Proteins at Interfaces: Conformational Behavior and Wear

Abstract

Proteins at interfaces: Conformational behavior and wear Emmanuel Louis Pierre Dumont Proteins at interfaces play a major role in biomaterials 1,2 and lab-on-a-chip devices. 3,4 Protein interactions with the surface change their conformations and therefore their ability to bind to their respective ligands. Another major area of interest surrounding biomaterials and lab-on-achip devices is the prediction and prevention of wear. 5,6 Wear is the progressive loss of material from an object caused by contact and relative movement of the contacting solid, liquid, or gas. 7-9 It is estimated that wear costs 1% of the gross domestic product (approximately $150 billion for the US). 7,10 With the emergence of drug-releasing implants and lab-on-the-chip devices, wear has also become a major concern in bioand nanotechnology. 11 In our laboratory, we use microtubules (filamentous proteins) gliding on kinesin motor proteins as transporters in biosensors. 12 This system, known as the motility assay, is ideal for studying how the conformation of kinesins impacts the gliding of microtubules and therefore the performance of the biosensor. The proposed studies seek to show that kinesins’ geometry changes with their grafting density following De Gennes’ scaling laws 13 for flexible polymers (Chapter2 , published in Langmuir as E.L.P. Dumont, H. Belmas, and H. Hess, Observing the mushroom-to-brush transition for kinesin proteins, 2013, 29 (49), 15142-15145) and that microtubules experience molecular wear due to their repeated interactions with kinesins (Chapter 3, under review for Nature Nanotechnology as E.L.P. Dumont and H. Hess, Molecular wear of microtubules propelled by surface-adhered kinesins). These two results permit the prediction of the lifetime of biosensors using kinesin-propelled microtubules (Chapter 4, to be submitted to Nano Letters as Y. JeuneSmith, E.L.P. Dumont and H. Hess, Wear and breakage combine to mechanically degrade kinesinpowered molecular shuttles). I also discuss the importance of mechanical fatigue for molecular machine design (Chapter 5, published as H. Hess and E.L.P. Dumont, Fatigue Failure and Molecular Machine Design, Small, 7, 1619-1623, 2011). Finally, and it is unrelated to the previous chapters, I developed Monte Carlo simulations for protein adsorption on polymer-coated surfaces (Chapter 6, to be submitted as E.L.P Dumont, A.V. Guillaume, A. Gore, and H. Hess, Random Sequential Adsorption of proteins on polymercovered surfaces: A simulation-based approach) and I explored a molecular model to explain the fracture of materials at low stresses (Chapter 7). Chapter 1: Microtubules, kinesins, and their interactions. In Chapter 1, I introduce the microtubules and kinesins, found in all living cells, and how they interact. We also introduce how these proteins are used in the “motility assay”, where microtubules glide on a kinesin-coated surface. Finally, we present existing litterature of molecular nanotribology (friction and wear) of microtubules and kinesins. Chapter 2: Observing the mushroom-to-brush transition for kinesin proteins. The height of polymers grafted to a surface is predicted to be constant at low densities (“mushroom” regime) and increase with the third root of the polymer surface density at high densities (“brush” regime). This mushroom-to-brush transition is explored with kinesin-1 proteins adhered to a surface at controlled densities. The kinesin height is measured by attaching fluorescently labeled microtubules to the kinesins and determining their elevation using fluorescence interference contrast microscopy. Our measurements are consistent with a mushroom and a brush regime and a transition near the theoretically predicted density. The mushroom-to-brush transition may play a role in protein behavior in crowded cellular environments and be exploited as a signal in intracellular regulation and mechanotransduction. Chapter 3: Molecular wear of microtubules propelled by surface-adhered kinesins. Wear, the progressive loss of material from a body caused by contact and relative movement, is a major concern not only in engineering but also in biology. 8,10,14,15 Advances in nanotechnology both enable the study of the origins of wear processes at the atomic and molecular scale and demand the prediction and control of wear in nanoscale systems. 11,16,17 Here, we show that wear occurs in an in vitro system consisting of microtubules gliding across a surface coated with kinesin-1 motor proteins, and that energetic considerations suggest a molecule-by-molecule removal of tubulin proteins. The wear rates show a complex dependence on sliding velocity and kinesin density, which – in contrast to the friction behavior between microtubules and kinesin 18 – cannot be explained by simple chemical reaction kinetics. Chapter 4: Wear and breakage combine to mechanically degrade kinesin-powered molecular shuttles. In this Chapter, I show how the combined wear of microtubules propelled by surface-adhered microtubules (Chapter 3) and their breaking (an already known phenomenon) permit the prediction of the failure of microtubule-based biosensors, similar to the failure of macroscopic machines. In macroscopic machines, failure as a result of activation is the result of breakage or wear. Breakage is a sudden and permanent phenomenon often caused by fatigue. Wear, the gradual removal of small amount of material, causes an increasing deviation of the part dimensions from the ideal. Unless breakage intervenes, any system will ultimately fail due to wear. Reducing breakage and wear is a major consideration in machine design. 19 Chapter 5: Fatigue Failure and Molecular Machine Design. Sophisticated molecular machines have evolved in nature, and the first synthetic molecular machines have been demonstrated. With our increasing understanding of individual operating cycles, the question of how operation can be sustained over many cycles comes to the forefront. In the design of macroscale machines, performance and lifetime are opposing goals. Similarly, the natural evolution of biological nanomachines, such as myosin motor proteins, is likely constrained by lifetime requirements. Rather than bond rupture at high forces, bond fatigue under repeated small stresses may limit the mechanical performance of molecular machines. Here we discuss the effect of cyclic stresses using single and double bonds as simple examples and demonstrate that an increase in lifetime requires a reduction in mechanical load and that molecular engineering design features such as polyvalent bonds capable of rebinding can extend the bond lifetime dramatically. A universal scaling law for the force output of motors is extrapolated to the molecular scale to estimate the design space for molecular machines. Chapter 6: Random Sequential Adsorption of proteins on polymer-covered surfaces: A simulation-based approach Non-fouling polymeric coatings enable the suppression of protein adsorption to surfaces, and their perfection is the objective of many recent experimental studies. 20-25 Obtaining a theoretical understanding of the functioning of these coatings and the prediction of residual protein adsorption as a function of the coating properties has similarly attracted significant interest. 26-35 A recent study developed a basic Random Sequential Adsorption (RSA) model for protein adsorption to non-fouling coatings, which was analytically solvable and yielded encouraging agreement with published experimental data. 36 The model assumed that polymer chains on the surface can be represented by hard spheres with a radius equal to their radius of gyration. These randomly distributed hard spheres obstruct the adsorption of proteins, again represented as hard spheres with a diameter equal to the diameter of the protein. The evolution of the protein density on the surface was calculated from the independent probabilities to penetrate the layer of adsorbed proteins and the layer of polymer chains. Here, we scrutinize this analytical model by conducting computer simulations of the adsorption process under the same assumptions. We find that the results of the computer simulations deviate significantly from the analytical solution, which indicates that the spatial distributions of proteins and polymers cannot be considered independently. Chapter 7: A molecular model to explain material fracture at low stresses. In this Chapter, I extend Zhurkov’s work on predicting the lifetime of materials with a simple molecular model. 37 In 1965, Zhurkov introduced a model 37 to predict the lifetime of materials under uniaxial tension. Zhurkov’s model connects the lifetime of the material and the uniaxial stress (macroscopic experimental values) to several microscopic constants such as the enthalpy of sublimation and the Boltzmann constant. I extend this model by introducing the possibility of rebinding. This new model enables the prediction of the lifetime of material at low stresses.