RUI: NMR Investigation of the Role of Protein Dynamics in the Selective Interactions between RGS and Galpha Signaling Proteins

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Intellectual Merit. Proteins are complex molecules that are the workhorses of biological organisms: they catalyze reactions, carry nutrients, provide structural support and transmit signals, just to name a few examples. The molecular structure of a protein is composed of a long string (polymer) of linked amino acids, with a variety of side chains that extend out from the backbone (similar to a charm bracelet). This string is then folded into a specifically-defined 3-dimensional structure that constitutes its functional form. Many critical signal transmission pathways are initiated by the activities of Galpha protein subunits, which are in turn controlled by regulators of G-protein signaling (RGS proteins). In particular, interactions between RGS proteins and their specific G proteins turn on and off pathways that influence brain development and function. Although much has been learned about the mechanisms of signal transmission, more research is required to obtain a thorough, molecular-level grasp of brain function. This research project focuses on two of the 20 proteins in the RGS protein family: RGS4 and RGS7. Both proteins can control the signaling of Gialpha1 (a common type of Galpha protein subunit), but the interaction between RGS4 and Gialpha1 is ~60 times stronger than that between RGS7 and Gialpha1. Normally, these differences are explained by variations in the types of amino acids that are found in the interface between the two proteins: one pair of interacting proteins may have more compatible and therefore stronger contacts than the other pair. However, when looking at the interfaces of RGS4 and RGS7 with Gialpha1, there is only one small difference between them and it cannot account for the large difference in binding preference.Other researchers have found evidence for changes in the three-dimensional structures of the protein backbones and/or the flexibility of the molecules in both RGS and Gialpha1 proteins when they interact. In addition, scientists have recently become more aware that these kinds of motions can have powerful influences over the functional capabilities of signaling proteins. This research therefore tests the hypothesis that internal movements within RGS proteins play critical roles in both their abilities to interact with various types of Galpha protein subunits and also their different degrees of preference. In order to investigate this we are using a nuclear magnetic resonance (NMR) spectrometer, which is a very powerful instrument that permits the detailed analysis of proteins; in fact, we can watch the activities of individual atoms within these molecules and thereby gain in-depth insight into their activities. The results from this work will fundamentally improve our awareness of the role of protein motions in molecular recognition and help explain the selectivity of RGS proteins for particular Galpha protein subunits, and accelerate advancements in our overall understanding of signaling mechanisms and control at the molecular level.Broader impacts. California State University, Northridge (CSUN) is a primarily undergraduate institution. Over half of the science students are from underrepresented minority groups; many students are the first in their family to attend college and are economically disadvantaged. There are few minority scientists doing protein biophysical research; this project should help to expand those numbers by drawing from the large pool of underrepresented students in the department. This project will also provide unprecedented opportunities to CSUN students to participate in cutting-edge research using modern methods and technology. For many students this will be the first (and possibly only) chance they will have to experience real research challenges and to learn troubleshooting and problem-solving skills. These new research skills will translate into increased self-confidence, improved learning in courses, and an increased likelihood that they will be inspired to pursue advanced degrees in Biochemistry or Structural Biology. Speaking more generally, a third major outcome of the project will be to illustrate to the broader public that high-quality, impactful research can be performed at a primarily undergraduate institution.