Steven M. Block

Stretching DNA with optical tweezers.

Force-extension (F-x) relationships were measured for single molecules of DNA under a variety of buffer conditions, using an optical trapping interferometer modified to incorporate feedback control. One end of a single DNA molecule was fixed to a coverglass surface by means of a stalled RNA polymerase complex. The other end was linked to a microscopic bead, which was captured and held in an optical trap. The DNA was subsequently stretched by moving the coverglass with respect to the trap using a piezo-driven stage, while the position of the bead was recorded at nanometer-scale resolution. An electronic feedback circuit was activated to prevent bead movement beyond a preset clamping point by modulating the light intensity, altering the trap stiffness dynamically. This arrangement permits rapid determination of the F-x relationship for individual DNA molecules as short as -1 micron with unprecedented accuracy, subjected to both low (approximately 0.1 pN) and high (approximately 50 pN) loads: complete data sets are acquired in under a minute. Experimental F-x relationships were fit over much of their range by entropic elasticity theories based on worm-like chain models. Fits yielded a persistence length, Lp, of approximately 47 nm in a buffer containing 10 mM Na1. Multivalent cations, such as Mg2+ or spermidine 3+, reduced Lp to approximately 40 nm. Although multivalent ions shield most of the negative charges on the DNA backbone, they did not further reduce Lp significantly, suggesting that the intrinsic persistence length remains close to 40 nm. An elasticity theory incorporating both enthalpic and entropic contributions to stiffness fit the experimental results extremely well throughout the full range of extensions and returned an elastic modulus of approximately 1100 pN.

Single kinesin molecules studied with a molecular force clamp.

Kinesin is a two-headed, ATP-driven motor protein that moves processively along microtubules in discrete steps of 8 nm, probably by advancing each of its heads alternately in sequence. Molecular details of how the chemical energy stored in ATP is coupled to mechanical displacement remain obscure. To shed light on this question, a force clamp was constructed, based on a feedback-driven optical trap capable of maintaining constant loads on single kinesin motors. The instrument provides unprecedented resolution of molecular motion and permits mechanochemical studies under controlled external loads. Analysis of records of kinesin motion under variable ATP concentrations and loads revealed several new features. First, kinesin stepping appears to be tightly coupled to ATP hydrolysis over a wide range of forces, with a single hydrolysis per 8-nm mechanical advance. Second, the kinesin stall force depends on the ATP concentration. Third, increased loads reduce the maximum velocity as expected, but also raise the apparent Michaelis–Menten constant. The kinesin cycle therefore contains at least one load-dependent transition affecting the rate at which ATP molecules bind and subsequently commit to hydrolysis. It is likely that at least one other load-dependent rate exists, affecting turnover number. Together, these findings will necessitate revisions to our understanding of how kinesin motors function.

Force and velocity measured for single kinesin molecules

We measured the force-velocity curves of single kinesin molecules attached to silica beads moving in an in vitro motility assay. Optical trapping interferometry was used to track movement with subnanometer precision and to apply calibrated, pN-sized forces to the beads. Velocity decreased linearly with increasing force, and kinesin molecules moved against applied loads of up to 5-6 pN. Comparison of force-velocity curves at limiting and saturating ATP concentrations suggests that the load-dependent diminution in kinesin velocity may be due to a decrease in the net displacement per molecule of ATP hydrolyzed, not simply to a slowing of the ATP turnover rate; kinesin would therefore appear to be a loosely coupled motor.

Direct observation of base-pair stepping by RNA polymerase

During transcription, RNA polymerase (RNAP) moves processively along a DNA template, creating a complementary RNA. Here we present the development of an ultra-stable optical trapping system with ångström-level resolution, which we used to monitor transcriptional elongation by single molecules of Escherichia coli RNAP. Records showed discrete steps averaging 3.7 ± 0.6 Å, a distance equivalent to the mean rise per base found in B-DNA. By combining our results with quantitative gel analysis, we conclude that RNAP advances along DNA by a single base pair per nucleotide addition to the nascent RNA. We also determined the force–velocity relationship for transcription at both saturating and sub-saturating nucleotide concentrations; fits to these data returned a characteristic distance parameter equivalent to one base pair. Global fits were inconsistent with a model for movement incorporating a power stroke tightly coupled to pyrophosphate release, but consistent with a brownian ratchet model incorporating a secondary NTP binding site.

Characterization of photodamage to Escherichia coli in optical traps.

Optical tweezers (infrared laser-based optical traps) have emerged as a powerful tool in molecular and cell biology. However, their usefulness has been limited, particularly in vivo, by the potential for damage to specimens resulting from the trapping laser. Relatively little is known about the origin of this phenomenon. Here we employed a wavelength-tunable optical trap in which the microscope objective transmission was fully characterized throughout the near infrared, in conjunction with a sensitive, rotating bacterial cell assay. Single cells of Escherichia coli were tethered to a glass coverslip by means of a single flagellum: such cells rotate at rates proportional to their transmembrane proton potential (Manson et al.,1980. J. Mol. Biol. 138:541-561). Monitoring the rotation rates of cells subjected to laser illumination permits a rapid and quantitative measure of their metabolic state. Employing this assay, we characterized photodamage throughout the near-infrared region favored for optical trapping (790-1064 nm). The action spectrum for photodamage exhibits minima at 830 and 970 nm, and maxima at 870 and 930 nm. Damage was reduced to background levels under anaerobic conditions, implicating oxygen in the photodamage pathway. The intensity dependence for photodamage was linear, supporting a single-photon process. These findings may help guide the selection of lasers and experimental protocols best suited for optical trapping work.

Kinesin hydrolyses one ATP per 8-nm step

Kinesin is a two-headed, ATP-dependent motor protein that moves along microtubules indiscrete steps of 8 nm. In vitro, single molecules produceprocessive movement, motors typically take ∼100steps before releasing from a microtubule . A central question relates tomechanochemical coupling in this enzyme: how many molecules ofATP are consumed per step? For the actomyosin system,experimental approaches to this issue have generated considerablecontroversy. Here we take advantage of theprocessivity of kinesin to determine the coupling ratio withoutrecourse to direct measurements of ATPase activity, which aresubject to large experimental uncertainties. Beads carrying singlemolecules of kinesin moving on microtubules were tracked with highspatial and temporal resolution by interferometry,. Statistical analysis of theintervals between steps at limiting ATP, and studies offluctuations in motor speed as a function of ATPconcentration, allow the coupling ratio to bedetermined. At near-zero load, kinesin moleculeshydrolyse a single ATP molecule per 8-nm advance. Thisfinding excludes various one-to-many andmany-to-one coupling schemes, analogous to thoseadvanced for myosin, and places severe constraints on models for movement.

Optical trapping of metallic Rayleigh particles

Metallic objects reflect light and have generally been considered poor candidates for optical traps, particularly with optical tweezers, which rely on a gradient force to provide trapping. We demonstrate that stable trapping can occur with optical tweezers when they are used with small metallic Rayleigh particles. In this size regime, the scattering pictures for metals and dielectrics are similar, and the larger polarizability of metals implies that trapping forces are greater. The latter fact makes the use of metal particles attractive for certain biological applications. Comparison of trapping forces for latex and gold spheres demonstrates that the gradient force is the major determinant of trapping strength and that competing effects, such as scattering or radiometric forces, are relatively minor.

Transcription Against an Applied Force

The force produced by a single molecule of Escherichia coli RNA polymerase during transcription was measured optically. Polymerase immobilized on a surface was used to transcribe a DNA template attached to a polystyrene bead 0.5 micrometer in diameter. The bead position was measured by interferometry while a force opposing translocation of the polymerase along the DNA was applied with an optical trap. At saturating nucleoside triphosphate concentrations, polymerase molecules stalled reversibly at a mean applied force estimated to be 14 piconewtons. This force is substantially larger than those measured for the cytoskeletal motors kinesin and myosin and exceeds mechanical loads that are estimated to oppose transcriptional elongation in vivo. The data are consistent with efficient conversion of the free energy liberated by RNA synthesis into mechanical work.

Estimating the Persistence Length of a Worm-Like Chain Molecule from Force-Extension Measurements

We describe a simple computation of the worm-like chain model and obtain the corresponding force-versus-extension curve. We propose an improvement to the Marko and Siggia interpolation formula of Bustamante et al (Science 1994, 265:1599-1600) that is useful for fitting experimental data. We apply it to the experimental elasticity curve of single DNA molecules. Finally, we present a tool to study the agreement between the worm-like chain model and experiments.

Force production by single kinesin motors

Motor proteins such as kinesin, myosin and polymerase convert chemical energy into work through a cycle that involves nucleotide hydrolysis. Kinetic rates in the cycle that depend upon load identify transitions at which structural changes, such as power strokes or diffusive motions, are likely to occur. Here we show, by modelling data obtained with a molecular force clamp, that kinesin mechanochemistry can be characterized by a mechanism in which a load-dependent isomerization follows ATP binding. This model quantitatively accounts for velocity data over a wide range of loads and ATP levels, and indicates that movement may be accomplished through two sequential 4-nm substeps. Similar considerations account for kinesin processivity, which is found to obey a load-dependent Michaelis–Menten relationship.