ABSTRACT Collagen is the major structural protein of our connective tissues. It provides integrit... more ABSTRACT Collagen is the major structural protein of our connective tissues. It provides integrity and mechanical strength through its hierarchical organization. Defects in collagen can lead to serious connective tissue diseases. Collagen is also widely used as a biomaterial. Given that mechanical properties are related to the structure of materials, the main goal of our research is to understand how molecular structure correlates with microscale mechanical properties of collagen solutions and networks. We use optical tweezers to trap and monitor thermal fluctuations of an embedded probe particle, from which viscoelastic properties of the solution are extracted. We find that elasticity becomes comparable to viscous behavior at collagen concentrations of 5mg/ml. Furthermore, by simultaneously neutralizing pH and adding salt, we observe changes in viscosity and elasticity of the solution over time. We attribute this to the self-assembly process of collagen molecules into fibrils with different mechanical properties. Self-assembly of collagen under these conditions is verified by turbidity measurements as well as electron microscopy. By comparing results from these local studies of viscoelasticity, we can detect spatial heterogeneity of fibril formation throughout the solution.
ABSTRACT In this talk, I will describe our senior undergraduate lab course in Biological Physics ... more ABSTRACT In this talk, I will describe our senior undergraduate lab course in Biological Physics at Simon Fraser University. Unique in Canada, this course combines modules that teach the students basic molecular and cell biology and leading-edge biophysical techniques with independent student projects. I'll outline the structure and components of the course, provide an overview of each experimental module, and describe student interest and feedback.
Probing relatively short molecules, such as the protein elastin (~200 nm), with optical tweezers ... more Probing relatively short molecules, such as the protein elastin (~200 nm), with optical tweezers requires manipulating trapped polystyrene beads at separations small compared to their micron dimensions. We discuss experimental complications arising from this proximity, such as hydrodynamic and optical interactions, and our efforts to minimize them.
Biomolecular motors have inspired the design and construction of artificial nanoscale motors and ... more Biomolecular motors have inspired the design and construction of artificial nanoscale motors and machines based on nucleic acids, small molecules, and inorganic nanostructures. However, the high degree of sophistication and efficiency of biomolecular motors, as well as their specific biological function, derives from the complexity afforded by protein building blocks. Here, we discuss a novel bottom-up approach to understanding biological
Journal of Optics A: Pure and Applied Optics, 2007
Motivated by recent attempts to use parametric resonance to calibrate the spring constant of a Br... more Motivated by recent attempts to use parametric resonance to calibrate the spring constant of a Brownian particle in an optical trap, we have looked systematically at the effects of modulating laser power on the motion of the trapped particle. We predict and find experimentally an increase in the particle's position variance at low laser modulation frequencies, but we find no evidence for any resonant effects in the extremely overdamped motion of the trapped particle. Our results can serve as a guide for designing multiple traps by the 'time-sharing' method.
Stretching and relaxing single protein molecules provides quantitative information on their elast... more Stretching and relaxing single protein molecules provides quantitative information on their elasticity and other mechanical properties. Optical tweezers can be used to perform such experiments, but require the ends of the protein to be chemically tethered to polystyrene beads used to manipulate the molecule and measure its response. Our goal is to use this method to study the human structural protein elastin, whose mechanical properties are directly related to its physiological function. Elastin's contour length is approximately 170 nm, so the trapped beads must be manipulated at separations small compared to their micron dimensions. The close proximity of the beads introduces experimental complications, primarily the result of optical and hydrodynamic interactions. We discuss these complications along with our efforts to minimize and account for them in our measurements. We then show preliminary evidence addressing a critical question of whether elastin has higher order struct...
We present an approach that will lead to an understanding of the operational characteristics of b... more We present an approach that will lead to an understanding of the operational characteristics of biological molecular motors by designing and modeling synthetic molecular motors that use a combination of natural and designed biological components. One such design is a protein-based rotationally diffusive motor dubbed the Tumbleweed (TW). This is a tri-pedal self-assembling complex in which each foot is able to bind to a unique binding site located periodically on an effective 1D track, and where the binding of each foot is controlled by a unique ligand in solution. The TW motor construct incorporates a combination of biological repressor proteins, a de-novo coiled-coil protein structure, and a dsDNA track. We present simulation results of the TW motor that exhibit unidirectional motion and a maximum theoretical speed of 0.01-0.1 mm/s, which is limited by rotational diffusion. Experimental limitations, such as the exchange of ligands in solution, will most likely reduce the motor speed to 10-100 nm/s, which is comparable to biological molecular motors.
Our understanding of biology stems from models at many resolutions, as we seek from detailed atom... more Our understanding of biology stems from models at many resolutions, as we seek from detailed atomic-scale interactions simpler emergent physical principles to support our understanding and to produce useful theoretical reductions and tractable simulations. In particular, multiscale methods coupling coarsegrained and atomic models are essential to modeling, predicting, and understanding the basic driving forces that operate across many biomolecular length and time scales. Yet, though coarse-graining strategies exist, it has been chal-
both hydrodynamic radii and optical properties were performed simultaneously. Such a dual-mode te... more both hydrodynamic radii and optical properties were performed simultaneously. Such a dual-mode technique may prove valuable for elucidating the mechanism of amyloid fibrillization and ultimately for designing possible diagnostic methods.
... Nancy R. Forde, Tanya L. Myers and Laurie J. Butler T he James Franck Institute and Departmen... more ... Nancy R. Forde, Tanya L. Myers and Laurie J. Butler T he James Franck Institute and Department of Chemistry, T he University of Chicago, Chicago, IL 60637 ... One must then analyze the remaining two-electron conÐguration interaction (Fo rster-and Dexter-type) matrix elements ...
In the past decade, the ability to manipulate and measure forces exerted by single biological mol... more In the past decade, the ability to manipulate and measure forces exerted by single biological molecules has transformed our understanding of their mechanical response, stability, and the mechanisms by which they operate. One of the essential tools in this revolution has been optical tweezers, which use a focused laser beam to ``trap'' (hold stably in three dimensions) micrometer-sized refractive particles. In this talk, I will briefly describe how optical tweezers work and how they can be used to manipulate and probe the mechanical response of single DNA and protein molecules. I will then describe the technique of holographic optical tweezers, which modify the phase of a laser beam in order to generate multiple optical traps at specified locations in three dimensions within a sample. I will discuss our work towards applying this technique to mechanical studies of protein-based biomaterials on the microscale.
The design of bioinspired nanostructures and materials of defined size and shape is challenging a... more The design of bioinspired nanostructures and materials of defined size and shape is challenging as it pushes our understanding of biomolecular assembly to its limits. In such endeavors, DNA is the current building block of choice because of its predictable and programmable self-assembly. The use of peptide-and protein-based systems, however, has potential advantages due to their more-varied chemistries, structures and functions, and the prospects for recombinant production through gene synthesis and expression. Here, we present the design and characterization of two complementary peptides programmed to form a parallel heterodimeric coiled coil, which we use as the building blocks for larger, supramolecular assemblies. To achieve the latter, the two peptides are joined via peptidic linkers of variable lengths to produce a range of assemblies, from flexible fibers of indefinite length, through large colloidal-scale assemblies, down to closed and discrete nanoscale objects of defined stoichiometry. We posit that the different modes of assembly reflect the interplay between steric constraints imposed by short linkers and the bulk of the helices, and entropic factors that favor the formation of many smaller objects as the linker length is increased. This approach, and the resulting linear and proteinogenic polypeptides, represents a new route for constructing complex peptide-based assemblies and biomaterials.
RNA polymerase (RNAP) is an essential enzyme for cellular gene expression. In an effort to furthe... more RNA polymerase (RNAP) is an essential enzyme for cellular gene expression. In an effort to further understand the enzyme's importance in the cell's response to temperature, we have probed the kinetic mechanism of Escherichia coli RNAP by studying the force-velocity behavior of individual RNAP complexes at temperatures between 7 and 45°C using optical tweezers. Within this temperature range and at saturating nucleotide concentrations, the pause-free transcription velocity of RNAP was independent of force and increased monotonically with temperature with an elongation activation energy of 9.7 ± 0.7 kcal/mol. Interestingly, the pause density at cold temperatures (7 to 21°C) was five times higher than that measured above room temperature. A simple kinetic model revealed a value of 1.29 ± 0.05 kcal/mol for the activation energy of pause entry, suggesting that pause entry is indeed a thermally accessible process. The dwell time distribution of all observable pauses was independent of temperature, directly confirming a prediction of the model recently proposed for Pol II in which pauses are diffusive backtracks along the DNA. Additionally, we find that the force at which the polymerase arrests (the arrest force) presents a maximum at 21°C, an unexpected result as this is not the optimum temperature for bacterial growth. This observation suggests that arrest could play a regulatory role in vivo, possibly through interactions with specific elongation factors.
ABSTRACT Collagen is the major structural protein of our connective tissues. It provides integrit... more ABSTRACT Collagen is the major structural protein of our connective tissues. It provides integrity and mechanical strength through its hierarchical organization. Defects in collagen can lead to serious connective tissue diseases. Collagen is also widely used as a biomaterial. Given that mechanical properties are related to the structure of materials, the main goal of our research is to understand how molecular structure correlates with microscale mechanical properties of collagen solutions and networks. We use optical tweezers to trap and monitor thermal fluctuations of an embedded probe particle, from which viscoelastic properties of the solution are extracted. We find that elasticity becomes comparable to viscous behavior at collagen concentrations of 5mg/ml. Furthermore, by simultaneously neutralizing pH and adding salt, we observe changes in viscosity and elasticity of the solution over time. We attribute this to the self-assembly process of collagen molecules into fibrils with different mechanical properties. Self-assembly of collagen under these conditions is verified by turbidity measurements as well as electron microscopy. By comparing results from these local studies of viscoelasticity, we can detect spatial heterogeneity of fibril formation throughout the solution.
ABSTRACT In this talk, I will describe our senior undergraduate lab course in Biological Physics ... more ABSTRACT In this talk, I will describe our senior undergraduate lab course in Biological Physics at Simon Fraser University. Unique in Canada, this course combines modules that teach the students basic molecular and cell biology and leading-edge biophysical techniques with independent student projects. I'll outline the structure and components of the course, provide an overview of each experimental module, and describe student interest and feedback.
Probing relatively short molecules, such as the protein elastin (~200 nm), with optical tweezers ... more Probing relatively short molecules, such as the protein elastin (~200 nm), with optical tweezers requires manipulating trapped polystyrene beads at separations small compared to their micron dimensions. We discuss experimental complications arising from this proximity, such as hydrodynamic and optical interactions, and our efforts to minimize them.
Biomolecular motors have inspired the design and construction of artificial nanoscale motors and ... more Biomolecular motors have inspired the design and construction of artificial nanoscale motors and machines based on nucleic acids, small molecules, and inorganic nanostructures. However, the high degree of sophistication and efficiency of biomolecular motors, as well as their specific biological function, derives from the complexity afforded by protein building blocks. Here, we discuss a novel bottom-up approach to understanding biological
Journal of Optics A: Pure and Applied Optics, 2007
Motivated by recent attempts to use parametric resonance to calibrate the spring constant of a Br... more Motivated by recent attempts to use parametric resonance to calibrate the spring constant of a Brownian particle in an optical trap, we have looked systematically at the effects of modulating laser power on the motion of the trapped particle. We predict and find experimentally an increase in the particle's position variance at low laser modulation frequencies, but we find no evidence for any resonant effects in the extremely overdamped motion of the trapped particle. Our results can serve as a guide for designing multiple traps by the 'time-sharing' method.
Stretching and relaxing single protein molecules provides quantitative information on their elast... more Stretching and relaxing single protein molecules provides quantitative information on their elasticity and other mechanical properties. Optical tweezers can be used to perform such experiments, but require the ends of the protein to be chemically tethered to polystyrene beads used to manipulate the molecule and measure its response. Our goal is to use this method to study the human structural protein elastin, whose mechanical properties are directly related to its physiological function. Elastin's contour length is approximately 170 nm, so the trapped beads must be manipulated at separations small compared to their micron dimensions. The close proximity of the beads introduces experimental complications, primarily the result of optical and hydrodynamic interactions. We discuss these complications along with our efforts to minimize and account for them in our measurements. We then show preliminary evidence addressing a critical question of whether elastin has higher order struct...
We present an approach that will lead to an understanding of the operational characteristics of b... more We present an approach that will lead to an understanding of the operational characteristics of biological molecular motors by designing and modeling synthetic molecular motors that use a combination of natural and designed biological components. One such design is a protein-based rotationally diffusive motor dubbed the Tumbleweed (TW). This is a tri-pedal self-assembling complex in which each foot is able to bind to a unique binding site located periodically on an effective 1D track, and where the binding of each foot is controlled by a unique ligand in solution. The TW motor construct incorporates a combination of biological repressor proteins, a de-novo coiled-coil protein structure, and a dsDNA track. We present simulation results of the TW motor that exhibit unidirectional motion and a maximum theoretical speed of 0.01-0.1 mm/s, which is limited by rotational diffusion. Experimental limitations, such as the exchange of ligands in solution, will most likely reduce the motor speed to 10-100 nm/s, which is comparable to biological molecular motors.
Our understanding of biology stems from models at many resolutions, as we seek from detailed atom... more Our understanding of biology stems from models at many resolutions, as we seek from detailed atomic-scale interactions simpler emergent physical principles to support our understanding and to produce useful theoretical reductions and tractable simulations. In particular, multiscale methods coupling coarsegrained and atomic models are essential to modeling, predicting, and understanding the basic driving forces that operate across many biomolecular length and time scales. Yet, though coarse-graining strategies exist, it has been chal-
both hydrodynamic radii and optical properties were performed simultaneously. Such a dual-mode te... more both hydrodynamic radii and optical properties were performed simultaneously. Such a dual-mode technique may prove valuable for elucidating the mechanism of amyloid fibrillization and ultimately for designing possible diagnostic methods.
... Nancy R. Forde, Tanya L. Myers and Laurie J. Butler T he James Franck Institute and Departmen... more ... Nancy R. Forde, Tanya L. Myers and Laurie J. Butler T he James Franck Institute and Department of Chemistry, T he University of Chicago, Chicago, IL 60637 ... One must then analyze the remaining two-electron conÐguration interaction (Fo rster-and Dexter-type) matrix elements ...
In the past decade, the ability to manipulate and measure forces exerted by single biological mol... more In the past decade, the ability to manipulate and measure forces exerted by single biological molecules has transformed our understanding of their mechanical response, stability, and the mechanisms by which they operate. One of the essential tools in this revolution has been optical tweezers, which use a focused laser beam to ``trap'' (hold stably in three dimensions) micrometer-sized refractive particles. In this talk, I will briefly describe how optical tweezers work and how they can be used to manipulate and probe the mechanical response of single DNA and protein molecules. I will then describe the technique of holographic optical tweezers, which modify the phase of a laser beam in order to generate multiple optical traps at specified locations in three dimensions within a sample. I will discuss our work towards applying this technique to mechanical studies of protein-based biomaterials on the microscale.
The design of bioinspired nanostructures and materials of defined size and shape is challenging a... more The design of bioinspired nanostructures and materials of defined size and shape is challenging as it pushes our understanding of biomolecular assembly to its limits. In such endeavors, DNA is the current building block of choice because of its predictable and programmable self-assembly. The use of peptide-and protein-based systems, however, has potential advantages due to their more-varied chemistries, structures and functions, and the prospects for recombinant production through gene synthesis and expression. Here, we present the design and characterization of two complementary peptides programmed to form a parallel heterodimeric coiled coil, which we use as the building blocks for larger, supramolecular assemblies. To achieve the latter, the two peptides are joined via peptidic linkers of variable lengths to produce a range of assemblies, from flexible fibers of indefinite length, through large colloidal-scale assemblies, down to closed and discrete nanoscale objects of defined stoichiometry. We posit that the different modes of assembly reflect the interplay between steric constraints imposed by short linkers and the bulk of the helices, and entropic factors that favor the formation of many smaller objects as the linker length is increased. This approach, and the resulting linear and proteinogenic polypeptides, represents a new route for constructing complex peptide-based assemblies and biomaterials.
RNA polymerase (RNAP) is an essential enzyme for cellular gene expression. In an effort to furthe... more RNA polymerase (RNAP) is an essential enzyme for cellular gene expression. In an effort to further understand the enzyme's importance in the cell's response to temperature, we have probed the kinetic mechanism of Escherichia coli RNAP by studying the force-velocity behavior of individual RNAP complexes at temperatures between 7 and 45°C using optical tweezers. Within this temperature range and at saturating nucleotide concentrations, the pause-free transcription velocity of RNAP was independent of force and increased monotonically with temperature with an elongation activation energy of 9.7 ± 0.7 kcal/mol. Interestingly, the pause density at cold temperatures (7 to 21°C) was five times higher than that measured above room temperature. A simple kinetic model revealed a value of 1.29 ± 0.05 kcal/mol for the activation energy of pause entry, suggesting that pause entry is indeed a thermally accessible process. The dwell time distribution of all observable pauses was independent of temperature, directly confirming a prediction of the model recently proposed for Pol II in which pauses are diffusive backtracks along the DNA. Additionally, we find that the force at which the polymerase arrests (the arrest force) presents a maximum at 21°C, an unexpected result as this is not the optimum temperature for bacterial growth. This observation suggests that arrest could play a regulatory role in vivo, possibly through interactions with specific elongation factors.
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