Study On Mechanics Of Single Bio-macromolecule Based On Atomic Force Microscopy

Bio-macromolecules play an important role in physiological activities,e.g.genetic information transcription,translation,protein expression and cellular signal transduction.The mechanical response of bio-macromolecules is closely related to biochemical reactions which they participate in.These reactions will further pose effects on inter-molecule interactions,cell-molecule interactions,cell-cell interactions,molecular motor functions and signal transduction process.Bio-macromolecules have also been widely used as scaffolds of biological materials for tissue engineering,immunotherapy,drug screening and transportation,etc.The mechanical nature of bio-macromolecules will not only affect biochemical reactions they participate in,but also regulate properties and functions of the biomaterials based on their assembly.Therefore,it is important to study mechanical properties of bio-macromolecules.The mechanical properties of bio-macromolecules like proteins and nucleic acids are determined by their three-dimensional topological structures formed by weak intramolecular interactions such as hydrogen bonding,hydrophobic interactions,electrostatic interactions,and Van der Waals forces.In addition,the synergistic effects between weak interactions including metal coordination bonds and benzene ring stacking can significantly improve the mechanical properties of elastin,metal ion-bound proteins,complex nucleic acid structures,membrane proteins,peptide fibers and other bio-macromolecules.Over the past three decades,atomic force microscopy has become a key platform for simultaneously performing morphological and mechanical characterization of biological systems on single-molecule level.We study the mechanical properties of bio-macromolecules,e.g.complex nucleic acid structures,metal ion-bound proteins,membrane proteins and peptide fibrils.We would like to obtain general principles about effects of weak intramolecular interactions and their synergistic effects on the mechanical properties of bio-macromolecules.We seek to deepen people's understanding of the mechanical properties of bio-macromolecules,and could rationally design biomimetic materials with specific mechanical properties based on our findings.The main research contents of this paper are as follows:In Chapter 1,we reviewed the atomic force microscopy-based single-molecule force spectroscopy technique and its principles.We made necessary clarifications and background introductions to our research projects including main principles,current research status and problems and challenges existed in these fields.In Chapter 2,we used the atomic force microscope to study the mechanical anisotropy of 3WJ-p RNA complex,which is the core structure of bacteriophage?29DNA packaging motor.Bacillus subtilis bacteriophage?29 uses a molecular motor driven by ATP to encapsulate virus'DNA into its capsid.3WJ-p RNA has strong thermodynamic stability and clinical application prospects.During its DNA encapsulation process,3WJ-p RNA showed high resistance to external force applied along its portal axis,which indicates that this structure has superior stability.We use atomic force microscope to attach different strands of 3WJ-p RNA to cantilever or substrate.And study its mechanical anisotropy by pulling from different directions in AFM experiments.We found that 3WJ-p RNA has excellent mechanical stability in the presence of 5m M Mg²⁺along its portal axis.The unfolding force of 3WJ-p RNA in this direction reaches up to 220 p N,this resistance even exceeds the mechanical stability of a variety of elastic protein.This extraordinary mechanical stability can be attributed to the synergistic effect of two Mg²⁺clamps located in the major groove between helix 1 and 3 of the 3WJ-p RNA complex.This rigidity allows the p RNA domain of the molecular motor to withstand high stress caused by DNA compression during the package process.While the lateral flexibility facilitates assembly of the p RNA and its binding to the capsid precursor.In addition,our findings also provide many new insights for designing tunable anisotropic biomaterials.In Chapter 3,we first studied the mechanical properties of gold-sulfur bonds in the gold-specific protein Gol B by combining X-ray crystallography and single-molecule force spectroscopy based on atomic force microscopy.The Gol B protein from Salmonella typhimurium binds toxic gold ions with high affinity without affecting the function of other copper transport proteins.In addition,Gol B must also transfer gold ions to the P-type ATPase Gol T,which acts as a gold transporter.We used atomic force microscopy to directly rupture single gold-sulfur bond in Gol B to detect its mechanical properties.And then we compared its mechanical properties with structural details obtained from X-ray crystallography to study the structure-function relationship of this chemical bond.We found that the rupture force of the gold-sulfur bond in Gol B protein is around 165 p N,and its mechanical stability is comparable to many non-covalent interactions such as streptavidin-biotin interactions and unfolding of elastin.We believe the low mechanical stability of the gold-sulfur bond in Gol B first originate from its chemical environment.The charge interactions between Cys10 and Cys13 and the backbone amino groups of the surrounding residues will neutralize the charges of the thiolates.Second,length of gold-sulfur bonds is longer than those in inorganic complex and other metal ion regulon.Our results highlight the effect of protein environment on the mechanical stability and kinetic properties of gold-sulfur bonds in a single-molecule environment.This is very valuable for understanding the biological function of Gol B protein,and may also hint at the general principle of metal ions transportation in vivo.In Chapter 4,we applied atomic force microscopy to study the mechanical properties of the outer membrane protein Bam A of Gram-negative bacteria and how it folds and inserts into the outer membrane.Bam A is the core component of the so-called?-barrel assembly machinery?BAM?complex,which could facilitate outer membrane proteins folding and insertion into the bacterial outer membrane.Our research shows that mechanical properties of Bam A depend on three factors related to its function,namely the POTRA domains,the membrane composition,and the lid-lock structure.We have found that the absence of the POTRA domains would lead to a decrease in the mechanical stability of the?-barrel domain.Outer membrane components could affect the mechanical stability of the?-barrel.There is coupling between the extracellular lid sealing the?-barrel and the seam stability of the Bam A?-barrel.These results provide a new perspective for the?-barrel metastable structure of Bam A present in the natural outer membrane.We also investigated the dynamics of Bam A folding and insertion into the outer membrane process,and the effects of molecular chaperones Sur A and Skp.In Chapter 5,we first directly measured the mechanical properties of individual fibrils in peptide hydrogel through the statistical analysis obtained from the images of the atomic force microscopy and material mechanics model.Our results showed that GFFY peptide motif could self-assemble and form hydrogel fibrils with outstanding bending rigidity and Young's modulus.Based on the structural analysis and molecular characterization of fibrils,we believe that intermolecular interactions play a considerable role on the mechanical properties of such individual fibrils.In addition,such fibrils possess mechanical properties resembling individual collagen fibers in the extracellular matrix.We believe that such mechanical properties and strong gelation property may make this peptide suitable for the application in cell culture and tissue engineering.