Dynamic Study On Conformational Transformations Of Biological Proteins Utilizing Silicon-Based Single-Molecule Devices

Single-molecule biophysics is a discipline that aims to probe and analyze the physical properties(static structural features or dynamic transition routines)of a single biomolecule at the microscopic scale.A detailed understanding of the physical characteristics and behavior of individual biomolecules can scientifically and delicately reveal many of the mysteries of life science.One-dimensional silicon nanowires are widely used in many biological fields because of their good size matching and biocompatibility.Therefore,in this thesis,the intrinsic conformational transition dynamics of proteins are studied on the scale of a single biomolecule based on the silicon nanowires single-molecule device platform.The current research work is as follows:(1)Using silicon nanowire biosensors with ultra-high sensitivity and spatio-temporal resolution in this work,we record the real-time response of a single Photosystem I Light-Harvesting Complex I(PSI-LHCI)supercomplex to various stress conditions(gradient changes in temperature,illumination,and electric field).Under the condition of temperature change,the physiological response process of PSI-LHCI complex protein has a two-state switching process related to the intrinsic thermal vibration behavior.When illumination and bias voltage are changed,we observe two additional shoulder states,which may be generated by the self-conformational regulation function of the protein in a physical environment.Therefore,relying on the real-time dynamic monitoring on a single PSI-LHCI supercomplex protein under different stress conditions,we successfully demonstrated the promising application of this nanotechnology in structural analysis of protein and functional mechanism understanding of photosynthesis.(2)Using single-molecule biosensors based on silicon nano wires,the conformational changes of the photosynthetic LH1-RC complex under physiological conditions were directly monitored in real time.The results show that the structural transition of the LH1-RC complex occurs between the four conformations with a strong temperature dependence.At the optimal temperature(55℃),state 2 and state 3 occupy the main conformation of the LH1-RC complex,and the conformational transition process mostly shows non-harmonic vibration mode,which is more conducive to the acquisition of photons and heat transport of the protein in a physical environment.At the same time,we also observed the effect of photoexcitation on the proportion of structural transformation,which may be caused by the vibration of the pigment molecules which are driven by light.These results demonstrate the reliability of this technique in revealing the mechanism of intrinsic physiological processes of various biomolecules in vitro.(3)Based on an ultra-high temporal resolution single-molecule biosensor i.e.,silicon nanowires field effect transistors,we examine in situ the physiological dynamic behavior of a single PNPase degrading enzyme when it functions as a catalytic degradation.This technique enables real-time monitoring on degradation process of RNA substrate molecules inside PNPase with a single-base resolution.The complete degradation process includes three stages:binding to an RNA substrate molecule,hydrolysis of a single nucleotide,and movement of a single base.At the same time,we also found a binding behavior between the enzyme(near the active site)and the nucleoside,which further interpreted the mechanism of RNA degradation.Relying on systematic analysis of independent read information,we can achieve approximately 80%accuracy in the identification of nucleoside information in the nucleotide sequences of an artificial RNA molecule and 79%accuracy in a celluar RNA molucule.Therefore,these results verify the potential value of this technology in realizing single molecule sequencing,which provides a new technical reference for realizing high-throughput real-time gene-information reading.(4)Through the high-gain silicon nanowire field effect transistor,we have successfully realized the real-time detection of the enzymatic reaction process of a single M-KF polymerase and found the bistable transition process of M-KF polymerase in the DNA catalytic polymerization process i.e.,corresponding to the closed conformation of M-KF polymerase in the low conductance state and the opened conformation in the high conductance state,respectively.Finally,we found the heterogeneous reaction kinetic behavior of M-KF polymerase in catalyzing polymerization of different template chains through control experiments.The results show that there is not only the chain growth process of polymerization reaction in the closed conformation,but also the hydrogen bond coordination between the nucleotide molecule and the base of the template chain,and there is no mutual interference between the two processes.Therefore,this work demonstrates the great potential of silicon nanowires-based biosensors in revealing protein conformational changes and structural transformation pathways in enzymatic reaction mechanisms,and provides a reliable technical platform for analyzing the catalytic mechanisms of various proteases in the field of single-molecule biology.