Principal Investigator Deborah Pheasant
The Biophysical Instrumentation Facility houses instrumentation for aiding in the elucidation of macromolecular structure. The facility was established by Dr. Barbara Imperiali and is located in the Dreyfus Chemistry Building (18-511). It is largely used by researchers in Chemistry, Biology and Biological Engineering but is available to anyone on campus and beyond. It is staffed 40 hours a week by Debby Pheasant who is available to train new users and provide advice to anyone considering potential applications of the equipment in the facility.
Beckman XL-I Analytical Ultracentrifuge -- Analytical ultracentrifugation is a classical method of biochemistry and molecular biology. Because analytical ultracentrifugation relies on the principal property of mass and the fundamental laws of gravitation, it has broad applicability. It is a primary technique requiring no standards for comparison. Sedimentation can be used to analyze the solution behavior of nearly any molecule over a wide range of solute concentrations and in a wide variety of solvents. Thus, while low concentration regimes are of interest for analyzing tight associations, the ability to characterize the thermodynamic behavior of a macromolecule at high concentrations makes ultracentrifugation a good adjunct for drug formulation studies, NMR, or crystallography. Add to these merits the fact that sedimentation is nondestructive, rapid, and simple, and it is easy to see why it has endured for more than 70 years.
Analytical ultracentrifugation provides two complementary views of solution behavior. Although the same instrument is used, different experimental protocols are employed. Sedimentation velocity provides first-principle, hydrodynamic information about the size and shape of a molecule, whereas sedimentation equilibrium provides first-principle, thermodynamic information about the solution molar mass, association constants, stoichiometries, and solution nonideality. For many questions, there is no substitute method of analysis.
Circular Dichroism Spectrometer -- Circular dichroism (CD) can monitor changes in the conformation of biopolymers and is used mainly for studying changes in the secondary and tertiary structure of proteins.
There are two requirements for a molecule or group of atoms in a molecule to exhibit a circular dichroism (CD) spectrum. The first is the presence of a chromophore—i.e., a group that can absorb radiation by virtue of the electronic configuration of its resting or ground state at room temperature. The energy absorbed results in a transition to a higher-energy or excited state, which has a different distribution of electrons around the nucleus. It can therefore interact with its environment in a way that differs from the ground state. In proteins, tryptophan, tyrosine, and phenylalanine are the main chromophores in the near-UV (240- to 320-nm) region; the peptide bond is the main chromophore in the far-UV (180- to 240-nm) region. Disulfide bonds and histidine residues are two other chromophores whose contribution to CD are, in general, less marked. Most chromophores exhibit more than one transition and their spectra are a composite of several absorption bands. It is an important feature of CD that, unlike optical rotation, the wavelength region within which spectra are observed is limited strictly to the wavelength region of the individual absorption bands…
The second requirement for CD is that the chromophore be in, or closely associated with, an optically asymmetric environment. The chromophores in proteins are themselves not chiral and exhibit no optical activity. The phenolic group of tyrosine, for example, exhibits a CD spectrum only because it is connected to an optically asymmetric carbon atom. However, when the same group is packed in a folded protein in an environment that is asymmetric with respect to polarity, or is interacting through the phenolic hydroxyl, it exhibits a different CD spectrum that is specific to the particular environmental influences acting on the chromophore.
In both the far- and near-UV regions, CD spectra can be used empirically as "fingerprints" of a particular protein, with the spectrum resulting from the aromatic residues being rather more specific and hence diagnostic. The far-UV spectra, however, can provide information about the protein conformation in terms of its secondary structure. As for fluorescence spectroscopy or any spectroscopic method, the sample needs to be chemically pure and homogeneous.
Microcal Isothermal Titration Calorimeter -- The VP-ITC Unit directly measures heat evolved or absorbed in liquid samples as a result of mixing precise amounts of reactants. A spinning syringe is utilized for injecting and subsequent mixing of reactants. Spin rates are user selectable.
Sample and reference cells are accessible for filling and cleaning through the top of the unit. The sample cell is on the right as one faces the front of the unit.
A pair of identical coin shaped cells is enclosed in an adiabatic Outer Shield (Jacket). Access stems travel from the top exterior of the instrument to the cells. Both the coin shaped cells and the access stems are totally filled with liquid during peration. This requires approximately 1.8 ml per cell even though the working volume of the cell is only 1.4 ml.
Temperature differences between the reference cell and the sample cell are measured, calibrated to power units and displayed to the user as well as saved to disk. The data channel is referred to as the DP signal, or the differential power between the reference cell and the sample cell. This signal is sometimes referred to as the "feedback" power used to maintain temperature equilibrium. Calibration of this signal is obtained electrically by administering a known quantity of power through a resistive heater element located on the cell.
Microcal VP-Differential Scanning Calorimeter -- The VP-DSC is a differential scanning microcalorimeter. The reference and sample cells are fixed in place lollipop shaped vessels with effective volumes of approximately 0.5 ml. The cells are constructed from a tantalum alloy and protrude out of the adiabatic chamber via their 1.5 mm (inner diameter) access tubes. The approximate volume of each cell stem is 0.085 ml.
The cell material (Tantaloy 61™) has chemical properties quite similar to glass. This alloy is immune to attack by almost all acids, having been tested under widely varying concentrations/temperatures involving chlorine, bromine, hydrochloric acid, nitric acid and sulfuric acid. Please refer to the dedicated manual outlining the chemical properties of Tantaloy 61™ for more detailed information.
The VP-DSC operates in the temperature range of -10° to 130° Celsius. It is capable of both upscanning and downscanning with no external heating/cooling source. Scanrates are user selectable and fall in the range of 0°/hr to +90°/hr in the upscan mode and 0°/hr to -60°/hr in the downscan mode. The VP-DSC can also be used at constant temperature for long periods of time (Isoscan) for shelf life studies and/or for evaluating the stability of drug or other chemical formulations. The duration of such a study is limited only by the available space on the PC hard disk.
DynaPro Titan Dynamic Light Scatterer -- The sample is illuminated by a semi-conductor laser of ~830 nm wavelength. The laser light passing through the cuvette is guided into a beam dump. An interlock switch automatically disables the laser if the MicroSampler lid is opened when the laser is operating. The light scattered by the sample in the cuvette is collected and guided via a fiber optic cable to an actively quenched, solid state Single Photon Counting Module (SPCM). The photons are then converted to electrical pulses and correlated.
The DynaPro Titan analyzes the time scale of the scattered light intensity fluctuations by a mathematical process called autocorrelation. To perform the very fast data manipulation necessary to obtain results in real time, the DynaPro Titan uses the latest generation of correlator running special algorithms. The translational diffusion coefficient (Dt) of the molecules in the sample cuvette is determined from the decay of the intensity autocorrelation data. The hydrodynamic radius (Rh) of the sample is then derived from Dt using the Stokes-Einstein equation.
The DynaPro Titan determines the uniformity of sizes through a monomodal (single particle) curve fit analysis called cumulants, which assumes a single particle size with a gaussian distribution. The SOS is the Sum Of Squares difference between the measured and the cumulants calculated intensity correlation curves, and is reported for each sample acquisition (a single correlation curve) in the Datalog Grid View of DYNAMICS. Low SOS values (<20) indicate reasonable agreement between the measured correlation curve and the cumulants fitted curve, and suggest the sample is likely monomodal with low polydispersity, i.e. a tight size distribution.
Perkin Elmer HTS 7000 Bio Assay Reader (Fluorescence Plate Reader) -- The HTS 7000 Bio Assay Reader is a versatile, optimized system for rapid, high-throughput screening of a large array of small-volume samples. It can read any plate type in any format from 6 to 384 wells, using luminescence (fluorescence, chemiluminescence, bioluminescence) from the top and bottom of wells or through-well absorbance down to 260 nm. Plates can be heated up to 40°C and shaken in orbital or linear modes.
Capillary Electrophoresis -- Capillary electrophoresis (CE) is a family of related techniques that employ narrow-bore (20-200 µm i.d.) capillaries to perform high efficiency separations of both large and small molecules. These separations are facilitated by the use of high voltages, which may generate electroosmotic and electrophoretic flow of buffer solutions and ionic species, respectively, within the capillary. The properties of the separation and the ensuing electropherogram have characteristics resembling a cross between traditional polyacrylamide gel electrophoresis (PAGE) and modern high performance liquid chromatography (HPLC).