Development of Collision Induced Unfolding for the Study of Large Multiprotein Complexes

Abstract

Protein complexes carry out numerous critical functions in cells and as such represent a key class of drug targets associated with myriad human diseases. Methods for rapid evaluation of protein complex structure and stability are, therefore, extremely important in ongoing efforts to discover new pharmaceuticals. While technologies such as ion mobility spectrometry-mass spectrometry (IM-MS) and collision induced unfolding (CIU) have been established as useful techniques for the rapid analysis of protein quaternary structure and stability using small amounts of unpurified sample, there are still significant challenges associated with the interpretation of such data, especially in the context of large multi-domain protein targets. In this dissertation, we develop CIU based approaches to target such multi-domain proteins in order to leverage IM-MS for future efforts focused on stability assessments, probing protein-substrate interactions, and next-generation protein engineering efforts. In Chapter 2, we study the CIU of HSA dimer ions as a model multiprotein complex unfolding. Through the novel combination of domain-specific chemical probes, domain-deleted protein constructs, CIU combined with electron capture dissociation (ECD), and steered MD, we were able to demonstrate, for the first time, a detailed mechanism for multiprotein complex CIU. Specifically, our data indicates that a single monomer within the complex is responsible for the CIU transitions observed for the dimer, and that the CIU observed is domain correlated. The remaining chapters contained within this thesis are concerned with developing IM-MS and CIU methods for the analysis of biosynthetic enzymes, a class of multi-protein complexes carrying out the synthesis of a host of natural products. In Chapter 3, we apply IM-MS and CIU to probe the transient physical association between co-dependent enzymes TamI (an iterative cytochrome P450 monooxygenases) and TamL (a flavin adenine dinucleotide-dependent oxidase), involved in late-stage oxidation of tirandamycin antibiotics. Our results demonstrate that TamI and TamL form a biocatalytically competent heterodimeric complex in vitro, and we utilize IM-MS to measure binding affinities between a range of tirandamycin antibiotics with TamI. Furthermore, we employ domain-specific chemical probes and CIU to mechanistically reveal that the loop region, which is the “lid” of the TamI substrate pocket unfolds at lower activation energies, while the heme binding pocket unfolds at higher CIU energies. In chapter 4, we turn our attention to type I polyketide synthases (PKS), which form an enzymatic assembly line for the production of polyketide natural products using a series of modules that include keto synthase (KS) and acyltransferase (AT) domains. we demonstrate the current limits of quantitative CIU technology by probing the stability of a ~280kDa PKS dimer protein complex, detecting evidence of stability shifts associated with substrate binding that account for <0.1% of the mass for the intact assembly. In Chapter 5, We use IM-MS to detect different conformational states of a 207kDa di-domain KS-AT dimer and are able to capture stability differences between these different conformations using CIU. Furthermore, through tracking these forms as a function of time, we elucidate a detailed disassembly pathway for KS-AT dimers for the first time. To conclude, this dissertation has focused on new developments in technology targeting the structure of gas-phase multiprotein complexes and its application in drug discovery. Significant progress has been made in advancing CIU technology for its application in structural biology.