Protein Structure and Dynamics Core | Biochemistry and Molecular Medicine | UC Davis School of Medicine

Knowing the structural dynamics of the proteins and biological membranes is key to understanding their mechanism of function. Most prominent methods for structural determination are X-ray crystallography and Nuclear Magnetic Resonance Spectroscopy. However crystallized samples select for one static conformation and are difficult to obtain for biomembranes or macromolecular assemblies (such as filaments). NMR methods are difficult for larger biomolecules and membranes, and also carry large sample demands, which may be difficult to obtain.

The Site Directed Spin Labeling Electron Paramagnetic Resonance (SDSL-EPR) spectroscopy offers a unique approach to elucidate the structure and dynamics of biomolecules in solution. The approach combines protein chemistry/molecular genetics to target spin probes into biomolecules (most commonly peptides and proteins) with EPR analysis using modifications that can accommodate small biological samples under a variety of conditions (pure protein, tissue, live cells, etc.).

The Circular Dichroism (CD) spectroscopy is the most common approach for studying the overall conformation of biopolymers, such as proteins and peptides. CD is a spectroscopic method based on the interaction of chiral centers in the polymer with circular polarized light, which is very sensitive to levels of regular order in the polymer backbone. CD offers a convenient method for identifying changes in the structure of proteins and peptides.

The EPR spectroscopy and CD spectroscopy is a unique combination of physical methodology used for determining the structural dynamics and molecular structure of proteins and other biomolecules. Such information is crucial for understanding the mechanism of biomolecules and for identifying drugs and other agents that can modulate the behavior of biomolecules involved in disease. The ability to measure structure and dynamics is also important for bioengineering efforts, such as designing platforms for molecular sensing and drug delivery.

Services include screening of the optimization of samples of specific interest and screening. Both qualitative and quantitative analysis will be provided.

Circular Dichroism

• buffer exchange of sample into compatible buffer and concentration of sample if needed
• loading sample into appropriate quartz cell and collection of CD spectra. Each sample has its own quartz cell specific.
• Recording the CD spectra with optimal parameters and temperatures (if temperature dependent studies needed).
• saving the spectra and plotting of data and computational analysis of secondary structure
• preparing report of results

A single, per sample charge will apply for the CD instrument.

Non-instrument CD services:

• CD Sample Prep. This optional service will provide buffer exchange (into a sodium-free buffer) and protein concentration of user-provided samples.
• CD data analysis. This option will provide data analysis of collected CD spectra. Data will be processed using a multi-component algorithm to estimate the fraction of secondary structural elements within a protein sample.

Electron Paramagnetic Resonance Spectroscopy

• spin-labeling and concentration of samples as needed
• loading samples into capillaries (quartz and TPX)
• Optimizing the Hamiltonian parameters and temperatures (in case of LT studies).
• Recording and collection of spectra
• plotting of spectra and measurement of line widths and determining the spin parameters.
• For power saturation measurements, each sample is examined under three conditions (air, N2, and NiEDDA). Collected spectral data is computationally processed for P1/2 and collisional frequency values.

The EPR instrument services are categorized under three different service types, each with a defined recharge rate:

• Simple room temperature (RT) scan. This is the lowest rate, as scans can be obtained in a matter of minutes with little instrumentation set-up time.
• Simple low temperature (LT) scan. This rate is higher than the RT scan due to the increased time for liquid nitrogen cooling and cryostat operation. Service also includes flash freezing.
• EPR power saturation scanning. This service will provide scanning of samples over successive power levels under three conditions (relaxer-free, hydrophilic relaxer, hydrophobic relaxer). This rate is the highest as it is the most time consuming.

Non-instrument EPR services:

• EPR Sample Prep and Spin-labeling. This optional protein chemistry service will provide spin-labeling and protein concentration of user-provided samples.
• EPR data analysis. This option will provide data analysis of collected EPR spectra. For protein samples, spectra will be fit by for correlation times of mobile components. For membrane samples, line-widths will be measured to estimate correlation times and order parameters. For power saturation samples, collision frequencies will be calculated for each relaxer.

Primary Contact E-mail: msbudamagunta@ucdavis.edu

Phone: 530-752-3164

Address:

School of Medicine
University of California
4108 Tupper Hall
One Shields Avenue
Davis, CA 95616