The College of Medicine provides a number of facilities for biomedical research including cell sorting, DNA synthesis and sequencing, and many other facilities for cell and molecular biology research. The Departments also have spectrometers and other routine instruments available. These are standard for any major medical school and are not enumerated or emphasized in this section. Instead we provide detailed descriptions of major Molecular Biophysics facilities important to the Training Program

Center for Synchrotron Biosciences
Director: Mark Chance

The Center for Synchrotron Biosciences of AECOM operates beamlines X9A, X28A, and X9B at the National Synchrotron Light Source for X-ray studies of various descriptions and U2B for infrared spectroscopy. The X-ray studies on X-9B and X-9A include x-ray absorption spectroscopy, macromolecular crystallography, and small-angle scattering. Each of the two beamlines is equipped with sagittal focusing monochromators and vertical focusing mirrors for high-flux bending magnet work (~5 x 10¹¹ photons per second in a spot size of 0.25 x 0.3 mm from 5-15 keV). Both X9A and X9B beamlines have cryogenic cooling and CCD detection available for macromolecular crystallography (see below). Time-resolved footprinting is carried out on X28A, which has white beam. The U2B station includes equipment for infrared microscopy and far-infrared spectroscopy.

The major equipment at the experimental floor of NSLS: large four circle for diffraction; 13-element Ge detector for spectroscopy, assorted test instrumentation, stopped flow for footprinting, Nicolet infrared microscope and Nicolet 550 spectroscopy bench; ATI He Displex refrigerator for EXAFS use on X9B. MAR 345 detector, Octane SGI., Oxford cryostream, 3 Neslab chillers, Surelite ND-YAG 5ns nanosecond laser with doubling and tripling optics, SRC digital delay generator, Janis "cold-finger" He cryostat, for use in infrared applications, Lakeshore temperature controller, ASDC 2k x 2K CCD, Mar 165 mm CCD, Oxford and MSC liquid nitrogen cryostream units for crystal freezing and cooling. Computation facilities at NSLS include 2 SGI Octane systems for data reduction at each beamline and separate computers linked to the CCD detectors for data acquisition. For footprinting, stopped flow and radiation safety equipment are available.

The Center is operated as a Biomedical Technology Research Resource (P41 grant). Several full-time staff are available to assist students with their experiments and in beamline training.

X-ray Diffraction Laboratory, including Molecular Graphics and Computation Facilities Director: Steve Almo

Drs. Almo, Roderick and Arnez of the Department of Biochemistry oversee this facility. It provides all the instrumentation to determine the three-dimensional structures of macromolecules, and includes over a dozen computer graphics workstations which can be used by trainees supported on this grant after appropriate training.

This facility includes two Rigaku RU2000 rotating anodes and Siemens X1000 and High Star Area Detectors. Liquid nitrogen crystal cooling is available for both data collection instruments. A recently successful NIH Shared Instrumentation Grant (Almo-PI) will allow for the acquisition of an R-Axis IV image plate detector, new generator and associated optics.

We also operate two beamlines at the National Synchrotron Light Source at Brookhaven National Laboratory on Long Island. The close proximity of this premiere facility provides the opportunity to collect data of the highest quality and resolution. The tunability and high flux of this source (~100-1000 times higher than conventional laboratory sources) allows for the most challenging structural problems to be addressed.

Laboratory for Macromolecular Analysis
Director: Ruth Angeletti 

The role of the Laboratory for Macromolecular Analysis (LMA) is to provide state-of-the-art protein analysis and synthesis to the AECOM research community, to help develop faculty programs to keep us at the forefront of these applications, and to plan and implement new technologies. Protein identification is carried out by mass mapping and database searches for sequences represented in databases. De novo high sensitivity protein sequencing is carried out by a combination of Edman chemistry and mass spectrometry. Enzymatic or chemical digests can be performed to identify proteins by mass mapping/database searching or to obtain internal sequence data. Two-dimensional gel analysis with immobilized pH gradients provides an interface to mass spectrometry for "proteomics" studies. Mass spectrometry is used to identify posttranslational modifications and disulfide bond pairs, as well as to verify the structure of mutant or recombinant proteins. Structures of carbohydrates and small molecules can also be determined. Electrospray ionization (ESI), matrix assisted, laser desorption ionization time-of-flight(MALDI-TOF), and ion trap mass spectrometers are available. The ESI mass spectrometers are interfaced to capillary HPLC to facilitate protein and peptide analysis. A nanospray device also assists in high sensitivity analysis of protein digests. Synthetic peptides for use as peptide antigens, enzyme substrates and affinity purifications are routinely prepared. Peptides can be made with posttranslational modifications such as phosphate and myristoyl groups, or with intact disulfide bonds. Biotin, fluorescent or photosensitive labeling groups can also be incorporated into peptides, as well as groups which can be easily radiolabeled. Longer peptides for structural studies can be made for specific research projects. The structures of allsynthetic peptides are verified by mass spectrometry. Analysis of solution structure parameters can be carried out with circular dichroism spectropolarimetry, biosensor technology, and limited hydrogen-deuterium exchange experiments. Student usage of instruments is facilitated by staff available to train them in use of mass spectrometers and student use of these instruments is common.

Densitometric Gel Electrophoresis Analysis Facility
Director: Michael Brenowitz

An image analysis facility has been established through the combined resources of an NIH SIG grant, the Cancer Center of the College of Medicine, and the NCRR supported synchrotron Center. Full support for footprinting research includes gel running, drying and image analysis. This facility includes optical and phosphor storage scanners and numerous computer workstations. This facility is available to all the trainers of this proposal and their students. Dr. M. Brenowitz provides overall supervision for this facility.

The W. M. Keck Foundation Biomolecular Laser Research Center housed in the Department of Physiology and Biophysics, contains ten different laser based work stations devoted to specific spectroscopic or kinetic measurement. The spectroscopic capabilities include Raman, fluorescence and absorption. Both steady state and time resolved measurements are possible. Femtosecond, picosecond and nanosecond lasers allow for a wide range of time resolved measurements. Available laser wavelengths extend from 200 nm to the mid-infrared. The combination of wavelength tunability and time resolution provides the necessary optical tools to probe protein structure and structural dynamics in a coherent and comprehensive fashion. The facility is well equipped with the necessary spectrometers, multichannel detectors, optical components, electronic devices, and cryostats to insure that a very wide range of laser based biophysical measurements can be carried out in a routine manner. Student use of the facilities is common.

The laser lab consists of argon and krypton mainframe cw lasers, one argon five watt continuous laser, a Spectra Physics model GCR-150 YAG laser (10 Hz), two Raman difference spectrometers with multichannel (CCD) detection, a Nicolet 760 FT-IR spectrometer, a SPEX fluorescence spectrometer, and a laser induced temperature jump nanosecond relaxation spectrometer system.

The Raman difference spectroscopic system in our lab can discern differences between two spectra at the level of one part in 1000 or better. We believe that these instruments are the most sensitive difference spectrometers anywhere. The Nicolet 760 FT-IR spectrometer has recently also been fitted with sample holders for difference spectroscopy, and it too can now perform IR difference spectroscopy at a level of one part in a thousand. Through the use of molecular editing procedures that have been developed in our lab, the vibrational (Raman and IR) spectra of specific molecular bonds within quite large macromolecular systems have been discerned. For example, the stretch vibrations of a specific phosphate group inside a 60 kDa protein have been measured with over 10/1 signal to noise.

In the laser-induced temperature jump apparatus, mid IR (1.54 microns for H20 and 1.9 microns for D20) light, produced by Raman shifting YAG laser light, is focused into water with a spot size of about 500 mm. Temperature jumps of 20-30 C within the irradiated volume are achieved with 100 mJ of power under these conditions. The temperature of the irradiated volume decays (by thermal diffusion) to ambient temperature with a half-life of about 30 msec depending on geometry. The change in sample temperature is monitored in real time by measurement of slightly sensitive water absorbance changes of near IR light produced by a diode laser. Structural changes in biological macromolecules are measured using uv-vis absorbance and/or fluorescence changes of chromophoric marker bands, also in real time. This approach measures changes with high precision because the zero value is determined very accurately by the signal before the Tjump is initiated. The resolution of the Tjump apparatus is about 10 nsec.

Rapid Kinetics Facility
Director: Syun-Ru Yeh and Denis L. Rousseau

The Rapid Kinetics Facility provides instrumentation for the study of rapid biological reactions. It consists of an integrated rapid mixing system that will allow stopped flow, continuous flow, freeze quench and chemical quench experiments to be carried out. In the stopped or continuous flow modes reactions can by studied by absorption, fluorescence, Raman scattering and circular dichroism. In the chemical and freeze quench modes reactive intermediates can be trapped and then characterized by electron paramagnetic resonance, nuclear magnetic resonance, or other spectroscopies. In the rapid-mix continuous flow modes, novel sub-millisecond mixing experiments are possible. The instrumentation is available to the Einstein scientific community and is part of the NIH supported Training Program in Molecular Biophysics. In general, the instrumentation will be applied to the study of protein and ribozyme folding, several enzyme substrate interactions, ligand binding in hemoglobins, protein-DNA interactions and proton translocation. The instrumentation is housed in the laboratory of D. L. Rousseau and S.-R. Yeh and maintained by them.

The Structural NMR Resource provides the faculty, postdocs, and students of Albert Einstein with state of the art facilities for the analysis of molecular structure, dynamics, and interactions by NMR. The Resource consists of two new 300 and 600 MHz spectrometers with five off-line graphics workstations, providing the instrumentation, computational resources, and expertise for the determination of molecular structures and dynamics by nuclear magnetic resonance techniques. Small molecules are routinely analyzed on the 2 channel 300 MHz instrument. Multinuclear 5 mm (quad - ¹H¹³C¹⁹F³¹P and inverse broadband) and 10 mm broadband probes are available. Macromolecular structure and dynamics are studied using the 600 MHz spectrometer. This 4-channel system can simultaneously manipulate ¹H¹³C¹⁵N and ³¹P spins in isotopically labeled samples in 1D through 4D experiments. Excellent solvent suppression and artifact elimination are provided by x,y,z-axis pulsed field gradients.

NMR data are processed and analyzed on five offline UNIX workstations. These graphics workstations are also used for the calculation, simulation and visualization of molecular structures. An array of NMR data processing, molecular simulation, and molecular graphics programs are available. The Resource is located in the Ullmann basement (B-08): The College of medicine is also part of the New York Structural Biology consortium, that will have access to 800 MHz NMR machines within the next year.

Physiological NMR Imaging Facility
Director: Linda Jelicks

The Physiological NMR Facilty houses three vertical bore NMR spectrometers: a Varian XL200, a Varian VXR 500, and a GE Omega 400 WB. The 200 MHz and 500 MHz Varian spectrometers are equipped with a variety of 5 and 10 mm broad band and 1H/19F probes which can be used to study isolated perfused tissues and cells in addition to tissue extracts and purified molecules. The 200 MHz spectrometer also has a 16 mm broad band probe capable of accommodating perfused rat hearts. The 400 MHz GE Omega spectrometer is equipped with 20 mm broad band and 1H/19F probes for spectroscopy studies of isolated perfused tissues, a microimaging accessory capable of accommodating mice and small rats, and a gating box used for cardiac gating of acquisitions. This system has 40 mm 1H and 1H/31P imaging coils and a 35 mm 1H imaging coil custom designed to permit imaging of rat brain. This spectrometer is interfaced to a Ponemah Physiology Suite and Gould amplifier that permits monitoring of electrocardiogram and interfaces to the cardiac gating box. The physiology system is internet connected for ease of data transfer. The system can also be used for respiratory gating in vivo and monitoring of physiological function of in vitro perfused organs. A new inhalation anesthesia apparatus and the perfusion apparatus for rat heart and mouse heart studies are available in the facility.

Pulsed EPR Spectroscopy
Director: Jack Peisach

The Biotechnology Resource in Pulsed EPR Spectroscopy, directed by Professor Jack Peisach, is housed in room G18 Forchheimer Building. It is an NIH funded facility that provides the investigator with a means for measuring hyperfine interactions between magnetic nuclei such as ¹H, ²H, ¹³C, ¹⁴N, ¹⁵N, ³¹P and ²³Na and paramagnetic centers encountered in biological samples, including Cu²⁺, Fe³⁺, Co²⁺, Mn²⁺, Ni³⁺, free radicals and iron-sulfur clusters, through the use of electron spin echo (ESE) envelope modulation techniques and electron-nuclear double resonance ENDOR spectroscopy. For both ESE and ENDOR, one can relate the observed spectroscopic splittings to the identity of the nuclei giving rise to the effect and can quantify the magnetic coupling between those nuclei and the paramagnetic center. These coupling parameters provide a means for characterizing ligand bonding and in some cases, the distance of a particular ligand nucleus from the paramagnetic center and the relative orientation of a ligand molecule with respect to a structural component of the paramagnetic center. In this way, data forthcoming from frozen solution samples provide information normally obtained from single crystal X-ray measurements. ESE envelope modulation studies provide additional information concerning ligand group identity and the number of such ligands bound. Therefore, these studies can yield information concerning the structure surrounding a paramagnetic center and how that structure is altered by chemical or biochemical processes. Linear electric field effect (LEFE) measurements using the ESE technique can provide information concerning crystal field symmetry for paramagnetic metalloproteins and transition metal model compounds.

ESE envelope modulation studies of metalloproteins, metal-drug complexes, transition metal model complexes, and radical species of biological importance, including single crystal studies are carried out at the Resource. Questions being addressed concern the ligation structure surrounding paramagnetic metal centers in these systems and how those structures change when various substrates or inhibitors are added. Model compound studies have focused on providing the framework for interpreting protein data and understanding ESE envelope modulation data from first principles. The development of computer software for analysis of these data and the development of pulsed EPR instrumentation to enhance sensitivity for the study of biological materials are also primary functions of the Resource.

The Resource includes a pulsed EPR spectrometer for performing ESEEM and pulsed ENDOR experiments in the range of 8-15 GHz excitation frequency and at temperatures spanning 300-1.2 K. CW spectrometers include a Varian E-112 (300 - 4 K), a Varian E-109 (300-77 K) and a Bruker ER 200 D with an EN-810 ENDOR accessory (300-4 K). (Construction of a high frequency (130 GHz) CW and pulsed spectrometer has begun.)

Computational Facilities Available for Biophysics Research
Director: Steve Schwartz