About Dmitry Fishman and UCI's Laser Spectroscopy Laboratories
Dr. Dmitry A. Fishman is as the Director of the Laser Spectroscopy Laboratories at the University of California, Irvine (UCI), a facility dedicated to research and education within the fields of light-matter interaction, linear and nonlinear spectroscopy and microscopy, and materials characterization. These laboratories have been providing expert research support and consultation to UCI scientists for over 20 years, and, as of 2017, have more than 400 registered and trained users. The laboratories assist with and provide access to optical setups for over 80 faculty members and research groups, representing 21 departments and 4 schools within UCI. In addition, the labs are currently offering services to several groups from UC Santa Barbara, UC San Diego, UC Riverside, California State University and the University of Southern California. This is a specialized central laboratory service with a large amount of varied research depending on it.
Choosing the perfect SEC-MALS detector
Numerous UCI faculty members recognize both dynamic light scattering (DLS) and static light scattering (SLS) as effective methods for investigating the oligomerization state, hydrodynamic radius, and molecular weight of biomolecules and biomimetic polymers in solution. Light scattering has proven extremely useful in investigating proteins and macromolecular complexes, enhancing the understanding of enzyme structure-function relationships, protein-protein and protein-ligand interactions, protein crystallization, and the formation of insoluble protein aggregates.
Together with a group of UCI professors, Dr. Fishman and his colleague Professor Rachel Martin were looking for a SEC-MALS (Size exclusion chromatography-multi angle light scattering) detector that would enable thorough characterization of the solution behavior and aggregation propensity of biological molecules, assist in investigations of protein-protein or protein-membrane complexes, enable fundamental studies of how pairwise interactions contribute to protein oligomerization, and also elucidate crystallization for structural biology. Despite the wealth of information known about the structures and bulk aggregation behavior of proteins, the early stages of aggregation, including its molecular driving forces, remain to be characterized.
After a thorough review of available detectors, the group at UCI concluded that the Malvern Panalytical Viscotek SEC-MALS 20 detector was the perfect solution for both protein and polymer analysis, to serve as many users of the laboratories as possible. Dr. Fishman says. “This detector measures the exact molecular weight, molecular size, and molecular structure of proteins and polymers between 1000 g/mol and 106 g/mol, and is also capable of determining the oligomeric state of proteins, with the use of size exclusion chromatography (SEC). What makes this detector different from other MALS detectors are the 20 custom photodiodes placed between 12° and 164° around the flow cell, whereas most other MALS instruments have fewer photodiodes. For this technique, access to more detection angles is important for precise measurements of the radius of gyration.”
The power and flexibility of light scattering technologies
Research of the mechanisms involved in protein aggregation and crystallization processes continues to lead to significant contributions in the research of cataracts, cancer, and the native states of membrane proteins, to name but a few areas of study. UCI’s Laser Spectroscopy Laboratories now have several Malvern Panalytical light scattering instruments: the Viscotek SEC-MALS 20, Zetasizer Nano and Zetasizer µV DLS systems, which have proved pivotal to numerous UCI research projects, including the below:
Professor Rachel Martin’s group uses the Viscotek SEC-MALS 20 to investigate the refractive indices, oligomerization states, and aggregation behavior of crystallins, the structural and refractive proteins of the eye lens. Cataract, an opacification of the lens, is caused by a loss of solubility of crystallin proteins. The World Health Organization estimates that 20 million of the 45 million people who are blind worldwide are affected by cataract.
Researchers in Professor Gregory Weiss’ group use the Viscotek SEC-MALS 20 to examine the refolding and oligomerization of proteins. This group is currently focused on examining the protein folding and aggregation dynamics of lysozyme, DNA polymerase, caveolin, and various bladder cancer biomarkers, which are proteins important to human health and disease.
Professor Thomas Poulos’ group is attempting to determine the crystal structures of components of the heme transport system, which enables gram-negative pathogenic bacteria to acquire various essential components from their host organisms. Crystallization trials in this area of study have proven challenging, but the ability provided by the Viscotek SEC-MALS 20 to determine accurate molecular weights and homogeneity is simplifying and accelerating this research.
Scientists in Professor Zhibin Guan’s group are working on functional biomaterials. They are interested in the design of biodegradable and functional polymers for biological applications, exploring new biomimetic strategies for materials design, and developing new methodologies for polymer synthesis. In each of these projects, the Viscotek SEC-MALS 20 and Zetasizer Nano are critical players, as the combination of multi-angle light scattering (MALS) and DLS elucidates the macromolecular topology for polymers made under different conditions, going beyond the capabilities of technologies such as nuclear magnetic resonance (NMR) and mass spectrometry (MS).
Professor Hartmut Luecke’s group focuses on understanding the molecular basis of urea transport by the human pathogen Helicobacter pylori, with the objective of designing new drugs targeting a pathogen-specific membrane protein. H. pylori infects about 50% of the world’s population, and is associated with several gastric diseases, including gastritis, peptic and duodenal ulcers, gastric carcinoma, and MALT lymphoma, as well as thrombocytopenic purpura. The Viscotek SEC-MALS 20 is expected to greatly assist in this research by enabling the determination of the molecular weights of protein-protein and small molecule-protein complexes of interest, as well as measuring A2 values to guide the process of finding successful crystallization conditions.
Dr. Fishman explains: “The Viscotek SEC-MALS 20 has enabled us to measure the osmotic second virial coefficient (A2) for proteins under physiologically-relevant solution conditions. In general, A2 is a measure of inter-protein interactions that can be obtained by SLS measurements as a function of concentration. A2 is quite sensitive to changing solution conditions, such as pH and ionic strength, but the Viscotek system makes it possible to investigate multiple pH values and salt concentrations for each protein in order to gain insight into the factors driving aggregation. Among the few experimental techniques for characterizing protein-protein interactions between well-dispersed proteins in solution, SLS is one of the most powerful, topped only by small-angle x-ray scattering, which requires a much more expensive system. Having the Viscotek SEC-MALS 20 in our laboratory enables us to characterize pairwise protein interactions in a way that was not previously possible.”
Working in partnership with Malvern Panalytical
In their search for a SEC-MALS detector, the UCI group, led by Dr. Fishman, considered a variety of manufacturers, directly comparing between the Viscotek SEC-MALS 20 and its closest competitors. They had previously worked with Malvern Panalytical systems, and again found that the quality of the SEC-MALS 20, in terms of both build and quality of data, and also the technical support offered by Malvern Panalytical were, as Dr. Fishman states, “an unbeatable argument in the decision-making process.” He continued, “I can say without hesitation that the level of customer support has been unprecedented. Malvern Panalytical specialists have been instrumental in our understanding of the concept of triple detection chromatography. They are constantly guiding our students and postdocs to develop experimental intuition, correct understanding of their data, and fine tuning of the experimental conditions. Their support is always available through countless phone and video conference calls, and same day email correspondence. It is no exaggeration to say that we consider them part of our research team.”
Dr. Fishman found that implementation of the detector was a quick and efficient process. “Malvern Panalytical scientists and engineers provided us with an extremely helpful 3-day workshop, which gave us efficient hands-on experience, with real measurements and data discussions. As a result of this excellent teaching, we have, in turn, been able to introduce the technique and the detector to more than 20 graduate and postgraduate students within the first year of ownership.”
Dr. Fishman continues, “It is hard to underestimate the direct effect of this technology. The Viscotek SEC-MALS 20 has made it possible to perform experiments that we could not previously undertake at our campus or at neighbouring institutions. Many research projects have finally been able to move on after few years of stagnation. As a result of this detector arriving at UCI approximately one year ago, we now have several papers in press, and several more manuscripts in progress.”