Characterization of Internal Dynamics in Vav1: Method Development, Mutual Coupling and Functional Relevance
| dc.contributor.advisor | Rosen, Michael K. | en |
| dc.creator | Li, Pilong | en |
| dc.date.accessioned | 2010-07-12T18:50:35Z | |
| dc.date.available | 2010-07-12T18:50:35Z | |
| dc.date.issued | 2009-09-04 | |
| dc.description.abstract | Protein motions are important to activity, but quantitative relationships between internal dynamics and function are not well understood. The Dbl homology (DH) domain of the proto-oncoprotein and guanine nucleotide exchange factor Vav1 is autoinhibited through interactions between its catalytic surface and a helix from an N-terminal acidic region. Phosphorylation of the helix relieves autoinhibition. Here I show by NMR spectroscopy that the autoinhibited DH domain (AD) exists in equilibrium between a ground state, where the active site is blocked by the inhibitory helix, and an excited state, where the helix is dissociated. Across a series of mutants that differentially sample these states, catalytic activity of the autoinhibited protein and its rate of phosphorylation are linearly dependent on the population of the excited state. Thus, internal dynamics are required for and control both basal activity and the rate of full activation of the autoinhibited DH domain. Vav1 belong to a class of multi-domain signaling proteins exhibit complex behaviors due to cooperative interactions between domains. In many such proteins there is a core regulatory interaction, involving binding of an inhibitory element to the active site of a functional domain like the inhibitory helix to DH in Vav1. The core interaction is cooperatively enhanced by additional intramolecular domain-domain contacts. The physical basis of this cooperativity, and thus the energetic construction of multi-domain systems, is not well understood. Dynamics analysis of AD reveals that the closed and open populations are about 10:1 for the core interaction in isolation. In the full five-domain regulatory fragment of Vav1, interactions between domains outside of the core further bias this inhibitory equilibrium ~10-fold toward the closed state, further suppressing activity. Thus, Vav1 is controlled by two, weakly biasing, but thermodynamically coupled equilibria--an energetic construction that is probably general among multi-domain proteins. The dynamic landscape of AD is composed of two ?s-ms time scale motions: one is the inhibitory helix binding to and dissociating from the DH domain and another is intrinsic to the DH domain. Interestingly relative populations and exchange rates of the second process are altered upon perturbations to the inhibitory helix, suggesting that the two dynamic processes are energetically and kinetically coupled. A strategy has been established to quantify the thermodynamic and kinetic coupling strengths between the two processes via direction parameterization of four-state equilibria using NMR Carr-Purcell-Meiboom-Gill measurement. The coupling strengths between the two dynamic processes in AD are 1.0~1.5 kcal M-1 comparable to the coupling strength between the modulatory interaction and the helix-DH interactions in the full five-domain regulatory fragment of Vav1. The coupling strength is relatively weak consistent with the coupling strengths reported for many other signaling proteins such as Src tyrosine kinase. These findings suggest that weakly coupling may be a common theme in regulatory molecules. | en |
| dc.format.digitalOrigin | born digital | en |
| dc.format.medium | Electronic | en |
| dc.format.mimetype | application/pdf | en |
| dc.identifier.oclc | 760314139 | |
| dc.identifier.uri | https://hdl.handle.net/2152.5/689 | |
| dc.language.iso | en | en |
| dc.subject | Proto-Oncogene Proteins c-vav | en |
| dc.subject | Mutagenesis, Site-Directed | en |
| dc.subject | Protein Structure, Tertiary | en |
| dc.title | Characterization of Internal Dynamics in Vav1: Method Development, Mutual Coupling and Functional Relevance | en |
| dc.type | Thesis | en |
| dc.type.genre | dissertation | en |
| dc.type.material | Text | en |
| thesis.date.available | 2011-09-04 | |
| thesis.degree.department | Graduate School of Biomedical Sciences | en |
| thesis.degree.discipline | Molecular Biophysics | en |
| thesis.degree.grantor | UT Southwestern Medical Center | en |
| thesis.degree.level | Doctoral | en |
| thesis.degree.name | Doctor of Philosophy | en |