Date

2016

Document Type

Dissertation

Degree

Doctor of Philosophy

Department

Mechanical Engineering

First Adviser

Zhang, Xiaohui Frank.

Other advisers/committee members

Oztekin, Alparslan; Webb, Edmund; Liu, Yaling; Cheng, Xuanhong

Abstract

Single-molecule experiments have been designed and carried out for investigating the mechanical and biological properties of proteins, and molecular interactions at a single molecular level [1, 2]. Since the first single-molecule technique, patch clamp, was developed in the 1970s [3], a variety of other techniques and instruments have been developed, including atomic force microscopy (AFM) [4–8], optical tweezers [9–19], magnetic tweezers [20–27], and biomembrane force probes (BFP) [28–30]. These optimized instruments are able to achieve high spatial resolution (nanometers), temporal resolution (microseconds), and force sensitivity (piconewtons). Furthermore, they allow us to carry out the single-molecule experiments in a force-clamp mode or force-ramp mode. In order to quantify the biological properties of the molecule, we can consider the molecule as an elastic chain and apply the worm-like chain (WLC) model [31, 32]. To characterize the mechanical properties of proteins or interactions, the Bell–Evans model [33] and Dudko model [34] are available, both of which are derived from Kramers theory [35].This thesis focuses on three projects of single-molecule force experiments using optical tweezers and AFM. In the first project, we performed single-molecule force experiments on the platelet mechanosensor glycoprotein (GP) Ib-IX complex. We successfully identified a juxtamembrane mechanosensory domain (MSD) in GPIb-IX, and further proposed a model of GPIb-IX mechanosensing (i.e., the “trigger model”). In this model, the force-induced unfolding of MSD is the step by which GPIb-IX converts a mechanical signal into a change of the protein conformation, a type of signal that could be recognized and transduced further. This has significant implications for the pathogenesis and treatment of related blood diseases.In the second project, we developed a novel SpyTag system for single-molecule experiments, inspired by the recent discovery of the SpyTag/SpyCatcher covalent bonds [36]. In a single-molecule experimental system, linking methods are usually used to capture, hold, and further apply an external force on the molecule. The traditional methods have many disadvantages, such as low mechanical stability and strength, or requiring the sample purification and reduction reaction. In our newly developed SpyTag system, we can perform the single-molecule pulling experiments efficiently since the SpyTag/SpyCatcher covalent bonds have high mechanical stability and mechanical strength. The minimal difference in the lifetimes of the C-terminal SpyTag/SpyCatcher and N-terminal SpyTag/SpyCatcher, indicates that we can fuse either terminal of the SpyTag to some other proteins without sacrificing the covalent bonds stability. This property makes the single-molecule experiments simpler. Furthermore, the new system simplifies the single-molecule experiments by eliminating the sample purification step, as well as providing a new approach to investigate complexed proteins, such as the integrin α4β7. In addition, the novel system does not involve any protein reduction procedure. This property makes the system promising for measuring the domain-domain interactions within a protein, such as the von Willebrand factor (VWF), where traditional methods are not practical since the sample reduction step would also break the abundant inner disulfide bonds of VWF. Finally, we aimed to investigate the domain-domain interactions within VWF, as well as the influence of the clotting factor VIII (FVIII) in VWF folding/unfolding, using the novel SpyTag system. Our experimental results showed that, there existed differences in the force spectrum of pulling VWF fragments, D’D3A1 or D’D3A1A2, in the absence and presence of 5 nM FVIII. We referred that FVIII binding to VWF altered the force-induced unfolding of D’D3A1A2 fragment, and possibly changed the normal A2 unfolding pathway where no FVIII existed. However, more unfolding events are still needed to clearly reveal the function of FVIII as well as the interaction between FVIII and D’D3A1 or D’D3A1A2. Meanwhile, we intended to investigate the influence of FVIII on the A2 unfolding pathways, by performing single-molecule experiments on the A2 protein, with or without FVIII. Besides, we need to use D’D3 generated by mammalian cells instead of bacterial cells, to repeat the single-molecule experiments. These projects have significant implications for the pathogenesis and treatment of related blood diseases.

Available for download on Sunday, June 10, 2018

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