| 1. |
- Boyle, Aimee L., et al.
(författare)
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Squaring the Circle in Peptide Assembly: From Fibers to Discrete Nanostructures by de Novo Design
- 2012
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 134:37, s. 15457-15467
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 2. |
- Bromley, Elizabeth H. C., et al.
(författare)
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The Tumbleweed: towards a synthetic protein motor
- 2009
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Ingår i: HFSP Journal. - : Informa UK Limited. - 1955-2068. ; 3:3, s. 204-212
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Tidskriftsartikel (refereegranskat)abstract
- 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 motors by considering the construction of synthetic protein motors. Specifically, we present a design for a synthetic protein motor that moves along a linear track, dubbed the "Tumbleweed." This concept uses three discrete ligand-dependent DNA-binding domains to perform cyclically ligand-gated, rectified diffusion along a synthesized DNA molecule. Here we describe how de novo peptide design and molecular biology could be used to produce the Tumbleweed, and we explore the fundamental motor operation of such a design using numerical simulations. The construction of this and more sophisticated protein motors is an exciting challenge that is likely to enhance our understanding of the structure-function relationship in biological motors. [DOI: 10.2976/1.3111282]
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| 3. |
- Korosec, Chapin S., et al.
(författare)
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Motility of an autonomous protein-based artificial motor that operates via a burnt-bridge principle
- 2024
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Ingår i: Nature Communications. - : Springer Nature. - 2041-1723. ; 15:1
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Tidskriftsartikel (refereegranskat)abstract
- Inspired by biology, great progress has been made in creating artificial molecular motors. However, the dream of harnessing proteins – the building blocks selected by nature – to design autonomous motors has so far remained elusive. Here we report the synthesis and characterization of the Lawnmower, an autonomous, protein-based artificial molecular motor comprised of a spherical hub decorated with proteases. Its “burnt-bridge” motion is directed by cleavage of a peptide lawn, promoting motion towards unvisited substrate. We find that Lawnmowers exhibit directional motion with average speeds of up to 80 nm/s, comparable to biological motors. By selectively patterning the peptide lawn on microfabricated tracks, we furthermore show that the Lawnmower is capable of track-guided motion. Our work opens an avenue towards nanotechnology applications of artificial protein motors.
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| 4. |
- Kovacic, Suzana, et al.
(författare)
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Construction and Characterization of Kilobasepair Densely Labeled Peptide-DNA
- 2014
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Ingår i: Biomacromolecules. - : American Chemical Society (ACS). - 1526-4602 .- 1525-7797. ; 15:11, s. 4065-4072
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Tidskriftsartikel (refereegranskat)abstract
- Directed assembly of biocompatible materials benefits from modular building blocks in which structural organization is independent of introduced functional modifications. For soft materials, such modifications have been limited. Here, long DNA is successfully functionalized with dense decoration by peptides. Following introduction of alkyne-modified nucleotides into kilobasepair DNA, measurements of persistence length show that DNA mechanics are unaltered by the dense incorporation of alkynes (similar to 1 alkyne/2 bp) and after click-chemistry attachment of a tunable density of peptides. Proteolytic cleavage of densely tethered peptides (similar to 1 peptide/3 bp) demonstrates addressability of the functional groups, showing that this accessible approach to creating hybrid structures can maintain orthogonality between backbone mechanics and overlaid function. The synthesis and characterization of these hybrid constructs establishes the groundwork for their implementation in future applications, such as building blocks in modular approaches to a range of problems in synthetic biology.
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| 5. |
- Kovacic, Suzana, et al.
(författare)
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Design and Construction of a One- Dimensional DNA Track for an Artificial Molecular Motor
- 2012
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Ingår i: Journal of Nanomaterials. - : Hindawi Limited. - 1687-4129 .- 1687-4110.
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Tidskriftsartikel (refereegranskat)abstract
- DNA is a versatile heteropolymer that shows great potential as a building block for a diverse array of nanostructures. We present here a solution to the problem of designing and synthesizing a DNA-based nanostructure that will serve as the track along which an artificial molecular motor processes. This one-dimensional DNA track exhibits periodically repeating elements that provide specific binding sites for the molecular motor. Besides these binding elements, additional sequences are necessary to label specific regions within the DNA track and to facilitate track construction. Designing an ideal DNA track sequence presents a particular challenge because of the many variable elements that greatly expand the number of potential sequences from which the ideal sequence must be chosen. In order to find a suitable DNA sequence, we have adapted a genetic algorithm which is well suited for a large but sparse search space. This algorithm readily identifies long DNA sequences that include all the necessary elements to both facilitate DNA track construction and to present appropriate binding sites for the molecular motor. We have successfully experimentally incorporated the sequence identified by the algorithm into a long DNA track meeting the criteria for observation of the molecular motor's activity.
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| 6. |
- Kovacic, Suzana, et al.
(författare)
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Design and Construction of the Lawnmower, An Artificial Burnt-Bridges Motor
- 2015
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Ingår i: IEEE Transactions on Nanobioscience. - 1558-2639. ; 14:3, s. 305-312
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Tidskriftsartikel (refereegranskat)abstract
- Molecular motors of the cell are protein-based, nanoscale machines, which use a variety of strategies to transduce chemical energy into mechanical work in the presence of a large thermal background. The design and construction of artificial molecular motors is one approach to better understand their basic physical principles. Here, we propose the concept of a protein-based, burnt-bridges ratchet, inspired by biological examples. Our concept, the lawnmower, utilizes protease blades to cleave peptide substrates, and uses the asymmetric substrate-product interface arising from productive cleavage to bias subsequent diffusion on the track (lawn). Following experimental screening to select a protease to act as the motor's blades, we chemically couple trypsin to quantum dots and demonstrate activity of the resulting lawnmower construct in solution. Accompanying Brownian dynamics simulations illustrate the importance for processivity of correct protease density on the quantum dot and spacing of substrates on the track. These results lay the groundwork for future tests of the protein-based lawnmower's motor performance characteristics.
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| 7. |
- Kuwada, Nathan J., et al.
(författare)
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Tuning the performance of an artificial protein motor
- 2011
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Ingår i: Physical Review E (Statistical, Nonlinear, and Soft Matter Physics). - 1539-3755. ; 84:3
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Tidskriftsartikel (refereegranskat)abstract
- The Tumbleweed (TW) is a concept for an artificial, tri-pedal, protein-based motor designed to move unidirectionally along a linear track by a diffusive tumbling motion. Artificial motors offer the unique opportunity to explore how motor performance depends on design details in a way that is open to experimental investigation. Prior studies have shown that TW's ability to complete many successive steps can be critically dependent on the motor's diffusional step time. Here, we present a simulation study targeted at determining how to minimize the diffusional step time of the TW motor as a function of two particular design choices: nonspecific motor-track interactions and molecular flexibility. We determine an optimal nonspecific interaction strength and establish a set of criteria for optimal molecular flexibility as a function of the nonspecific interaction. We discuss our results in the context of similarities to biological, linear stepping diffusive molecular motors with the aim of identifying general engineering principles for protein motors.
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| 8. |
- Linke, Heiner, et al.
(författare)
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Synthetic biology approaches to dissecting linear motor protein function : towards the design and synthesis of artificial autonomous protein walkers
- 2020
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Ingår i: Biophysical Reviews. - : Springer Science and Business Media LLC. - 1867-2450 .- 1867-2469. ; 12:4, s. 1041-1054
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Forskningsöversikt (refereegranskat)abstract
- Molecular motors and machines are essential for all cellular processes that together enable life. Built from proteins with a wide range of properties, functionalities and performance characteristics, biological motors perform complex tasks and can transduce chemical energy into mechanical work more efficiently than human-made combustion engines. Sophisticated studies of biological protein motors have provided many structural and biophysical insights and enabled the development of models for motor function. However, from the study of highly evolved, biological motors, it remains difficult to discern detailed mechanisms, for example, about the relative role of different force generation mechanisms, or how information is communicated across a protein to achieve the necessary coordination. A promising, complementary approach to answering these questions is to build synthetic protein motors from the bottom up. Indeed, much effort has been invested in functional protein design, but so far, the “holy grail” of designing and building a functional synthetic protein motor has not been realized. Here, we review the progress made to date, and we put forward a roadmap for achieving the aim of constructing the first artificial, autonomously running protein motor. Specifically, we propose to break down the task into (i) enzymatic control of track binding, (ii) the engineering of asymmetry and (iii) the engineering of allosteric control for internal communication. We also propose specific approaches for solving each of these challenges.
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| 9. |
- Niman, Cassandra, et al.
(författare)
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Controlled microfluidic switching in arbitrary time-sequences with low drag.
- 2013
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Ingår i: Lab on a Chip. - : Royal Society of Chemistry (RSC). - 1473-0189 .- 1473-0197. ; 13:12, s. 2389-2396
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Tidskriftsartikel (refereegranskat)abstract
- The ability to test the response of cells and proteins to a changing biochemical environment is of interest for studies of fundamental cell physiology and molecular interactions. In a common experimental scheme the cells or molecules of interest are attached to a surface and the composition of the surrounding fluid is changed. It is desirable to be able to switch several different biochemical reagents in any arbitrary order, and to keep the flow velocity low enough so that the cells and molecules remain attached and can be expected to retain their function. Here we develop a device with these capabilities, using U-shaped access channels. We use total-internal reflection fluorescence microscopy to characterize the time-dependent change in concentration during switching of solutions near the device surface. Well-defined fluid interfaces are formed in the immediate vicinity of the surface ensuring distinct switching events. We show that the experimental data agrees well with Taylor-Aris theory in its range of validity. In addition, we find that well-defined interfaces are achieved also in the immediate vicinity of the surface, where analytic approaches and numerical models become inaccurate. Assisted by finite-element modelling, the details of our device were designed for use with a specific artificial protein motor, but the key results are general and can be applied to a wide range of biochemical studies in which switching is important.
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| 10. |
- Niman, Cassandra, et al.
(författare)
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Fluidic switching in nanochannels for the control of Inchworm: a synthetic biomolecular motor with a power stroke.
- 2014
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Ingår i: Nanoscale. - : Royal Society of Chemistry (RSC). - 2040-3372 .- 2040-3364. ; 6:24, s. 15008-15019
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Tidskriftsartikel (refereegranskat)abstract
- Synthetic molecular motors typically take nanometer-scale steps through rectification of thermal motion. Here we propose Inchworm, a DNA-based motor that employs a pronounced power stroke to take micrometer-scale steps on a time scale of seconds, and we design, fabricate, and analyze the nanofluidic device needed to operate the motor. Inchworm is a kbp-long, double-stranded DNA confined inside a nanochannel in a stretched configuration. Motor stepping is achieved through externally controlled changes in salt concentration (changing the DNA's extension), coordinated with ligand-gated binding of the DNA's ends to the functionalized nanochannel surface. Brownian dynamics simulations predict that Inchworm's stall force is determined by its entropic spring constant and is ∼0.1 pN. Operation of the motor requires periodic cycling of four different buffers surrounding the DNA inside a nanochannel, while keeping constant the hydrodynamic load force on the DNA. We present a two-layer fluidic device incorporating 100 nm-radius nanochannels that are connected through a few-nm-wide slit to a microfluidic system used for in situ buffer exchanges, either diffusionally (zero flow) or with controlled hydrodynamic flow. Combining experiment with finite-element modeling, we demonstrate the device's key performance features and experimentally establish achievable Inchworm stepping times of the order of seconds or faster.
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