Friday, November 22, 2024
11/22/2024
PROJECT SUMMARY/ABSTRACT Structural biology plays a central role in modern molecular bioscience, enabling both a greater understanding and new mechanisms of manipulation of biomolecular action. However, despite tremendous development in tools for the generation of high resolution molecular models, large families of biomolecules and biomolecular complexes are still poorly represented in databases of protein structure due to limitations of current technology, and methods for probing protein structure within mammalian tissue are few. One method that has been used successfully to qualitatively study the structure of several of these families is hydroxyl radical protein footprinting (HRPF), an emerging technology that has been used to study changes in protein topography by measuring changes in the apparent rate of reaction between hydroxyl radicals generated in situ and amino acid side chains on the protein surface. Our initial work has developed HRPF into a quantitative measurement of protein topography at the individual amino acid level, accurately measuring the average solvent accessible surface areas (<SASA>) of many individual amino acids in a single experiment. In this renewal, we will expand our technology into structural systems that change dynamically with time, including protein posttranslational modification systems, large heteromeric protein complexes, and protein:carbohydrate complexes. The core technology we will develop to enable these studies is high performance liquid chromatography coupled inline with amino acid resolution HRPF (LC-HR-HRPF). Inline liquid chromatography allows the separation of protein conformers and immediate quantitative measurement of the purified conformers’ topographies by HR-HRPF before the dynamic system has a chance to re-equilibrate, freezing the structural information in the stable chemical footprint. We will also develop technology for analysis of protein structure within mammalian whole blood, enabling the study of protein structure and interactions within highly complex native systems. We will develop flow systems to precisely and carefully deliver hydrogen peroxide to blood for protein labeling without damaging cells, and will demonstrate the technology with the structural analysis of monoclonal antibodies dosed into a mouse model. Finally, we will develop technologies to probe the topography of complex carbohydrates, enabling us to measure which parts of carbohydrates mediate interactions with proteins, even in complex mixtures of glycans. We will develop both reducing-end specific and non-specific labeling strategies for probing carbohydrate topography. Together, these advances represent potential transforming technologies for the structural analysis of biomedically important and highly challenging systems.
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