Solution Characterization Facility

The goal of this Facility is to provide state of the art equipment and expertise in the solution-state analysis of proteins, nucleic acids and macromolecular complexes. Our capabilities include the acquisition and analysis of sedimentation velocity and sedimentation equilibrium data from two Beckman Analytical Ultracentrifuges, which are housed in the Facility. Further, the Facility has a Jasco 720 Spectropolarimeter [Circular Dichroism (CD) Spectrometer] for the acquisition and analysis of measurements of the 2º and 3º structures of proteins and complexes. In combination, AUC and CD offer valuable information regarding the degree of folding of a macromolecule or complex, and the size, shape and aggregation state (oligomerization state) of these same types of samples.

Too often, recombinant proteins are characterized in in vitro functional assays without a thorough understanding of their solution state behavior. For example, imagine performing a kinetic analysis of an enzyme without first knowing whether the protein was appropriately folded, or if the protein was aggregated. Also, consider trying to understand the product of a complex between a DNA-binding protein and a nucleic acid, or the formation and stoichiometry of a protein-protein complex without first knowing the oligomerization state (monomer, dimer, etc) of the reactants. A rigorous understanding of the solution state properties of these reagents is essential.

The Analytical Ultracentrifuge (AUC) is the perfect union between a preparative, high-speed centrifuge and a dual-beam spectrophotometer. This union allows for detection of the movement of bio-molecules in real time. The velocity with which a molecule moves in an aqueous solution depends on its size, shape and buoyancy. We can determine a 'sedimentation velocity' (the sedimentation coefficient, a Svedberg ) for a particular particle (protein or DNA molecule, or a larger nucleo-protein complex) by subjecting it to an extremely high centrifugal force (at up to 60,000 RPM). This measurement gives very detailed information about the size and conformation of the particle or complex. Importantly, such a measurement allows for a determination of the homogeneity (purity) of the material, thus this technique is often a prelude to a crystallographic, 3-D structure determination, which requires highly pure materials. This technique will reveal if the protein self-associates, forming dimers, trimers or larger oligomers.

Alternatively, we can subject the particle or complex to a lesser centrifugal force for many hours and days, and allow its natural buoyancy to counteract the centrifugal force. When this ‘sedimentation equilibrium’ is reached, the distance the particle or complex has traveled is dependent on its’ molecular size, which is readily obtained by fitting the data to the appropriate model. Such experiments also allow for the determination of the self-association scheme of a protein, which often is an important factor in the biological function of the protein.

Practically, the sample solution is loaded into the sample cell, which allows the illumination source (absorbance, interference or fluorescence) to strike the sample while the rotor is spinning. The samples cells are inserted into a titanium rotor capable of very high-speed rotation. The rotor-cell assembly is placed in the AUC with the optical detector. The data is acquired for the appropriate time and at the appropriate speed, and the data is processed at remote computer with the analytical software installed. The software for analysis of AUC data are generally freeware, and have evolved rapidly over the past 10 years. They allow ready determination of hydrodynamic parameters, and the molecular mass can now be determined from well-behaved proteins by sedimentation velocity AUC.

In the Fall of 2005, our facility became only the second facility in the world to obtain an AUC retro-fitted with the AVIV fluorescence optical system (there are now ~10 world-wide). The optics excite the sample at 490 nm and collects emission data at >505 nm, thus is compatible with many modern fluors (Sybr Green, Green Fluorescent Protein (GFP), Alexa Fluor 488). This system will increase the detection sensitivity by at least two orders of magnitude (to low nanomolar concentrations), and allow for detection of single, labeled molecules from within complex, impure mixtures such as nuclear lysates or serum.

The Circular Dichroism spectrometer (CD) utilizes circularly polarized light over the near- and far-UV spectrum to illuminate the polypeptide chain. The angle at which left and right circularly polarized light are differentially re-oriented (Theta, θ) is indicative of the conformation of the peptide backbone, and reveals the secondary and tertiary structures of proteins. The Solution Characterization Facility houses a Jasco 720 instrument, which has an optical system which is superior to many, more modern instruments. Additionally, the Facility has dozens of sample holders of varied pathlengths, to accommodate samples of a wide range of concentration and absorbance properties.

In the lab, CD is used to determine whether a protein of interest is properly folded, or is aggregated or denatured. Many important proteins have recently been recognized as containing many and often long stretches of disordered amino acids, that is, they lack true secondary protein structures. These regions have been shown to play important roles in the function of the protein, creating a disconnect within the structure-function dogma. CD and limited proteolysis are important tools for probing not only structured domains but also unstructured domain. To collect a CD spectrum, a sample (of accurately determined concentration) is loaded into a temperature-controlled sample cuvette, and illuminated with the laser light source within the instrument. These spectra are baseline (buffer) subtracted and normalized. Prominent spectral features (peaks, both negative and positive), such as the minima at 208 and 222nm indicating the presence of a-helix, are readily seen in the resulting spectra. Shifts in the position and/or intensity of these peaks can be induced by ligand binding, conformational changes or denaturation by heat or chaotrope.

These spectra may be further deconvoluted using software such as those available though CDPro and Dichroweb. These analyses allow for the determination of the fraction of secondary structures within each sample, and will suggest what fraction of amino acids lack true secondary structure, thus are intrinsically disordered.