Spectroscopy and Biophysics


Javier Seravalli, PhD
E157 Beadle Center

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The role of the Spectroscopy and Biophysics Core is to provide instrumentation, training and support for any type of experimental work involving biophysical and spectroscopic measurements. Although most of the instruments listed below are configured for use on small molecule and protein characterization, the services that our core provides can be extended to partially purified proteins, whole cell assays and analysis of tissues. The instrumentation is located in rooms E155 and N113B of the Beadle Center and includes an Inductively Coupled Plasma Mass Spectrometer (Agilent 7500 cx), an HPLC with diode array detector (Agilent LC1200), a 96-well plate autosampler (Elemental Scientific Inc), a Stopped Flow rapid kinetics instrument (Hi Tech) with fluorescence and Diode Array Detection, a differential scanning calorimeter (Microcal), an isothermal calorimeter (Microcal) and a Spectrofluorimeter (Varian, Cary Eclipse) plus a mini SPR from Forte Bio (Blitz).

Microcal Differential Scanning Calorimeter and ancillary computer.
Services Offered by the RBC Spectroscopy Facility:

  • Determination of protein stability and thermodynamic parameters for protein conformational changes by means of differential scanning calorimetry (DSC). During a DSC experiment, the temperature of the sample cell and a reference cell is ramped up - usually in a linear fashion, and the difference in heat that is necessary reach that 

  • temperature is continuously monitored. The heat measurement is translated into enthalpies of transitions and heat capacities.

Microcal Differential Scanning Calorimeter and ancillary computer.
  • Ligand-protein and protein-protein binding studies by means of isothermal microcalorimetry. A titrator syringe adds a concentrated solution to a sample of protein. The heat evolved or taken during the binding process or a chemical reaction is monitored at a constant temperature set by the user. After correction for heat of dilution of the titrant the enthalpy of binding can be accurately estimated. Studies at several temperatures allow the measurement of entropies and free energies ligand-macromolecule interactions and macromolecule-macromolecule interactions such and protein-protein and protein-DNA complexes.

  • Stopped flow spectrophotometry. Rapid mixing can be achieved in 0.2 milliseconds. Measurements in absorbance, fluorescence and chemiluminescence are routinely performed with detection in single wavelength and diode array (512 nm per scan) configurations. Accessories included are double mixing experiments, a chemical quench 6 port valve (Q-pod) and nitrogen gas attachments for anaerobic experiments.

Hi-Tech Stopped flow
Hi-Tech Stopped flow showing the sample handling unit, monochromator, photomultiplier power supply and diode array detector.
  • Standard Spectrofluorimeter equipped with up to 6 cuvets, a 96-well plate holder and a Peltier thermostated single cuvet holder for fluorescence, phosphorescence and chemiluminescence experiments in spectral and kinetic modes.

Cary Eclipse Spectrofluorimeter
Cary Eclipse Spectrofluorimeter with 96-well plate holder in place.
  • Elemental Analysis by means of Inductively Coupled Plasma Mass Spectrometry. Almost any element of the periodic chart can be determined down to the parts per trillion (ng per kg or picomolar) concentration except C,H, O, N, F, Cl and noble gases. Samples can be as small as 100 microliters and can be in either liquid or solid states. An HPLC unit is coupled with this instrument which allow the separation of elements in distinct chemical environments (chemical speciation) or the reduction of interferences by reverse phase or ion chromatography. In addition, isotope ratio and spectral semi-quantitative analysis can be performed.

Agilent ICP-MS 7500cx
Agilent ICP-MS 7500cx with the autosampler.
Beckman Analytical Ultracentrifugation
Beckman Analytical Ultracentrifugation
  • Analytical Ultracentrifugation. Determination of the protein quaternary structure of proteins using equilibrium analytical ultracentrifugation of protein solutions in water and deuterium oxide.

JASCO CD spectrometer Model J815
JASCO CD spectrometer Model J815
  • This spectrophotometer is routinely used to record Circular Dichroism (CD) spectra of Biomolecules in solution, but it can also be used for studies of protein, peptide and protein/ligand complex stability, rapid (stopped flow) conformational changes, fluorescence excitation and emission spectra, fluorescence-detected CD, fluorescence polarization/anisotropy and time and/or temperature dependent studies of these phenomena.  The instrument is equipped with detection between 167 and 900 nm, monochromators for both excitation (polarized light) and emission, Peltier thermal sample holder (10 to 90 C) and a stopped flow for monitoring rapid CD-changes.

    Sciex 4000 QTrap
    Sciex 4000 QTrap Triple Quadrupole Mass Spectrometer
  • This mass spectrometer can operate in 12 different scanning modes for structural and quantitative workflows. The first and third quadrupoles have a resolution of > 0.4 Da with scan rates up to 4000 m/z units per second (Trap mode). The instrument is equipped with the standard ESI, APCI and nanoSpray nanosources. It is interfaced with 2 HPLC stacks from Agilent: an LC-1200 series with column thermostated compartment, thermostated capillary Autosampler with 96-well plate capability and capillary quad-pump; an LC-1200 series HPLC with standard binary pump, thermostated autosampler and diode-array detector.

Recent Publications:

Oseeyi I. Daudu, Kaylen R. Meeks, Lu Zhang, Javier Seravalli, John J. Tanner, and Donald F. Becke. ACS Omega, 2023. Jan 2023. doi.org/10.1021/acsomega.2c07788. Functional Impact of a Cancer-Related Variant in Human Δ1‑Pyrroline-5-Carboxylate Reductase-1.

Eva Nývltová, Jonathan V Dietz, Javier Seravalli, Oleh Khalimonchuk, Antoni Barrientos. Nat.Commun., 2022, Jun 24;13(1):3615. doi.org/10.1038/s41467-022-31413-1. Coordination of metal center biogenesis in human cytochrome c oxidase.

Sagar M. Patel, Javier Seravalli, Kyle M. Stiers, John J. Tanner, Donald F. Becker. Amino Acids, 2021 Dec; 53(12):1863-1874.doi.org/10.1007/s00726-021-03095-4. Kinetics of human pyrroline-5-carboxylate reductase in L-thioproline metabolism.

Yizi Mao, Javier Seravalli, Thomas G Smith, Martha Morton, John J. Tanner, Donald F. Becker.Biochemistry, 2021, 30;60(47):3610-3620. doi.org/10.1021/acs.biochem.1c00625. Evidence for Proline Catabolic Enzymes in the Metabolism of Thiazolidine Carboxylates.

Jonathan Herskovitz, Mahmudul Hasan, Jatin Machhi, Insiya Mukadam, Brendan M Ottemann, James R Hilaire, Christopher Woldstad, JoEllyn McMillan, Yutong Liu, Javier Seravalli, Anandakumar Sarella, Howard E. Gendelman, Bhavesh D. Kevadiya. Nanotheranostics. 2021 5(4):417-430. doi.org/10.7150/ntno.59568. eCollection 2021. Europium sulfide nanoprobes predict antiretroviral drug delivery into HIV-1 cell and tissue reservoirs.

Sagar M. Patel, Javier Seravalli, Xinwen Liang, John J. Tanner, Donald F Becker. Arch Biochem Biophys. 2021 30;703:108852. doi.org/10.1016/j.abb.2021.108852. Epub 2021 Mar 24. Disease variants of human Δ 1-pyrroline-5-carboxylate reductase 2 (PYCR2).

Lise Pingault, Nathan A Palmer, Kyle G Koch, Tiffany Heng-Moss, Jeffrey D Bradshaw, Javier Seravalli, Paul Twigg, Joe Louis, Gautam Sarath Int.J.Mol.Sci. 2020 21(21):7966. doi.org/10.3390/ijms21217966. Differential Defense Responses of Upland and Lowland Switchgrass Cultivars to a Cereal Aphid Pest.

Sagar M Patel, Thomas G Smith, Martha Morton, Kyle M Stiers, Javier Seravalli, Stephen J Mayclin, Thomas E Edwards, John J Tanner, Donald F Becker. Biochemistry 2020 Sep 15; 59 (36):3285-3289. . Cautionary Tale of Using Tris(alkyl) phosphine Reducing Agents with NAD +-Dependent Enzymes.

Kyle G Koch, Nathan A Palmer, Teresa Donze-Reiner, Erin D Scully, Javier Seravalli, Keenan Amundsen, Paul Twigg, Joe Louis, Jeffrey D Bradshaw, Tiffany Marie Heng-Moss, Gautam Sarath. Front.Plant.Sci. 2020, Jul 30;11:1145. doi.org/10.3389/fpls.2020.01145. Aphid-Responsive Defense Networks in Hybrid Switchgrass.

Bohan Zhang, Dmitriy I Podolskiy, Marco Mariotti, Javier Seravalli, Vadim N Gladyshev. Aging Cell 2020 May;19(5):e13119. doi.org/10.1111/acel.13119. Epub 2020 Apr 23. Systematic age-, organ-, and diet-associated ionome remodeling and the development of ionomic aging clocks.