Biophysical Studies

Supporting biophysical studies are often invaluable both for sample characterization before starting structural studies as well as for investigating structure-function relationships after structural information is available. In addition, a plethora of questions can be addressed through biophysical studies in the absence of any structural information.

Sample characterization

Samples for structural studies need to be:

• chemically homogeneous (pure)
• structurally homogeneous
• stable
• soluble
• functionally active (ideally)

It is generally worthwhile to invest energy in the characterization of proteins expressed in heterologous systems or obtained from native tissues before embarking on structural studies. Ascertaining that samples are in fact amenable to structural studies is crucial to avoid wasting resources, time, and money in futile experiments. See below for a list of methods suitable for characterizing these aspects.

Elucidation of structure-function relationships

Once high-resolution structural information has been obtained, the fun really begins, and biophysical studies are at the heart of it all!

Structure-function-relationship studies include:

• Interactions between macromolecules
• Interactions with small molecules, such as substrates, products, inhibitors and effectors
• Folding & stability
• Protein engineering
• Spectroscopic studies
• Reaction mechanisms

Typical biophysical aspects and appropriate methods

High-end experimental equipment and expertise for biophysical studies are available in the Center for Structural Biology and/or other core facilities on the UNC-Chapel Hill campus, most notably the Macromolecular Interactions Facility (Mac-In-Fac).

Please contact the CSB director for further guidance, such as experiment planning and design as well as for the interpretation of results and the integration with structural and biological information.

Chemical homogeneity (purity), concentration determination
Electrophoresis, UV/vis absorption spectroscopy

Solubility
Light scattering, analytical ultracentrifugation, size-exclusion chromatography

Oligomeric status
Analytical ultracentrifugation, size-exclusion chromatography, dynamic & static light scattering, fluorescence anisotropy

Folding, stability
Circular dichroism (CD) spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, fluorescence spectroscopy, differential scanning calorimetry (DSC), infrared (IR) spectroscopy

Interactions
Isothermal titration calorimetry (ITC), fluorescence spectroscopy, fluorescence anisotropy, surface-plasmon resonance (SPR), analytical ultracentrifugation

Kinetics
Stopped flow, surface-plasmon resonance (SPR)

Spectroscopy
UV/vis absorption, CD, fluorescence, IR spectroscopy