Showing papers by "Carlos Bustamante published in 2000"

Single-molecule studies of the effect of template tension on T7 DNA polymerase activity

Abstract: T7 DNA polymerase1,2 catalyses DNA replication in vitro at rates of more than 100 bases per second and has a 3′→5′ exonuclease (nucleotide removing) activity at a separate active site. This enzyme possesses a ‘right hand’ shape which is common to most polymerases with fingers, palm and thumb domains3,4. The rate-limiting step for replication is thought to involve a conformational change between an ‘open fingers’ state in which the active site samples nucleotides, and a ‘closed’ state in which nucleotide incorporation occurs3,5. DNA polymerase must function as a molecular motor converting chemical energy into mechanical force as it moves over the template. Here we show, using a single-molecule assay based on the differential elasticity of single-stranded and double-stranded DNA, that mechanical force is generated during the rate-limiting step and that the motor can work against a maximum template tension of ∼34 pN. Estimates of the mechanical and entropic work done by the enzyme show that T7 DNA polymerase organizes two template bases in the polymerization site during each catalytic cycle. We also find a force-induced 100-fold increase in exonucleolysis above 40 pN.

Pulling a single chromatin fiber reveals the forces that maintain its higher-order structure

Abstract: Single chicken erythrocyte chromatin fibers were stretched and released at room temperature with force-measuring laser tweezers. In low ionic strength, the stretch-release curves reveal a process of continuous deformation with little or no internucleosomal attraction. A persistence length of 30 nm and a stretch modulus of ≈5 pN is determined for the fibers. At forces of 20 pN and higher, the fibers are modified irreversibly, probably through the mechanical removal of the histone cores from native chromatin. In 40–150 mM NaCl, a distinctive condensation-decondensation transition appears between 5 and 6 pN, corresponding to an internucleosomal attraction energy of ≈2.0 kcal/mol per nucleosome. Thus, in physiological ionic strength the fibers possess a dynamic structure in which the fiber locally interconverting between “open” and “closed” states because of thermal fluctuations.

Grabbing the cat by the tail: manipulating molecules one by one.

Abstract: Methods for manipulating single molecules are yielding new information about both the forces that hold biomolecules together and the mechanics of molecular motors. We describe here the physical principles behind these methods, and discuss their capabilities and current limitations.

of the effect of template tension on T7 DNA polymerase activity

Abstract: T7 DNA polymerase catalyses DNA replication in vitro at rates of more than 100 bases per second and has a 3′→5′ exonuclease (nucleotide removing) activity at a separate active site. This enzyme possesses a ‘right hand’ shape which is common to most polymerases with fingers, palm and thumb domains. The rate-limiting step for replication is thought to involve a conformational change between an ‘open fingers’ state in which the active site samples nucleotides, and a ‘closed’ state in which nucleotide incorporation occurs. DNA polymerase must function as a molecular motor converting chemical energy into mechanical force as it moves over the template. Here we show, using a single-molecule assay based on the differential elasticity of single-stranded and double-stranded DNA, that mechanical force is generated during the rate-limiting step and that the motor can work against a maximum template tension of ∼34 pN. Estimates of the mechanical and entropic work done by the enzyme show that T7 DNA polymerase organizes two template bases in the polymerization site during each catalytic cycle. We also find a force-induced 100-fold increase in exonucleolysis above 40 pN.

Single-molecule study of transcriptional pausing and arrest by E. coli RNA polymerase.

Abstract: Using an optical-trap/flow-control video microscopy technique, we followed transcription by single molecules of Escherichia coli RNA polymerase in real time over long template distances. These studies reveal that RNA polymerase molecules possess different intrinsic transcription rates and different propensities to pause and stop. The data also show that reversible pausing is a kinetic intermediate between normal elongation and the arrested state. The conformational metastability of RNA polymerase revealed by this single-molecule study of transcription has direct implications for the mechanisms of gene regulation in both bacteria and eukaryotes.

Solid-state synthesis and mechanical unfolding of polymers of T4 lysozyme

Abstract: Recent advances in single molecule manipulation methods offer a novel approach to investigating the protein folding problem. These studies usually are done on molecules that are naturally organized as linear arrays of globular domains. To extend these techniques to study proteins that normally exist as monomers, we have developed a method of synthesizing polymers of protein molecules in the solid state. By introducing cysteines at locations where bacteriophage T4 lysozyme molecules contact each other in a crystal and taking advantage of the alignment provided by the lattice, we have obtained polymers of defined polarity up to 25 molecules long that retain enzymatic activity. These polymers then were manipulated mechanically by using a modified scanning force microscope to characterize the force-induced reversible unfolding of the individual lysozyme molecules. This approach should be general and adaptable to many other proteins with known crystal structures. For T4 lysozyme, the force required to unfold the monomers was 64 ± 16 pN at the pulling speed used. Refolding occurred within 1 sec of relaxation with an efficiency close to 100%. Analysis of the force versus extension curves suggests that the mechanical unfolding transition follows a two-state model. The unfolding forces determined in 1 M guanidine hydrochloride indicate that in these conditions the activation barrier for unfolding is reduced by 2 kcal/mol.

Solvent Accessibility and Purifying Selection Within Proteins of Escherichia coli and Salmonella enterica

Abstract: The neutral theory of molecular evolution predicts that variation within species is inversely related to the strength of purifying selection, but the strength of purifying selection itself must be related to physical constraints imposed by protein folding and function. In this paper, we analyzed five enzymes for which polymorphic sequence variation within Escherichia coli and/or Salmonella enterica was available, along with a protein structure. Single and multivariate logistic regression models are presented that evaluate amino acid size, physicochemical properties, solvent accessibility, and secondary structure as predictors of polymorphism. A model that contains a positive coefficient of association between polymorphism and solvent accessibility and separate intercepts for each secondary-structure element is sufficient to explain the observed variation in polymorphism between sites. The model predicts an increase in the probability of amino acid polymorphism with increasing solvent accessibility for each protein regardless of physicochemical properties, secondary-structure element, or size of the amino acid. This result, when compared with the distribution of synonymous polymorphism, which shows no association with solvent accessibility, suggests a strong decrease in purifying selection with increasing solvent accessibility.

Mechanical manipulation of single titin molecules with laser tweezers.

Abstract: Titin (also known as connectin) is a giant filamentous polypeptide of multi-domain construction spanning between the Z- and M-lines of the vertebrate muscle sarcomere. The molecule is significant in maintaining sarcomeric structural integrity and generating passive muscle force via its elastic properties. Here we summarize our efforts to characterize titin’s elastic properties by manipulating single molecules with force-measuring laser tweezers. The titin molecule can be described as an entropic spring in which domain unfolding occurs at high forces during stretch and refolding at low forces during release. Statistical analysis of a large number (>500) of stretch-release experiments and comparison of experimental data with the predictions of the wormlike chain theory permit the estimation of unfolded titin’s mean persistence length as 16.86 A (±0.11 SD). The slow rates of unfolding and refolding compared with the rates of stretch and release, respectively, result in a state of non-equilibrium and the display of force hysteresis. Folding kinetics as the source of non-equilibrium is directly demonstrated here by the abolishment of force hysteresis in the presence of chemical denaturant. Experimental observations were well simulated by superimposing a simple domain folding kinetics model on the wormlike chain behavior of titin and considering the characteristics of the compliant laser trap. The original video presentation of this paper may be viewed on the web at http://www.pote.hu/ mm/prezentacio/mkpres/mkpres. htm.URL