Investigating Electron Transfer Complexes Using Single Molecule Force Spectroscopy

• Main Author: Mayneord, Guy E
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-2019
• Subjects: Cytochrome; Proteins; Spectrum analysis

Small diffusible redox proteins play a ubiquitous role in facilitating electron transfer (ET) in respiration and photosynthesis by shuttling electrons between large, relatively immobile membrane bound complexes. In order to sustain high ET turnover rates, the association between the cognate partners must be highly specific, yet also sufficiently weak to promote rapid post-ET separation and thus avoid ‘product inhibition’. ET complexes have been investigated extensively using a variety of bulk phase structural and spectroscopic methods. While these ensemble studies have provided useful information on the general factors that facilitate efficient electron transport, the averaging involved obscures the heterogeneity inherent within the system. In contrast, single molecule techniques allow distinct states within a heterogenous system to be accessed and through repeated observation robust statistics are acquired that provide new insights into stochastic processes. Single molecule force spectroscopy (SMFS), often performed by atomic force microscopy (AFM), has been used previously to interrogate the strength and specificity of a range of protein-protein interactions. In such experiments one protein is attached to the AFM probe and is scanned over a surface to which its binding partner is attached, and the force-distance curves measured at each point provide quantitative information on the interaction. However, classical SMFS is limited by the millisecond surface dwell times and low repetition rates which are unsuitable for investigating ET complexes which turnover on a microsecond timescale. Recently, the application of a faster AFM technique called PeakForce-quantitative nanomechanical mapping (PF-QNM) has allowed SMFS on rapid transient interactions to be investigated for the first time. In this work, PF-QNM was applied to investigate the cytochrome b6f : plastocyanin and photosystem I : plastocyanin ET complexes from spinach, and the RC-LH1 : cytochrome c2 ET complex from Rhodobacter sphaeroides. The results provide new information on how the redox state of the participants in biological ET reactions determines their association and dissociation and allowed the forces involved to be quantified. Moreover, the work described identifies and resolves a number of commonly encountered issues with attaching ET complexes to inorganic surfaces and manipulating their stability and functional state.