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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.

The separation of nanoparticles from micron size particles in high conductance buffers was achieved using an AC dielectrophoretic (DEP) microarray device with hydrogel over-coated microelectrodes. While nanoparticles could be selectively concentrated into high field regions directly over the platinum microelectrodes, micro-bubbling and electrode darkening was also observed. For similar experiments using un-coated microelectrodes, SEM analysis showed severe erosion of the platinum microelectrodes and fusion of nanoparticles due to the aggressive electrochemistry.

DNA looping occurs in many important protein-DNA interactions, including those regulating replication, transcription, and recombination. Recent theoretical studies predict that tension of only a few piconewtons acting on DNA would almost completely inhibit DNA looping. Here, we study restriction endonucleases that require interaction at two separated sites for efficient cleavage. Using optical tweezers we measured the dependence of cleavage activity on DNA tension with 15 known or suspected two-site enzymes (BfiI, BpmI, BsgI, BspMI, Cfr9I, Cfr10I, Eco57I, EcoRII, FokI, HpaII, MboII, NarI, SacII, Sau3AI, and SgrAI) and six one-site enzymes (BamHI, EcoRI, EcoRV, HaeIII, HindIII, and DNaseI). All of the one-site enzymes were virtually unaffected by 5 pN of tension, whereas all of the two-site enzymes were completely inhibited. These enzymes thus constitute a remarkable example of a tension sensing ''molecular switch.'' A detailed study of one enzyme, Sau3AI, indicated that the activity decreased exponentially with tension and the decrease was Ϸ10-fold at 0.7 pN. At higher forces (Ϸ20 -40 pN) cleavage by the one-site enzymes EcoRV and HaeIII was partly inhibited and cleavage by HindIII was enhanced, whereas BamHI, EcoRI, and DNaseI were largely unaffected. These findings correlate with structural data showing that EcoRV bends DNA sharply, whereas BamHI, EcoRI, and DNaseI do not. Thus, DNA-directed enzyme activity involving either DNA looping or bending can be modulated by tension, a mechanism that could facilitate mechanosensory transduction in vivo.DNA looping ͉ protein-DNA interactions ͉ single-molecule manipulation R estriction endonucleases (REases) are prokaryotic enzymes that act to ''restrict'' invasion of foreign DNA by cleaving phosphodiester bonds (1). These enzymes also serve as indispensable tools in molecular biology research and are used in procedures such as DNA cloning, fingerprinting, mapping, and sequencing (2). From the perspective of molecular biophysics, these enzymes are excellent model systems for studying basic principles of protein-DNA interactions (3, 4).The most commonly studied REases are of the type II variety, which cleave within or near specific recognition sites, usually require Mg 2ϩ ions as a cofactor, and do not hydrolyze ATP. More than 3,500 different type II REases having Ͼ200 different binding specificities have been identified (5). Of particular interest in our present study are the many unorthodox type II REases that do not cleave DNA efficiently if the template contains only one recognition site (6, 7). Efficient cleavage is only observed with templates containing two or more sites, suggesting that the active complex binds at two sites and the intervening DNA is looped out (7). This phenomenon of DNA looping is of broad importance in molecular biology and plays a role in many key processes including DNA transcription, replication, recombination, and repair (8-13). Looping of DNA by the Lac and Gal repressor proteins in Escherichia coli, for example, is well demonstrated and h...

Proteins interacting at multiple sites on DNA via looping play an important role in many fundamental biochemical processes. Restriction endonucleases that must bind at two recognition sites for efficient activity are a useful model system for studying such interactions. Here we used single DNA manipulation to study sixteen known or suspected two-site endonucleases. In eleven cases (BpmI, BsgI, BspMI, Cfr10I, Eco57I, EcoRII, FokI, HpaII, NarI, Sau3AI and SgrAI) we found that substitution of Ca2+ for Mg2+ blocked cleavage and enabled us to observe stable DNA looping. Forced disruption of these loops allowed us to measure the frequency of looping and probability distributions for loop size and unbinding force for each enzyme. In four cases we observed bimodal unbinding force distributions, indicating conformational heterogeneity and/or complex binding energy landscapes. Measured unlooping events ranged in size from 7 to 7500 bp and the most probable size ranged from less than 75 bp to nearly 500 bp, depending on the enzyme. In most cases the size distributions were in much closer agreement with theoretical models that postulate sharp DNA kinking than with classical models of DNA elasticity. Our findings indicate that DNA looping is highly variable depending on the specific protein and does not depend solely on the mechanical properties of DNA.

The functioning of cytochrome c oxidases involves orchestration of long-range electron transfer (ET) events among the four redox active metal centers. We report the temperature dependence of electron transfer from the Cu(A)(r) site to the low-spin heme-(a)b(o) site, i.e., Cu(A)(r) + heme-a(b)(o) → Cu(A)(o) + heme-a(b)(r) in three structurally characterized enzymes: A-type aa(3) from Paracoccus denitrificans (PDB code 3HB3 ) and bovine heart tissue (PDB code 2ZXW ), and the B-type ba(3) from T. thermophilus (PDB codes 1EHK and 1XME ). k,T data sets were obtained with the use of pulse radiolysis as described previously. Semiclassical Marcus theory revealed that λ varies from 0.74 to 1.1 eV, H(ab), varies from ∼2 × 10(-5) eV (0.16 cm(-1)) to ∼24 × 10(-5) eV (1.9 cm(-1)), and βD varies from 9.3 to 13.9. These parameters are consistent with diabatic electron tunneling. The II-Asp111Asn Cu(A) mutation in cytochrome ba(3) had no effect on the rate of this reaction whereas the II-Met160Leu Cu(A)-mutation was slower by an amount corresponding to a decreased driving force of ∼0.06 eV. The structures support the presence of a common, electron-conducting "wire" between Cu(A) and heme-a(b). The transfer of an electron from the low-spin heme to the high-spin heme, i.e., heme-a(b)(r) + heme-a(3)(o) → heme-a(b)(o) + heme-a(3)(r), was not observed with the A-type enzymes in our experiments but was observed with the Thermus ba(3); its Marcus parameters are λ = 1.5 eV, H(ab) = 26.6 × 10(-5) eV (2.14 cm(-1)), and βD = 9.35, consistent also with diabatic electron tunneling between the two hemes. The II-Glu15Ala mutation of the K-channel structure, ∼24 Å between its CA and Fe-a(3), was found to completely block heme-b(r) to heme-a(3)(o) electron transfer. A structural mechanism is suggested to explain these observations.

Mechanical manipulation of single DNA molecules can provide novel information about DNA properties and protein-DNA interactions. Here we describe and characterize a useful method for manipulating desired DNA sequences from any organism with optical tweezers. Molecules are produced from either genomic or cloned DNA by PCR using labeled primers and are tethered between two optically trapped microspheres. We demonstrate that human, insect, plant, bacterial and viral sequences ranging from $10 to 40 kilobasepairs can be manipulated. Forceextension measurements show that these constructs exhibit uniform elastic properties in accord with the expected contour lengths for the targeted sequences. Detailed protocols for preparing and manipulating these molecules are presented, and tethering efficiency is characterized as a function of DNA concentration, ionic strength and pH. Attachment strength is characterized by measuring the unbinding time as a function of applied force. An alternative stronger attachment method using an aminocarboxyl linkage, which allows for reliable DNA overstretching, is also described.

Looping and cleavage of single DNA molecules by the two-site restriction endonuclease Sau3AI were measured with optical tweezers. A DNA template containing many recognition sites was used, permitting loop sizes from approximately 10 to 10,000 basepairs. At high enzyme concentration, cleavage events were detected within 5 s and nearly all molecules were cleaved within 5 min. Activity decreased approximately 10-fold as the DNA tension was increased from 0.03 to 0.7 pN. Substituting Ca(2+) for Mg(2+) blocked cleavage, permitting measurement of stable loops. At low tension, the initial rates of cleavage and looping were similar (approximately 0.025 s(-1) at 0.1 pN), suggesting that looping is rate limiting. Short loops formed more rapidly than long loops. The optimum size decreased from approximately 250 to 45 basepairs and the average number of loops (in 1 min) from 4.2 to 0.75 as tension was increased from 0.03 to 0.7 pN. No looping was detected at 5 pN. These findings are in qualitative agreement with recent theoretical predictions considering only DNA mechanics, but we observed weaker suppression with tension and smaller loop sizes. Our results suggest that the span and elasticity of the protein complex, nesting of loops, and protein-induced DNA bending and wrapping play an important role.