for a revised model in which the D2 loop contacts the D1 sensor 1 domain in the ATP-bound form of the complex. These data provide the first direct experimental support for the nucleotide-dependent D2 loop conformational change previously proposed to mediate substrate translocation.

Structural Characterization of HIV gp41 with the Membrane-proximal External Region

Human immunodeficiency virus, type 1 (HIV-1) envelope glycoprotein (gp120/gp41) plays a critical role in virus infection and pathogenesis. Three of the six monoclonal antibodies considered to have broadly neutralizing activities (2F5, 4E10, and Z13e1) bind to the membrane-proximal external region (MPER) of gp41. This makes the MPER a desirable template for developing immunogens that can elicit antibodies with properties similar to these monoclonal antibodies, with a long term goal of developing antigens that could serve as novel HIV vaccines. In order to provide a structural basis for rational antigen design, an MPER construct, HR1-54Q, was generated for x-ray crystallographic and x-ray footprinting studies to provide both high resolution atomic coordinates and verification of the solution state of the antigen, respectively. The crystal structure of HR1-54Q reveals a trimeric, coiled-coil six-helical bundle, which probably represents a postfusion form of gp41. The MPER portion extends from HR2 in continuation of a slightly bent long helix and is relatively flexible. The structures observed for the 2F5 and 4E10 epitopes agree well with existing structural data, and enzyme-linked immunosorbent assays indicate that the antigen binds well to antibodies that recognize the above epitopes. Hydroxyl radical-mediated protein footprinting of the antigen in solution reveals specifically protected and accessible regions consistent with the predictions based on the trimeric structure from the crystallographic data. Overall, the HR1-54Q antigen, as characterized by crystallography and footprinting, represents a postfusion, trimeric form of HIV gp41, and its structure provides a rational basis for gp41 antigen design suitable for HIV vaccine development.

Installation and testing of a focusing mirror at beamline X28C for high flux x-ray radiolysis of biological macromolecules

The NSLS X28C white-light beamline has been upgraded with a focusing mirror in order to provide increased x-ray density and a wide selection of beam shapes at the sample position. The cylindrical single crystal silicon mirror uses an Indalloy 51 liquid support bath as both a mechanism for heat transfer and a buoyant support to counter the effects of gravity and correct for minor parabolic slope errors. Calorimetric measurements were performed to verify that the calculated more than 200-fold increase in flux density is delivered by the mirror at the smallest beam spot. The properties of the focused beam relevant to radiolytic footprinting, namely, the physical dimensions of the beam, the effective hydroxyl radical dose delivered to the sample, and sample heating upon irradiation, have been studied at several mirror angles.

Characterization of metalloproteins by high-throughput X-ray absorption spectroscopy

High-throughput X-ray absorption spectroscopy was used to measure transition metal content based on quantitative detection of X-ray fluorescence signals for 3879 purified proteins from several hundred different protein families generated by the New York SGX Research Center for Structural Genomics. Approximately 9% of the proteins analyzed showed the presence of transition metal atoms (Zn, Cu, Ni, Co, Fe, or Mn) in stoichiometric amounts. The method is highly automated and highly reliable based on comparison of the results to crystal structure data derived from the same protein set. To leverage the experimental metalloprotein annotations, we used a sequence-based de novo prediction method, MetalDetector, to identify Cys and His residues that bind to transition metals for the redundancy reduced subset of 2411 sequences sharing <70% sequence identity and having at least one His or Cys. As the HT-XAS identifies metal type and protein binding, while the bioinformatics analysis identifies metal- binding residues, the results were combined to identify putative metal-binding sites in the proteins and their associated families. We explored the combination of this data with homology models to generate detailed structure models of metal-binding sites for representative proteins. Finally, we used extended X-ray absorption fine structure data from two of the purified Zn metalloproteins to validate predicted metalloprotein binding site structures. This combination of experimental and bioinformatics approaches provides comprehensive active site analysis on the genome scale for metalloproteins as a class, revealing new insights into metalloprotein structure and function.

Transmission-mode diamond white-beam position monitor at NSLS

Two transmission-mode diamond X-ray beam position monitors installed at National Synchrotron Light Source (NSLS) beamline X25 are described. Each diamond beam position monitor is constructed around two horizontally tiled electronic-grade (p.p.b. nitrogen impurity) single-crystal (001) CVD synthetic diamonds. The position, angle and flux of the white X-ray beam can be monitored in real time with a position resolution of 500 nm in the horizontal direction and 100 nm in the vertical direction for a 3 mm × 1 mm beam. The first diamond beam position monitor has been in operation in the white beam for more than one year without any observable degradation in performance. The installation of a second, more compact, diamond beam position monitor followed about six months later, adding the ability to measure the angular trajectory of the photon beam.

Structural Analysis of a Highly Glycosylated and Unliganded gp120-Based Antigen Using Mass Spectrometry †

Structural characterization of the HIV-1 envelope protein gp120 is very important for providing an understanding of the proteins immunogenicity and its binding to cell receptors. So far, the crystallographic structure of gp120 with an intact V3 loop (in the absence of a CD4 coreceptor or antibody) has not been determined. The third variable region (V3) of the gp120 is immunodominant and contains glycosylation signatures that are essential for coreceptor binding and entry of the virus into T-cells. In this study, we characterized the structure of the outer domain of gp120 with an intact V3 loop (gp120-OD8) purified from Drosophila S2 cells utilizing mass spectrometry-based approaches. We mapped the glycosylation sites and calculated the glycosylation occupancy of gp120-OD8; 11 sites from 15 glycosylation motifs were determined as having high-mannose or hybrid glycosylation structures. The specific glycan moieties of nine glycosylation sites from eight unique glycopeptides were determined by a combination of ECD and CID MS approaches. Hydroxyl radical-mediated protein footprinting coupled with mass spectrometry analysis was employed to provide detailed information about protein structure of gp120-OD8 by directly identifying accessible and hydroxyl radical-reactive side chain residues. Comparison of gp120-OD8 experimental footprinting data with a homology model derived from the ligated CD4-gp120-OD8 crystal structure revealed a flexible V3 loop structure in which the V3 tip may provide contacts with the rest of the protein while residues in the V3 base remain solvent accessible. In addition, the data illustrate interactions between specific sugar moieties and amino acid side chains potentially important to the gp120-OD8 structure.

Probing the structure of ribosome assembly intermediates in vivo using DMS and hydroxyl radical footprinting

The assembly of the Escherichia coli ribosome has been widely studied and characterized in vitro. Despite this, ribosome biogenesis in living cells is only partly understood because assembly is coupled with transcription, modification and processing of the pre-ribosomal RNA. We present a method for footprinting and isolating pre-rRNA as it is synthesized in E. coli cells. Pre-rRNA synthesis is synchronized by starvation, followed by nutrient upshift. RNA synthesized during outgrowth is metabolically labeled to facilitate isolation of recent transcripts. Combining this technique with two in vivo RNA probing methods, hydroxyl radical and DMS footprinting, allows the structure of nascent RNA to be probed over time. Together, these can be used to determine changes in the structures of ribosome assembly intermediates as they fold in vivo.

Pixelated transmission-mode diamond X-ray detector

Fabrication and testing of a prototype transmission-mode pixelated diamond X-ray detector (pitch size 60-100 µm), designed to simultaneously measure the flux, position and morphology of an X-ray beam in real time, are described. The pixel density is achieved by lithographically patterning vertical stripes on the front and horizontal stripes on the back of an electronic-grade chemical vapor deposition single-crystal diamond. The bias is rotated through the back horizontal stripes and the current is read out on the front vertical stripes at a rate of ∼ 1 kHz, which leads to an image sampling rate of ∼ 30 Hz. This novel signal readout scheme was tested at beamline X28C at the National Synchrotron Light Source (white beam, 5-15 keV) and at beamline G3 at the Cornell High Energy Synchrotron Source (monochromatic beam, 11.3 keV) with incident beam flux ranges from 1.8 × 10(-2) to 90 W mm(-2). Test results show that the novel detector provides precise beam position (positional noise within 1%) and morphology information (error within 2%), with an additional software-controlled single channel mode providing accurate flux measurement (fluctuation within 1%).

Laser patterning of diamond. Part I. Characterization of surface morphology

As the use of diamond as a material for electronic and research applications increases, methods of patterning diamond will be required. In this work, single- and polycrystalline synthetic diamond samples were exposed to laser beams of different energies, wavelengths, and pulse durations. The effects of this exposure were characterized using optical microscopy, scanning electron microscopy, and atomic force microscopy. The threshold ablation energy density for 266 nm radiation with ∼30 ps pulse duration was measured to be ∼14 J/cm2. The threshold for ∼10 ns pulses at the same wavelength was similar, but the ablated area displayed larger surface damage. The surface damage and the threshold energy increase significantly for 532 and 1064 nm radiations. Ablation performed using 213 nm radiation produced the most uniform surface. Changes in the ablated surface are presented in detail.

Laser patterning of diamond. Part II. Surface nondiamond carbon formation and its removal

As diamond becomes more prevalent for electronic and research applications, methods of patterning diamond will be required. One such method, laser ablation, has been investigated in a related work. We report on the formation of surface nondiamond carbon during laser ablation of both polycrystalline and single-crystal synthetic diamonds. Near edge x-ray absorption fine structure spectroscopy was used to confirm that the nondiamond carbon layer formed during the ablation was amorphous, and Fourier transform infrared absorption spectroscopy (FTIR) was used to estimate the thickness of this layer to be ∼60 nm. Ozone cleaning was used to remove the nondiamond carbon layer.