Event Information
Novel Mass Spectrometry-Based Strategies for Biophysical Analysis of Proteins and Protein-Ligand Complexes
- Abstract:
Biophysical analyses of proteins and protein-ligand complexes are important for understanding how proteins fold and function in vitro and in vivo. The sensitivity and speed of mass spectrometry make it an attractive technique for protein analysis. Presented in this thesis is the development of a MALDI mass spectrometry- and H/D exchange-based technique for measuring the solution-phase thermodynamic properties of proteins and protein-ligand complexes. The technique, termed SUPREX (Stability of Unpurified Proteins from Rates of H/D Exchange), is designed to evaluate the folding free energy, Gf, and m-value of a protein’s overall folding/unfolding reaction. It can also be used to detect and quantify protein-ligand binding.
Three new SUPREX-based protocols were developed and applied to selected model proteins in proof-of-principle studies. Two of the protocols expand the scope of SUPREX for the biophysical property characterization of large and/or multi-domain proteins. The third protocol helps overcome back-exchange issues that can limit high-throughput SUPREX applications and those that require highly multiplexed analyses.
In the first protocol, a surrogate ligand was employed to increase the global protection of amide protons in the proteins under study, thereby facilitating SUPREX analyses, which require such global protection. The surrogate ligand protocol was developed and validated in the context of ligand-binding studies with calmodulin, and bovine carbonic anhydrase II (BCAII) using calcium, and zinc as the surrogate ligand, respectively.
The second protocol described here incorporates a limited protease digestion step into the conventional SUPREX protocol after the H/D exchange step. The protease digestion protocol facilitates domain-specific thermodynamic stability and ligand-binding affinity measurements when the domains are in the intact protein. The protocol was developed and tested using calmodulin and a linked SH3-SH2 domain construct as model systems, and applied to the biophysical analysis of a molecular chaperone protein, Hsp33.
The third protocol developed here is a SUPREX-like protocol that employs a chemical modification reaction (i.e., oxidation reaction) in place of the amide H/D exchange reaction in SUPREX. This SUPREX-like protocol, termed SPROX (Stability of Proteins from Rates of Oxidation) was developed and tested to detect and quantify protein-ligand binding using cyclophilin A and BCAII as model systems.
Ph.D. Defense Examination Seminar
Student Exams Seminar