Single-molecule studies of nucleic acid motors

Nucleic acid motors comprise a variety of structurally, mechanistically and functionally very different enzymes. These motor proteins have in common the ability to directionally move DNA or RNA, or to move along DNA or RNA using a chemical energy source such as ATP. Recently, it became possible to study the action of a single motor on single DNA or RNA molecules in real time; this has provided unprecedented insight into the behavior and mechanism of these motors. As a result, the past few years have witnessed an enormous increase in such single-molecule studies of a variety of different motor systems. Particular highlights have included the investigation of the sequence-dependent behavior and helical tracking of motors, and the attainment of the ultimate (i.e. single base pair) resolution, which enables the detection of individual single base motor steps.     

Kinetics: a tool to study molecular motors

Molecular motors are enzymes that couple the energy from nucleoside triphosphate hydrolysis to movement along a filament lattice. The three cytoskeletal motor superfamilies include myosin, dynein, and kinesin. However, in the last decade it has become apparent that the nucleic acid-based enzymes (DNA and RNA polymerases as well as the DNA helicases) share a number of mechanistic features in common with the microtubule and actin motors despite the fact that their cellular functions are so different. This review addresses the mechanistic approaches that have been used to study molecular motors. We discuss the basic biochemical techniques used to characterize a protein preparation, including active site determination and steady-state kinetics. In addition, we present the transient-state kinetic approaches used to define a mechanochemical cycle. We attempt to integrate the information obtained from kinetic studies within the context of motility results to provide a better understanding of the contribution of each approach for dissecting unidirectional force generation.     

Single-molecule fluorescence imaging: Generating insights into molecular interactions in virology

Single-molecule fluorescence methods remain a challenging yet information-rich set of techniques that allow one to probe the dynamics, stoichiometry and conformation of biomolecules one molecule at a time. Viruses are small (nanometers) in size, can achieve cellular infections with a small number of virions and their lifecycle is inherently heterogeneous with a large number of structural and functional intermediates. Single-molecule measurements that reveal the complete distribution of properties rather than the average can hence reveal new insights into virus infections and biology that are inaccessible otherwise. This article highlights some of the methods and recent applications of single-molecule fluorescence in the field of virology. Here, we have focused on new findings in virus–cell interaction, virus cell entry and transport, viral membrane fusion, genome release, replication, translation, assembly, genome packaging, egress and interaction with host immune proteins that underline the advantage of single-molecule approach to the question at hand. Finally, we discuss the challenges, outlook and potential areas for improvement and future use of single-molecule fluorescence that could further aid our understanding of viruses.     

Single-molecule fluorescence resonance energy transfer

Fluorescent resonance energy transfer (FRET) is a powerful technique for studying conformational distribution and dynamics of biological molecules. Some conformational changes are difficult to synchronize or too rare to detect using ensemble FRET. FRET, detected at the single-molecule level, opens up new opportunities to probe the detailed kinetics of structural changes without the need for synchronization. Here, we discuss practical considerations for its implementation including experimental apparatus, fluorescent probe selection, surface immobilization, single-molecule FRET analysis schemes, and interpretation.     

Single-molecule fluorescence of nucleic acids

Less than a decade old, single-molecule fluorescence of nucleic acids has rapidly become an important tool in the arsenal of biological probes. A variety of novel approaches to investigate conformational dynamics, catalytic mechanisms, folding pathways and protein-nucleic-acid interactions have recently been devised for nucleic acids using this technique. Combined with biomechanical tools and ensemble measurements, single-molecule fluorescence methods extend our ability to observe and understand biomolecules and complex biological processes.     

Single-molecule fluorescence methods for the study of nucleic acids.

Single-molecule fluorescence methods and biomechanical tools provide exciting new opportunities to probe biochemical processes in unprecedented detail. The detection and spectroscopy of single fluorophores have recently been used to observe conformational changes and biochemical events involving nucleic acids. A number of fluorescence observables, including localization, quenching, polarization response and fluorescence resonance energy transfer, have been utilized. An exciting new opportunity of combining fluorescence methods and biomechanical tools to study the structural changes and functions of enzymes that participate in nucleic acid metabolism has also arisen.     

Single-molecule fluorescence characterization in native environment

Single-molecule detection (SMD) with fluorescence is a widely used microscopic technique for biomolecule structure and function characterization. The modern light microscope with high numerical aperture objective and sensitive CCD camera can image the brightly emitting organic and fluorescent protein tags with reasonable time resolution. Single-molecule imaging gives an unambiguous bottom-up biomolecule characterization that avoids the "missing information" problem characteristic of ensemble measurements. It has circumvented the diffraction limit by facilitating single-particle localization to ~1 nm. Probes developed specifically for SMD applications extend the advantages of single-molecule imaging to high probe density regions of cells and tissues. These applications perform under conditions resembling the native biomolecule environment and have been used to detect both probe position and orientation. Native, high density SMD may have added significance if molecular crowding impacts native biomolecule behavior as expected inside the cell.     

Single-molecule fluorescence polarization study of conformational change in archaeal group II chaperonin

Group II chaperonins found in archaea and in eukaryotic cytosol mediate protein folding without a GroES-like cofactor. The function of the cofactor is substituted by the helical protrusion at the tip of the apical domain, which forms a built-in lid on the central cavity. Although many studies on the change in lid conformation coupled to the binding and hydrolysis of nucleotides have been conducted, the molecular mechanism of lid closure remains poorly understood. Here, we performed a single-molecule polarization modulation to probe the rotation of the helical protrusion of a chaperonin from a hyperthermophilic archaeum, Thermococcus sp. strain KS-1. We detected approximately 35° rotation of the helical protrusion immediately after photorelease of ATP. The result suggests that the conformational change from the open lid to the closed lid state is responsible for the approximately 35° rotation of the helical protrusion.     

Single-molecule fluorescence observed with mercury lamp illumination

We demonstrate that it is possible to observe single fluorescent molecules using a standard fluorescence microscope with mercury lamp excitation and an inexpensive cooled charge-coupled device (CCD) camera. With this equipment, we have been able to observe single molecules of tetramethyl-rhodamine, rhodamine 6G, fluorescein isothiocyanate and green fluorescent protein. Immobilized molecules were observed both in air and in aqueous solution.     

Linking molecular motors to Alzheimer's disease.

It is currently thought that Alzheimer's disease develops due to aberrant generation of amyloid-beta peptides. However, the mechanisms underlying the aberrant generation of amyloid-beta peptides remain unknown. An emerging concept suggests that impaired axonal transport may play a pivotal role in the aberrant generation of amyloid-beta peptides. Here we review and discuss advances in understanding AD with the primary focus on the possible role of molecular motors and axonal transport in its pathogenesis.     

Single-molecule fluorescence measurements of ribosomal translocation dynamics

We employ single-molecule fluorescence resonance energy transfer (smFRET) to study structural dynamics over the first two elongation cycles of protein synthesis, using ribosomes containing either Cy3-labeled ribosomal protein L11 and A- or P-site Cy5-labeled tRNA or Cy3- and Cy5-labeled tRNAs. Pretranslocation (PRE) complexes demonstrate fluctuations between classical and hybrid forms, with concerted motions of tRNAs away from L11 and from each other when classical complex converts to hybrid complex. EF-G?GTP binding to both hybrid and classical PRE complexes halts these fluctuations prior to catalyzing translocation to form the posttranslocation (POST) complex. EF-G dependent translocation from the classical PRE complex proceeds via transient formation of a short-lived hybrid intermediate. A-site binding of either EF-G to the PRE complex or of aminoacyl-tRNA?EF-Tu ternary complex to the POST complex markedly suppresses ribosome conformational lability.     

Single-molecule fluorescence studies from a bioinorganic perspective

In recent years, single-molecule methods have enabled many innovative studies in the life sciences, which generated unprecedented insights into the workings of many macromolecular machineries. Single-molecule studies of bioinorganic systems have been limited, however, even though bioinorganic chemistry represents one of the frontiers in the life sciences. With the hope to stimulate more interest in applying existing and developing new single-molecule methods to address compelling bioinorganic problems, this review discusses a few single-molecule fluorescence approaches that have been or can be employed to study the functions and dynamics of metalloproteins. We focus on their principles, features and generality, possible further bioinorganic applications, and experimental challenges. The fluorescence quenching via energy transfer approach has been used to study the O₂-binding of hemocyanin, the redox states of azurin, and the folding dynamics of cytochrome c at the single-molecule level. Possible future applications of this approach to single-molecule studies of metalloenzyme catalysis and metalloprotein folding are discussed. The fluorescence quenching via electron transfer approach can probe the subtle conformational dynamics of proteins, and its possible application to probe metalloprotein structural dynamics is discussed. More examples are presented in using single-molecule fluorescence resonance energy transfer to probe metallochaperone protein interactions and metalloregulator-DNA interactions on a single-molecule basis.     

Three approaches to assembling nano-bio-machines using molecular motors

Efforts to use protein molecular motors as nanoactuators are making rapid progress. For instance, it is now possible to carry out directional transport of small cargo along microtracks or microchannels using kinesin-microtubule systems, which could be the basis of micro-conveyor belts or molecular shuttles. However, the applicability of protein-based devices is limited by their poor stability in artificial environments. In addition, assembly of complex, intelligent microdevices or systems will likely require bottom-up self-assembly, and we still do not have sufficient knowledge to rationally design self-assembling protein-based microdevices or systems. One approach to solving the problems associated with protein-based systems is to use DNA-based nanodevices, which are amenable to rational design. Indeed, ingenious design has enabled realization of DNA-based nanoactuators and self-assembled micropatterns of various shapes. One also could use cells, organelles, or tissues as preassembled motile units, and several motile devices have already been realized using this approach. In addition to being less prone to the assemaly problems, cell-based microdevices have the advantage that the motile units reproduce themselves, and genetically encoded functional modifications can be replicated effortlessly. These protein-based, DNA-based, and cell-based systems each have distinct advantages and disadvantages, so that hybrid devices combining the best characteristics of all three would seem the most likely to succeed.     

构建“小”的艺术——从分子开关到分子马达

生物体的复杂结构和功能为我们提供了启发和挑战,即如何在分子水平构建纳米结构,并控制它的功能。通过＂自下而上＂的路线,我们着重在以下方面进行了探索：组装对外界刺激具有响应性的纳米体系,其中关键是在分子和超分子水平控制它的动态过程。分子开关是受外界刺激控制的单元,并可作为分子存储和电子元件。本文介绍了一类受光控制的分子开关,由于光控的简单性和高效性,此类光分子开关有望在分子水平的信息存储领域展现重要的应用价值。此外,通过将另一类光分子开关螺吡喃与通道蛋白结合,成功实现了蛋白孔的光控开关,可以被称为＂分子阀门＂,对未来的可控药物释放提供了很好的模型。分子马达是对纳米科学的巨大挑战,并将是未来分子机器的核心组成部分。本文介绍了新型的光驱动分子马达,对外界能量的利用使马达能够进行循环的定向运动,并且对马达分子结构的设计,能够构建出速度更快的第二代光驱动分子马达。然后,通过化学方法能够将该马达固定在纳米颗粒以至宏观界面的表面上,马达仍然能够受光驱动而高效运行。最后,本文展示了分子马达的一些应用,例如,马达的运动能够引起与之结合的高分子体系、液晶,甚至宏观物体的变化。     

Single-molecule multiparameter fluorescence spectroscopy reveals directional MutS binding to mismatched bases in DNA

Mismatch repair (MMR) corrects replication errors such as mismatched bases and loops in DNA. The evolutionarily conserved dimeric MMR protein MutS recognizes mismatches by stacking a phenylalanine of one subunit against one base of the mismatched pair. In all crystal structures of G:T mismatch-bound MutS, phenylalanine is stacked against thymine. To explore whether these structures reflect directional mismatch recognition by MutS, we monitored the orientation of Escherichia coli MutS binding to mismatches by FRET and anisotropy with steady state, pre-steady state and single-molecule multiparameter fluorescence measurements in a solution. The results confirm that specifically bound MutS bends DNA at the mismatch. We found additional MutS-mismatch complexes with distinct conformations that may have functional relevance in MMR. The analysis of individual binding events reveal significant bias in MutS orientation on asymmetric mismatches (G:T versus T:G, A:C versus C:A), but not on symmetric mismatches (G:G). When MutS is blocked from binding a mismatch in the preferred orientation by positioning asymmetric mismatches near the ends of linear DNA substrates, its ability to authorize subsequent steps of MMR, such as MutH endonuclease activation, is almost abolished. These findings shed light on prerequisites for MutS interactions with other MMR proteins for repairing the appropriate DNA strand.     

Kinesin motors as molecular machines

Molecular motor proteins, fueled by energy from ATP hydrolysis, move along actin filaments or microtubules, performing work in the cell. The kinesin microtubule motors transport vesicles or organelles, assemble bipolar spindles or depolymerize microtubules, functioning in basic cellular processes. The mechanism by which motor proteins convert energy from ATP hydrolysis into work is likely to differ in basic ways from man-made machines. Several mechanical elements of the kinesin motors have now been tentatively identified, permitting researchers to begin to decipher the mechanism of motor function. The force-producing conformational changes of the motor and the means by which they are amplified are probably different for the plus- and minus-end kinesin motors.     

Millennial musings on molecular motors

In a universe that is dominated by increasing entropy, living organisms are a curious anomaly. The organization that distinguishes living organisms from their inanimate surroundings relies upon their ability to execute vectorial processes, such as directed movements and the assembly of macromolecules and organelle systems. Many of these phenomena are executed by molecular motors that harness chemical potential energy to perform mechanical work and unidirectional motion. This article explores how these remarkable protein machines might have evolved and what roles they could play in biological and medical research in the coming decades.     

Transport characteristics of molecular motors

Properties of transport of molecular motors are investigated. A simplified model based on the concept of Brownian ratchets is applied. We analyze a stochastic equation of motion by means of numerical methods. The transport is systematically studied with respect to its energetic efficiency and quality expressed by an effective diffusion coefficient. We demonstrate the role of friction and non-equilibrium driving on the transport quantifiers and identify regions of a parameter space where motors are optimally transported.