NANO Nano Reviews Vol. 1, February 2010, pp. 1–2 sici xxx PUBMED Abbreviation Nano Reviews Perspective Obtaining a nano-view of single molecules in actions In 1959, Nobel laureate Richard Feynman suggested that ‘there is plenty of room at the bottom,’ predicting the possibility of single-molecule detection and studies. Over the last 20 years, we have witnessed a rapid development in single-molecule spectroscopy. Single-molecule spectroscopy and imaging have been demonstrated to be a powerful molecular analytical approach to studying the complex and inhomogeneous chemical, biological, and physical processes involved in protein dynamics, protein–protein interactions, protein–DNA interaction dynamics, biological and chemical catalyses, and interfacial dynamics 12345. The discipline of single-molecule spectroscopy has been expanded across a broad range, including optical imaging, optical spectroscopy of fluorescence and Raman, atomic force spectroscopy, and various forms of scanning probe microscopy 34. A significant feature of this exciting development is that single-molecule spectroscopy is developing hand-in-hand with the recent advancements in nanotechnology, imaging technologies, ultrafast dynamics technologies, theoretical modeling and analyses, and computational technologies 25.Molecular sciences in condensed phase, including chemistry, biophysics, and biology, have been traditionally conducted at the ensemble-averaged level. For complex and inhomogeneous systems such as proteins, and molecular processes at the chemical and biological interfaces, the averaged properties are often insufficient for a molecule-level understanding of the temporal and spatial behaviors of the systems. The complexity of the molecular interaction and the inherent inhomogeneity of the molecular structure and dynamics often hinder the efficiency and specificity in the exploration of a fundamental understanding of the systems. It is now technically possible to directly probe single-molecule dynamics in real time, even single-molecule spectroscopic and topographic properties simultaneously 12345. By studying one molecule at a time, the static and dynamic inhomogeneities of the complex molecular systems can be identified, characterized, and/or resolved. Single-molecule spectroscopy reveals statistical distributions correlated with microscopic parameters and their fluctuations, which are often hidden in ensemble-averaged measurements. Single molecules and molecular complexes are observed in real time as they traverse a range of energy states, and the effect of this ever-changing ‘system configuration’ on chemical reactions and other dynamic processes can be mapped. Some of the most exciting research fields of single-molecule spectroscopy and biophysics are the dynamics of proteins and interfacial chemical and biological processes. For example, single-molecule spectroscopy has been applied to the study of photochemical reactions and photo-induced catalytic reactions in solar energy conversion, light harvesting, and catalyses 5.Following the success of the human genome project, it is increasingly clear that protein dynamics is critical to the molecular understanding of human health and diseases. Subtle conformational changes play a crucial role in protein functions, and protein conformations are highly dynamic rather than static. Using only a static structure characterization from an ensemble-averaged measurement at equilibrium is often inadequate for predicting dynamic conformations and understanding correlated functions of proteins in real time. The new paradigm of the protein structure–function relationship is that the dynamics of protein structures play critical roles in protein functions. Thus, understanding protein conformational dynamics is critical for manipulating the biological functions of proteins and structure-based drug/vaccine design. To characterize and study such dynamics, single-molecule approaches, which reach beyond ensemble-averaged approaches, are particularly powerful and informative. Capable of probing and analyzing protein conformational changes, single-molecule spectroscopy holds high promise for obtaining a mechanistic understanding of enhanced or inhibited activities.One of the most informative single-molecule protein dynamics researches is single-molecule enzymology. Enzymes involve many critical biological processes, and enzymes can change the biological reaction pathways and accelerate the reaction rate by thousands and even millions of times. It is the enzyme–substrate interactions and complex formation that often play a critical role in defining the enzymatic reaction landscape, including reaction potential surface, transition states of chemical transformation, and reaction pathways. Static structural characterization, from an ensemble-averaged measurement at equilibrium, is often inadequate in predicting dynamic conformations and understanding correlated enzyme functions in real time involving non-equilibrium, multiple-step, multiple-conformation complex chemical interactions and transformations. Single-molecule enzymology has provided a unique and powerful molecular-level analysis and nanoscale characterization of each step in the enzymatic reaction turnover cycle in real time. Notably, in recent years, the development of combined single-molecule spectroscopy and atomic force microscopy (AFM) approaches to study individual enzyme proteins under reaction conditions provide a nano-view of many local sites, one at a time. This will enable us to probe and record single-molecule reaction temporal and spatial trajectories without requiring synchronization or other approaches for deconvolution of static inhomogeneity.In recent years, there have been significant developments and demonstrations on manipulating and analyzing protein conformations, and biomolecular interactions by AFM tip force pulling, and by the manipulations of optical and magnetic tweezers 123. These approaches expanded the reach of single-molecule science and technology to eventually change molecules at nanoscale. AFM topographic imaging can also characterize single-molecule proteins in membranes and biological surfaces. Combined AFM-fluorescence and near-field scanning microscopy are able to obtain correlated spectroscopic and topographic imaging at single-molecule level, which have demonstrated significant applications in studies of chemical and biological systems. In the future, one of the most promising advancements in single-molecule researches will be topographic and optical manipulation of single-molecule protein conformations to explore protein function and structure and analyze such exclusive states in real time at an extreme molecular sensitivity. New, exciting developments demonstrate the advancement of probing molecular processes in living cells at single-molecule sensitivities 4. Ultimately, combining real-time measurements of protein conformation with methods for the external manipulation of protein structures will help in the development of molecular-level understanding of human diseases and medical treatments.H. Peter Lu
Department of Chemistry
Center for Photochemical Sciences
Bowling Green State University
Bowling Green, OH 43403, USA
Email: hplu@bgsu.edu
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