Complementary Mass Spectrometry Methods for Characterizing Protein Folding, Structure, and Dynamics

Proteins are involved in virtually every biochemical process. A comprehensive characterization of factors that govern protein function is essential for understanding the biomedical aspects of human health. This dissertation aims to develop complementary mass spectrometry-based methods and apply them to solve problems pertaining to the area of protein structure, folding and dynamics. ‎Chapter 1 uses fast photochemical oxidation of proteins (FPOP) to characterize partially disordered conformers populated under semi-denaturing conditions. In FPOP, ·OH generated by laser photolysis of H2O2 introduces oxidative modifications at solvent accessible side chains. By contrast, buried sites are protected from radical attack. Using apomyoglobin (aMb), it was demonstrated that under optimized conditions undesired can be almost completely eliminated and detailed structural information can be obtained. ‎Chapter 3 combines FPOP with submillisecond mixing to enable studying early events in protein folding. aMb served as a model system for these measurements. Spatially-resolved changes in solvent accessibility follow the folding process. Data revealed that early aMb folding events are driven by both local and sequence-remote docking of hydrophobic side chains. Assembly of a partially formed scaffold after 0.2 ms is followed by stepwise consolidation that ultimately yields the native state. The submillisecond mixer used improved the time resolution by a factor of 50 compared to earlier FPOP experiments. Submillisecond mixing in conjunction with slower mixing techniques help monitor completes folding pathways, from fractions of a millisecond all the way to minutes. ‎Chapter 4 uses ion mobility mass spectrometry (IM-MS) to explore the structural relationship between semi folded solution and gas phase protein conformers. Collision cross sections (CCSs) provide a measure of analyte size. Mb was used as model system because it follows a sequential unfolding pathway that comprises two partially disordered states. IM-MS data showed that the degree of gas phase unfolding is not strongly correlated with the corresponding solution. Gas phase unfolding as well as collapse events can lead to disparities between gaseous and solution structures for partially unfolded proteins. IM-MS data on non-native conformers should therefore be interpreted with caution. ‎Chapter 5 uses HDX-MS to examine the role of conformational dynamics for the function of multi-protein molecular machines such as FoF1 ATP synthase. HDX-MS monitors backbone deuteration kinetics in the presence of D2O. Disordered segments exchange more rapidly than those in tightly folded regions. Measurements of spatially-resolved deuterium are performed using LC-MS. It was found that the H-bonding network of key power transmission elements is insensitive to PMF-induced mechanical stress. Unexpectedly, HDX-MS reveals a pronounced destabilization of the g C-terminus during rotational catalysis under PMF. The behavior of g is attributed to kinetic friction within the apical rotor bearing

Changes in Enzyme Structural Dynamics Studied by Hydrogen Exchange-Mass Spectrometry: Ligand Binding Effects or Catalytically Relevant Motions?

It is believed that enzyme catalysis is facilitated by conformational dynamics of the protein scaffold that surrounds the active site, yet the exact nature of catalytically relevant protein motions remains largely unknown. Hydrogen/deuterium exchange (HDX) mass spectrometry (MS) reports on backbone H-bond fluctuations. HDX/MS thus represents a promising avenue for probing the relationship between enzyme dynamics and catalysis. A seemingly straightforward strategy for such studies involves comparative measurements during substrate turnover and in the resting state. We examined the feasibility of this approach using rabbit muscle pyruvate kinase (rM1-PK) which catalyzes the conversion of phosphoenolpyruvate and Mg-ADP to pyruvate and Mg-ATP. HDX/MS revealed that catalytically active rM1-PK undergoes significant rigidification in the active site. This finding is counterintuitive, considering the purported correlation between dynamics and catalysis. Interestingly, virtually the same rigidification was seen upon exposing rM1-PK to substrates or products in the absence of turnover. These data imply that the active site dynamics during turnover are dominated by protein-ligand binding interactions. These interactions stabilize H-bonds in the vicinity of the active site, thereby masking subtle dynamic features that might be uniquely associated with catalysis. Our data uncover an inherent problem with side-by-side turnover/resting state measurements, i.e., the difficulty to design a suitable reference state against which the working enzyme can be compared. Comparative HDX/MS experiments on enzyme dynamics should therefore be interpreted with caution

Evidence for a Partially Stalled γ Rotor in F

F1-ATPase uses ATP hydrolysis to drive rotation of the γ subunit. The γ C-terminal helix constitutes the rotor tip that is seated in an apical bearing formed by α3β3. It remains uncertain to what extent the γ conformation during rotation differs from that seen in rigid crystal structures. Existing models assume that the entire γ subunit participates in every rotation. Here we interrogated E. coli F1-ATPase by hydrogen-deuterium exchange (HDX) mass spectrometry. Rotation of γ caused greatly enhanced deuteration in the γ C-terminal helix. The HDX kinetics implied that most F1 complexes operate with an intact rotor at any given time, but that the rotor tip is prone to occasional unfolding. A molecular dynamics (MD) strategy was developed to model the off-axis forces acting on γ. MD runs showed stalling of the rotor tip and unfolding of the γ C-terminal helix. MD-predicted H-bond opening events coincided with experimental HDX patterns. Our data suggest that in vitro operation of F1-ATPase is associated with significant rotational resistance in the apical bearing. These conditions cause the γ C-terminal helix to get stuck (and unfold) sporadically while the remainder of γ continues to rotate. This scenario contrasts the traditional greasy bearing model that envisions smooth rotation of the γ C-terminal helix. The fragility of the apical rotor tip in F1-ATPase is attributed to the absence of a c10 ring that stabilizes the rotation axis in intact FoF1. Overall, the MD/HDX strategy introduced here appears well suited for interrogating the inner workings of molecular motors

ClpP protease activation results from the reorganization of the electrostatic interaction networks at the entrance pores

Bacterial ClpP is a highly conserved, cylindrical, self-compartmentalizing serine protease required for maintaining cellular proteostasis. Small molecule acyldepsipeptides (ADEPs) and activators of self-compartmentalized proteases 1 (ACP1s) cause dysregulation and activation of ClpP, leading to bacterial cell death, highlighting their potential use as novel antibiotics. Structural changes in Neisseria meningitidis and Escherichia co ClpP upon binding to novel ACP1 and ADEP analogs were probed by X-ray crystallography, methyl-TROSY NMR, and small angle X-ray scattering. ACP1 and ADEP induce distinct conformational changes in the ClpP structure. However, reorganization of electrostatic interaction networks at the ClpP entrance pores is necessary and sufficient for activation. Further activation is achieved by formation of ordered N-terminal axial loops and reduction in the structural heterogeneity of the ClpP cylinder. Activating mutations recapitulate the structural effects of small molecule activator binding. Our data, together with previous findings, provide a structural basis for a unified mechanism of compound-based ClpP activation2CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP306943/2015-8; 420567/2016-099999.004913/2015-092015/15822-1; 2012/01953-9; 2016/05019-0; 2012/50161-8Precision Medicine Initiative (PRiME) at the University of Toronto internal fellowship [PMRF2019-007]; Canadian Institutes of Health Research (CIHR) postdoctoral fellowshipCanadian Institutes of Health Research (CIHR); CNPq-Brazil fellowship [202192/2015-6]; Saskatchewan Health Research Foundation postdoctoral fellowship; Ontario Graduate Scholarship (OGS)Ontario Graduate Scholarship; Department of Biochemistry at the University of Toronto; Centre for Pharmaceutical Oncology (University of Toronto); CIHR Training Program in Protein Folding and Interaction Dynamics: Principles and Diseases fellowshipCanadian Institutes of Health Research (CIHR) [TGF-53910]; University of Toronto Fellowship from the Department of Biochemistry; OGS fellowship; NSERC PGS-D2 fellowship; CIHR Emerging Team Grants from the Institute of Infection and ImmunityCanadian Institutes of Health Research (CIHR) [XNE-86945]; CIHR Project grantCanadian Institutes of Health Research (CIHR) [PJT-148564]; Global Affairs Canada (Canada); CAPES (Brazil)CAPES [99999.004913/2015-09]; NSERCNatural Sciences and Engineering Research Council of Canada [RGPIN-2015-04877, DG-20234]; Canada Research Chairs ProgramCanada Research Chairs; CIHR new investigator programCanadian Institutes of Health Research (CIHR); FAPESPFundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) [2015/15822-1, 2012/01953-9, 2016/05019-0, 2012/50161-8]; CNPqNational Council for Scientific and Technological Development (CNPq) [306943/2015-8, 420567/2016-0]; AbbVie [1097737]; BayerBayer AG [1097737]; Boehringer IngelheimBoehringer Ingelheim [1097737]; Genome Canada through Ontario Genomics Institute GrantGenome Canada [1097737, OGI-055]; GlaxoSmithKlineGlaxoSmithKline [1097737]; JanssenJohnson & Johnson USAJanssen Biotech Inc [1097737]; Lilly CanadaEli Lilly [1097737]; MerckMerck & Company [1097737]; Novartis Research Foundation [1097737]; Ontario Ministry of Economic Development and Innovation [1097737]; PfizerPfizer [1097737]; TakedaTakeda Pharmaceutical Company Ltd [1097737]; Wellcome Trust GrantWellcome Trust [1097737, 092809/Z/10/Z]; Canada Foundation for InnovationCanada Foundation for Innovation; NSERCNatural Sciences and Engineering Research Council of Canada; University of Saskatchewan; Government of Saskatchewan; Western Economic Diversification Canada; National Research Council Canada; CIHRCanadian Institutes of Health Research (CIHR

Effect of Photochemical and Mechanical Degradation Mechanisms on Polyethylene

As polyethylene (PE) products become more prevalent in a wide variety of engineering applications, it is critical to understand their chemical and mechanical degradation to prevent premature failure. Sunlight radiation has been recognized as the most severe environmental factor causing chemical degradation in PE. Furthermore, PE is also susceptible to mechanical failure through creep rupture and stress cracking. This dissertation focuses on the impact of these degradation mechanisms on the lifetime of PE. PE products designed for sunlight exposure are required to be stabilized with antioxidant (AO) to retard polymer degradation. In this dissertation, photochemical depletion of AO in PE samples exposed to artificial sunlight was monitored by the oxidative induction time (OIT) testing. A mathematical model was developed by implementing the photochemical reaction scheme, radiation attenuation, and diffusion of AO and oxygen. The model successfully described the AO depletion profiles and obtained a relationship between radiation intensity and AO depletion rate, based on which an equation was proposed to predict the AO depletion in PE in exposure to natural sunlight. Mechanical degradation of PE was studied by investigating creep rupture and stress cracking in thin-wall PE tubes. A test setup was designed to apply static and cyclic internal pressures to thin-wall PE tubes and to record their failure times. The pressure tests were accelerated using elevated temperatures. The failure time (and number of cycles) was predicted using two shifting methods for the dry-cooling applications. Furthermore, this dissertation investigated wave formation of PE geomembranes undergoing thermal expansion during installation. Finite element method (FEM) was used to simulate the formation of waves and quantify the tensile strains along the deformed geomembrane. The model was validated by previous experimental studies. Additionally, the effect of overburden pressure on waves was studied.Ph.D., Civil engineering -- Drexel University, 201

Predicting the depletion of antioxidants in high density polyethylene (HDPE) under sunlight using the reciprocity law

Depletion of antioxidants in HDPE subjected to sunlight exposure was studied. Sunlight radiation was simulated using a laboratory xenon light weatherometer at three irradiation levels. Oxidative induction time (OIT) test was performed on different layers along the thickness of the test coupons to establish the antioxidant depletion throughout the exposure duration. The highest drop in OIT was obtained for the surface layer facing the radiation, followed by the backside layer which was exposed to indirect radiation reflected from the wall of the weatherometer. The core section showed a slower decrease under the same exposure conditions. Furthermore, the OIT depletion rate in the surface layer increased with radiation intensity. The study proved that the sunlight degradation of the tested polyethylene can be accelerated by increasing the irradiation intensity based on the reciprocity law

Sunlight Degradation of Polymeric Detectable Warning Surface Products

Detectable warning surface (DWS) is a panel product installed at the edge of curb ramps to warn visually impaired pedestrians about the proximity of the roadway. Most DWS products are made of polymers and are subjected to outdoor weathering. Therefore, sunlight degradation is an important factor that affects their service life. In this study, the effects of sunlight on material degradation in four DWS products made from polyester, neopentylglycol (NPG), polyurethane, and polyolefin were evaluated by exposing DWS test coupons in a xenon weatherometer. The exposure conditions were largely based on ASTM D 2565 for a duration of 3,000 h. Color change induced by radiation was measured using a spectro-colorimeter. The fastest discoloration was measured in DWS made from polyurethane. The largest color change occurred between 500 and 1,000 h for polyurethane and between 1,000 and 3,000 h for other polymers. The changes of surface appearance observed under a digital microscope were consistent with the discoloration. The reinforcing fibers in DWS products made from polyester and polyurethane were revealed after 3,000 h exposure. Decrease in surface wear resistance was obtained in tested coupons that were exposed to the highest irradiance level. The product made from NPG exhibited the greatest decrease in wear resistance, while that made from polyolefin showed the least change