MINIREVIEW PROLOGUE
Biochemistry at the Single-molecule Level: Minireview Series*

Kensal E. van HoldeDagger

From the Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331-7305

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The most profound scientific revolutions are those that provide an entirely new way of viewing and studying a field. These are the ones that provoke new questions and question old answers and in the end give us a new understanding of what we thought we understood. Often, they are occasioned by the invention of novel instruments or techniques; the telescope, the microscope, and x-ray diffraction come to mind. It may be that such a revolution is occurring in biochemistry today through the development of methods that allow us to investigate the dynamics of single macromolecules in real time.

For the entire history of our science, we (like all chemists) have had to be content to study the average behavior of enormous assemblages of molecules. Much has been learned in this way, and we have been assured by statistical mechanicians that it was meaningful; by following the ensemble-average of a very large number of particles, we could learn the time-average behavior of a typical particle. However, it has always been obvious that if we could watch one molecule go through a chemical or physical process, or interact with another molecule, we might observe nuances and complexities of behavior that are simply smoothed out and lost in the average behavior of the mass.

Until very recently, it was impossible to observe the dynamics of individual molecules, even macromolecules. To be sure, we have been able for decades to visualize macromolecules (via electron microscopy or x-ray diffraction, for example) but only in a static state. No techniques existed that would allow us to study the actions and interactions of individual protein or nucleic acid molecules in solution. In the past decade a series of new methods has emerged (scanning microscopy, confocal fluorescence microscopy, and optical tweezers, to name a few) that allow us to not only observe and follow the behavior of individual macromolecules in an aqueous environment but in some cases to manipulate them as well.

This field of single-molecule biochemistry is in its infancy, but the things that can already be accomplished are quite amazing. We can watch a polymerase move along a template and observe directly details of its motion that could only be guessed at from macroscopic studies. We can measure the force generated by an individual molecular motor, under varying load and varying constraints, and measure the displacement corresponding to one ATP expenditure. We can measure the force of interaction between two macromolecules as they are allowed to approach one another along defined trajectories. We can watch an individual enzyme molecule in action and monitor its conformational changes as it accepts substrate and releases product.

Even at this early stage, it seems that the information obtainable from single-molecule studies will yield results not anticipated on the basis of ensemble-averaged methods. For example, it appears that the interactions between enzymes and their substrates may be much more complex than we would have expected from macroscopic kinetic studies. Individual protein molecules appear to undergo conformational fluctuations that modify their ability, at any instant, to bind or process substrate molecules. Furthermore, there is evidence for unexpected "memory" effects in reactions involving biological macromolecules. We should be prepared for many surprises.

The following series of minireviews is intended to provide a sampling of current experimentation using single-molecule techniques and the application of these methods to a range of biochemical problems. The series begins with an overview, by Amit Mehta, Matthias Rief, and James A. Spudich, of studies of motor proteins by these techniques. This is appropriate, for it is in this field that some of the earliest single-molecule studies were carried out and where the techniques have been developed to a high level of sophistication. We then turn to studies of one-dimensional diffusion on DNA in a paper by Nobuo Shimamoto. Here a very simple technique is used to raise fundamental questions about processes at the molecular level. The third paper, by X. Sunney Xie and H. Peter Lu, will extend the focus to enzymology, with some surprising results. Finally, the series will conclude with a paper, by Carlos Bustamante, Martin Guthold, Xingshu Zhu, and Guoliang Yang, describing how different possible modes of protein motion on DNA can be analyzed and distinguished using scanning force microscopy. The reader will find that the scope of some of these reviews is rather narrow; this is because these areas are so new that few investigators are yet working in each. Furthermore, the series is not intended to be inclusive or even fully representative. Rather, it is my hope that these short papers, by illustrating the kinds of things that can be done, will inspire the application of these methods to other problems. The technology involved is neither terribly difficult nor unreasonably expensive, and the range of possibilities seems limited only by the ingenuity of its practitioners.

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* This minireview will be reprinted in the 1999 Minireview Compendium, which will be available in December, 1999. 

Dagger To whom correspondence should be addressed. Tel.: 541-737-2178; Fax: 541-737-2179; E-mail: vanholde{at}asbmb.faseb.org.


Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.