Home > Research > NIBIB Intramural Labs > Laboratory of Bioengineering and Physical Science

Molecular Interactions

Contents

• Specialized Instrumentation
• Computational Analysis Capabilities
• Initiating New Collaborations and Projects
  • Concentration
  • Buffer
  • Purity
  • Volume
  • What else do we need?
• Meet the MI Staff

The Molecular Interactions (MI) group provides collaborative research support in the biophysical characterization of macromolecules and their reversible interactions. The major techniques and areas of expertise are analytical ultracentrifugation, light scattering and calorimetry. Besides performing collaborative research, we develop and share methodology for advanced experimentation in analytical ultracentrifugation and light scattering. The purpose of our work is the characterization of macromolecules with respect to their mass and hydrodynamic shape, and the in-depth characterization of their reversible interactions, in terms of binding stoichiometry, kinetics, and particularly thermodynamics. The reliable determination of these parameters frequently reveals information on the structure/function relationship of the macromolecules. Examples for interactions that have been studied include a number of protein-protein, protein-peptide, peptide-peptide, protein-nucleic acid, receptor-ligand, and antigen-antibody interactions.

• Two analytical ultracentrifuges with absorbance and interference optics (Beckman Optima XL-A and XL-I)
• An isothermal titration microcalorimeter (Microcal)
• A dynamic light scattering instrument (DynaPro, Protein Solutions)
• A Minidawn multi-angle light scattering instrument for SEC-MALLS (Wyatt)
• A CD spectrophotometer (Jasco)

These instruments enable us to:

• Perform analytical ultracentrifugation: This allows us to characterize macromolecules in solutions and measure the binding constants of reversible interactions in sedimentation equilibrium that range, on average, between 50 nM and 1 mM, although specific cases permit exceeding these limits. This solution method is based on first thermodynamic principles. The hydrodynamic shapes of proteins and their complexes can be measured using the sedimentation velocity technique.
• Measure diffusion coefficients via analysis of the fluctuations using dynamic light scattering: This solution method is also based on first principles. With our instrument, we can measure translational diffusion coefficients of proteins in small sample volumes, which gives rapid information on the state of aggregation of the macromolecules. New instrumentation nearing functional applicability offers the same capabilities as equilibrium ultracentrifugation, but offers the advantage of much greater speed, which is critical when sample thermal stability is a problem.

The combination of these techniques can be very powerful since they provide complementary information on the macromolecules. Our objective is not only to continuously refine experimental techniques presently used to provide the most reliable analyses possible, but also to add new techniques in order to increase the complexity of the systems that can be investigated. In addition to providing collaborative services in molecular interactions, MI has ancillary instrumentation, such as an Anton-Paar densitometer, for the performance of high-precision solution density measurements.

Back to top

Computational Analysis Capabilities

Computational analyses of genomic DNA sequences can be performed to predict the location and strength of acceptor and donor sites for pre-mRNA splicing. Effects of several types of mutations can be calculated using an information-theory based model. Alternative splicing results can be calculated in both wild type and mutated sequences. Strengths of binding to protein sites can be calculated, as well.

Data required for analyses

We need genomic DNA sequences encompassing the regions of interest, preferably in GenBank format. Other formats can be processed, but generally require more time and effort to use. The actual exon structure is desirable, if available. If for some reason limited data are available, it is important to realize that at least 26 bases of intron are required for analysis of acceptor sites (-25 to +2 with coordinate zero being the first base on the intron side of the junction) and 7 bases of intron are required for analysis of donor sites (-3 to +6 with coordinate zero being the first base on the intron side of the junction). In general, it is best to provide all of the known sequence.

Back to top 

Initiating New Collaborations and Projects: Analytical Ultracentrifugation Sample Preparation Considerations

• Concentration
• Buffer
• Purity
• Volume
• What else do we need?

Important Note: Before preparing and sending any materials, the exact composition, volume, and concentration of the samples should be discussed with us.

Back to top 

The analytical ultracentrifuge (AUC) is basically a dual-beam spectrophotometer integrated into an ultracentrifuge. Therefore, the sample concentration required can be most easily specified in terms of the absorbance of the sample. For example, experiments with proteins would make use of the 280 nm absorbance of the aromatic amino acids, and protein concentrations giving absorbancies of between 0.1 OD and 1 OD at 280 nm (or up to 1 OD at 250 nm) would be required. If there are no substances in the sample that absorb in the far UV at 230 nm (such as DTT, Hepes, etc.), then the measurement can be performed at 230 nm, increasing the sensitivity approximately five-fold. It is generally better to send more concentrated protein, which we can dilute as needed into larger volumes. Protein-nucleic acid interactions are best studied by multi-wavelength analysis between 230 nm and 246 nm. Appropriate labeling techniques are optimal for studies of protein-peptide interactions.

Back to top

Buffer

There are two considerations with respect to the buffer: First, it should be transparent at the wavelength that is to be used for detection. For example, Hepes absorbs at 230 nm and cannot be used as a buffer in this range. At 280 nm, it will be fine, though the best buffers in terms of UV transparency are, in our experience, phosphate buffer (e.g., PBS) and acetate. These work well, as long as there are no other additives that might absorb (e.g., DTT, or other protease inhibitors) in the buffer. It is very easy to check the suitability of the buffer with a spectrophotometer by scanning from 230 nm to 320 nm.

Detergents are, in general, no problem as long as they do not absorb at critical wavelengths. Sucrose is problematic, and glycerol is even more troublesome. Although they don't absorb, they create major problems since they markedly increase the viscosity of the solution, and therefore, may increase the experimental time beyond reasonable limits. If glycerol is mandatory for the stability of a given protein, the glycerol concentration should not exceed 2%. The second consideration is the density of the buffer.

What we measure in the concentration profiles is the buoyant molar mass of the proteins. According to the principle of Archimedes, we have to know the density of the buffer. While, for a large number of buffers and salts, the density increments are very well known, and we can calculate the buffer density (e.g., for phosphate, most common salts, etc.). Our preference is to measure buffer densities with the Anton Paar density meter, which is both very rapid and exceptionally precise over the temperature range of interest. Ill-defined buffers obtained with a column eluted with a gradient of solutes may present significant problems.

Back to top

This is usually a critical factor. Since we are measuring absorbance, all impurities that absorb will contribute to the signal. Particularly problematic are small molecule impurities (a couple of thousand Da or larger, such as small peptides), which because their presence can be difficult to distinguish from buffer absorbance, can lead to highly misleading data interpretations. Since gel electrophoresis frequently does not detect these small molecules, and since the ability to stain is not necessarily a good measure for the concentration of impurities, gel electrophoresis is not sufficient as a control for sample purity in AUC. The best, and generally, the most reliable, is the use of size-exclusion chromatography as the last step in the sample preparation. This separates the protein to be studied from small molecules and from large aggregates, and it is a simple way of changing the buffer to one suitable for UV absorbance measurements.

Back to top

Volume requirements depend on the experiment. Usually, most of the sample can be recovered. The minimal volume that can be used in any AUC experiment is 30 µl. The sample volume used in a single-band centrifugation experiment gives information about heterogeneity and sedimentation coefficient (hydrodynamic shape). In general, the most informative experiments are sedimentation equilibrium experiments with 5 mm columns that require 180 µl per single experiment. Usually, it is necessary to run multiple experiments at different loading concentrations (this is crucial for interacting systems), and we prefer to make use of all 7 positions in the rotor. Therefore, 500 µl at fairly high concentration (absorbance) is convenient, and a reasonable minimum is probably 200 µl.

Back to top

If possible, we need the amino acid composition (preferably in electronic form) for calculation of the partial specific volume of the proteins. Also, we need at least 10 ml additional buffer for dilution of the protein in order to use it as an optical reference in the double sector cells. If the measurement of the solution density is necessary, 25 ml of buffer is desirable.

Meet the MI Staff

Marc Lewis, Ph.D. - Research Chemist, Chief

James Ellis, Ph.D. - Electronics Engineer

Jacob Lebowitz, Ph.D. - Biophysical Chemist, Special Volunteer

Back to top