The Bio-Mechanical Properties Of Mussel Foot Protein Revealed By Single Molecule Force Spectroscopy

The application scale and effects of artificial materials on wet surfaces are severely limited by the hydration,high salt and environmental pH.While marine mussels utilize their byssals to get them firmly affixed on the sea bottom,rocks and even on a moving ship to resist the fierce sea flows rushing them away from their habitats.Experimental evidences have proved that a post-translational amino acid DOPA is responsible for the versatile binding ability of mussel foot protein.DOPA can not only adhere to surfaces with different chemical properties,but also can chelate with Fe3+ to form a protective cuticle over the byssals.Using surface force apparatus(SFA),Waite et al.systematically studied the mechanism of mussel foot protein adhesion ability and DOPA-Fe3+ coordination.However,limited by the scanning range and force resolution of SFA,it is not clear that how DOPA interacts with surface,the influence of physical and chemical factors on DOPA adhesion as well as DOPA-metal coordination on single molecule level.Based on atomic force microscope,single-molecule technic has been widely used for detecting protein mechanics,donor-receptor interaction and chemical bonds strength.Exploited single-molecule measurements,we can measure the single DOPA-surface interaction and the influence of surface atom arrangement as well as adjacent charge on DOPA adhesion ability.Using our recently designed 'multi-fishhook' technic,we can measure the DOPA-Fe3+ coordination interactions efficiently.Our study not only illustrates the molecule mechanism of mussel foot protein on surface adhesion and its environmental effects,but also provide new insights for rational design of new generation bio-inspired materials,surface modification and surface adhesives.In Chapter ?,we briefly introduced the principle of AFM based single-molecule force spectroscopy technic and the recent studies as well as challenges on mussel foot protein inspired adhesives.In Chapter ?,we used single-molecule 'multi-fishhook' method to reveal the adhesion force between single DOPA molecule and surfaces with different chemical properties.By conjugating DOPA to a polymer chain,we can measure the adhesion force with surfaces efficiently.We found the detaching force of DOPA with different nine organic as well as inorganic surfaces are in the range of 60-90 pN under the pulling speed of 1000 nm s-1,which indicates DOPA can strongly adhere to variety surfaces.Moreover,by constructing the free energy landscape for the rupture events,we revealed several distinct binding modes between DOPA and different surfaces.These results explain the molecular origin of the versatile binding ability of DOPA.Moreover,we could quantitatively predict the relationship between DOPA contents and the binding strength based on the measured rupture kinetics.We found that the binding strength increases rapidly at low DOPA contents and the increase of the binding strength becomes much shallower at higher DOPA contents.These results serve as the basis for the quantitative prediction of the relationship between DOPA contents and adhesion strength to different wet surfaces,which is important for the design of novel DOPA based materials.In Chapter ?,we studied the effects of surface atom arrangement on DOPA adhesion.We used rutile(110),(011),(111)and(100)surface as model surfaces.By conjugating dopamine to AFM cantilever via a PEG linker,we can perform our single-molecule measurement.Our results showed even for chemically well-defined crystal surfaces,DOPA can bind to them with multiple binding modes.The binding forces for DOPA to different rutile surfaces can vary in a broad range from 40-800 pN at a pulling speed of 1000 nm s-1 and are largely dependent on the surface properties.Our finding indicates that local chemical environments could greatly affect DOPA adhesion.Our results give new insight to the principles of DOPA surface interaction and could guide for the applications of DOPA containing adhesive materials.In Chapter ?,we studied the synergistic adhesion of DOPA and lysine in mussel foot protein.Previous studies revealed that mussel adhesive proteins are also rich of positively charge lysine residues which commonly occur in close proximity to Dopa along the protein backbone.Surface force apparatus measurements proved that DOPA and lysine can bind to surface synergistically.However,it remains elusive that how DOPA and lysine adhere synergistically.Two dipeptides(lysine-DOPA and DOPA-lysine)were synthesized for this study.The dipeptides were flanked to the AFM cantilever tip via a PEG linker.Then single-molecule measurements were exploited to detect the interaction between the dipeptides and titania and mica surface.We found that the binding forces for lysine-DOPA dipeptide are significantly higher when the positive charge of lysine is unprotected on both surfaces.However,such synergistic effect is absent when the sequence is reversed.We attribute such different synergistic effects to their distinct structure for load distribution within the molecules,which may represent a general principle for synergistic strong adhesion.In Chapter ?,we studied the coordination interaction between Dopa and Fe3+.Mussel foot proteins are shielded from abrasion in wave-swept habitats protected by cuticle layer,which contains large amount of DOPA-Fe3+ coordinating structure.The coordinating structures not only provide sufficient toughness,but also dissipate shocking energy,which protecting mussel foot protein from fracture.Experimental evidence showed that DOPA and Fe3+ can form mono,bis and tris complex,and this stoichiometry is dependent on environmental pH and Fe3+ concentration.In our experimental design,dopamine was first conjugated to hyaluronic acid chain to form HA-DOPA molecule.By adding desired amount of FeCl3 solution,we can prepare HA-DOPA-Fe3+ nano-particles.Then we can fish HA-DOPA-Fe3+nano-particle to measure DOPA-Fe3+ coordination interaction.Varing pH and Fe3+concentration,we revealed the binding force of Bis-DOPA-Fe3+ and Tris-DOPA-Fe3+complex.Our study provides the nanoscale mechanistic understanding of the coordination bond-mediated mechanical properties of biogenetic materials,and could guide future rational design and regulation of the mechanical properties of synthetic materials.In this thesis,we revealed the adhesion ability of DOPA to different surface,and quantitively measured the DOPA-Fe3+ coordinating interactions.These findings will not only explain the versatile binding ability of mussel foot protein,but also guide the design of new generation bio-inspired materials.