Martin Karplus

Department of Chemistry and Chemical Biology, Cambridge, MA 02138, USA and Laboratoire de Chimie Biophysique, ISIS, Universite Louis Pasteur, 67000 Strasbourg, France
Received 30 May 2002; accepted 30 July 2002

Molecular Dynamics of Biological Macromolecules: A Brief History and Perspective

Abstract: A description of the origin of my interest in and the development of molecular dynamics simulations of biomolecules is presented with a historical overview, including the role of my interactions with Shneior Lifson and his group in Israel. Some early applications of the methodology by members of my group are summarized, followed by a description of examples of recent applications and some discussion of possible future directions. 2003 Wiley Periodicals, Inc. Biopolymers 68: 350 358, 2003 Keywords: NMR; molecular dynamics simulations; trajectory calculations; proteins; conformational change; channels

PROLOGUE
Since I was very young, I had been interested in biology and progressed from being passionate about birdwatching1 to a desire to understand biological processes at the level of chemistry and physics. However, for many years after my thesis research with Linus Pauling, I concentrated on chemical physics and biological applications remained in the background. After moving to Harvard in the late 1960s, I concluded that if I was ever to return to biology I had to make a break with a very busy research program. For this purpose, I decided to take a six-month leave from Harvard and go somewhere with a good library and people to talk to about my biological interests. I decided that the Weizmann Institute in Rehovot would be a good place, in part because I had always
Correspondence to: Martin Karplus; email: marci@tammy. harvard.edu Contract grant sponsor: National Science Foundation and the National Institutes of Health Biopolymers, Vol. 68, 350 358 (2003) 2003 Wiley Periodicals, Inc.

wanted to visit Israel and friends had told me the Weizmann had an excellent library. So the questions arose as to what group I should join. I was aware of Shneior Lifsons work on polymer theory and his reputation for being an open-minded scientist, as well as a wonderful storyteller. So I wrote Shneior in 1968 asking whether I could come for a semester. He kindly invited me to join his group and I was able to obtain a one-semester research leave from Harvard, not always an easy thing to do. The stay in Rehovot was a great success from my point of view. I had the time I needed to read and was able to nd a number of areas where I thought I might be able to do constructive research by applying my expertise in theoretical chemistry to biological problems. Among others, they included vision and particularly the application of semiempirical quantum mechanics

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to the visual chromophore, retinal.2 This is an area in which I had been interested since my undergraduate days at Harvard when I had many discussions of the problem with George Wald and Ruth Hubbard.3 Another question that appeared ready for investigation was the origin of hemoglobin cooperativity. Although the phenomenological model of Monod, Wyman, and Changeux4 had provided many insights, it did not make contact with the detailed structure of the molecule. The needed information was being obtained by x-ray diffraction in the laboratory of Max Perutz,5 and I was hopeful that it soon would be possible to connect the plethora of available thermodynamic data with the atomic interactions involved.6,7 A focus on the mechanism of protein folding arose from a visit of Chris Annsen to the Lifsons group. Annsen was a regular visitor to the Weizmann and we had many discussions of his experiments8 on protein folding in solution. What most impressed me was that he showed a lm of the folding of a protein with ickering helices forming and dissolving and coming together to form stable substructures. Of course, the lm was purely imaginary, but it led to my asking him whether he had thought of taking the ideas in the lm and translating them into a quantitative model. He said that he did not really know how he would do this, but to me it seemed clear that such a model could be based on straightforward physical concepts. When David Weaver joined my group at Harvard while he was on a sabbatical leave from Tufts, we developed what is now known as the diffusion-collision model for protein folding.9,10 Although it is a simplied description of the folding process, it was the rst model that made possible the calculation of folding rates based on meaningful physical parameters. In Shneiors group at the time there was considerable excitement about developing empirical potential energy functions for small molecules. The important new idea was to use a functional form that could serve not only for calculating vibrational frequencies, as did the expansions of the potential about a known or assumed minimum energy structure, but also for determining that structure. The so-called consistent force eld (CCF) of Lifson and his co-workers, particularly Arieh Warshel, included nonbonded interaction terms so that the minimum energy structure could be found after the energy terms had been appropriately calibrated.10 The possibility of using such energy functions for larger systems, such as proteins, struck me as very exciting, though I did not begin working on this for a while. However, I did invite Arieh Warshel to join my group as a postdoctoral fellow.

When I returned to Harvard, biologically related research began in earnest. The rst two systems to be investigated were the visual pigment retinal and the cooperative mechanism in hemoglobin, both of which I had thought about in Rehovot, as I already mentioned. An initial study of retinal, which presaged quantum mechanical/molecular mechanical (QM/MM) calculations with a Huckel -electron energy and a pairwise nonbonded energy2 was made by Barry Honig, who continued to do fundamental research on the problem of vision for many years. Barrys calculation predicted a 12-s-cis geometry for 11-cis retinal, the active chromophore of rhodopsin; this prediction was subsequently conrmed by experiment. In a review of this work,12 I noted, Theoretical chemists tend to use the word prediction rather loosely to refer to any calculation that agrees with experiment, even when the latter was done before the former. This is as true today as it was then. When Arieh Warshel came to Harvard, he worked on improving the methodology for the calculation of the structure and spectra of -electron systems, particularly polyenes, including retinal.13 The -electrons were treated by molecular mechanics, essentially as in the CFF program, and the -electrons by a rened version of the PariserParrPople method. Useful results came out of applications of this methodology,12 although the rhodopsin structure was not known, so that the specic effect of the protein on the conformation and spectrum could not be calculated at that time. The next step was the investigation of hemoglobin. Attila Szabo had just nished a statistical mechanical model of hemoglobin cooperativity6 that was based on crystallographic studies and their interpretation by Max Perutz.5 This work raised a number of questions concerning the energetics of ligand binding in hemoglobin and its coupling to protein structural changes involved in the transition from the unliganded to the liganded state (the T to R transition). The best means to study such a problem was to have available a way of calculating the energy of the protein as a function of the atomic positions, but we did not have such a program. Bruce Gelin had begun theoretical research in my group in 1967 and started by studying the application of the random phase approximation to two-electron problems. He was collaborating with Neal Ostlund, who was a postdoctoral fellow at Harvard at the time. However, after two years Bruce was drafted and ended up in a laboratory as an MP concerned with drug usage (LSD, etc.) in the U.S. Army. This aroused his interest in living systems and when he returned to nish his degree, he wanted to change his area of research to biologically related problems. Bruce and I

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decided it was time to try to develop a program that would make it possible to take a given amino acid sequence (e.g., that of the hemoglobin chain) and a set of coordinates (e.g., those obtained from the x-ray structure of deoxy hemoglobin), and to use this information to calculate the energy of the system and its derivatives as a function of the atomic positions. This was a major task, but Bruce had just the right combination of abilities to carry it out.14 The result was Pre-CHARMM (it did not have a name at that time). Although not trivial to use, the program was applied to a variety of problems, including Bruces pioneering study of aromatic ring ips in the bovine pancreatic trypsin inhibitor (BPTI),15 as well as Bruces primary project to introduce the effect of ligand binding on the heme group as a perturbation (undoming of the heme) and to use energy minimization to determine the response of the protein.7 To do such a calculation on the available computers (an IBM 7090 at Columbia University was our workhorse at the time) required considerable courage. Another application was Dave Cases analysis of ligand escape after photodissociation from myoglobin.16 Bruce would have had a very difcult time constructing such a program if there had not been prior work by other groups on protein energy calculations. Although many persons have contributed to the development of empirical potentials, the two major inputs to our work came from Schneiors group in Rehovot and Harold Scheragas group at Cornell University.17 As I already mentioned, Arieh Warshel was at Harvard and had brought his CFF program with him. His presence and the availability of the CFF program was an important resource for Bruce, who was also aware of Michael Levitts and Shneior Lifsons pioneering energy calculations for proteins.18 Unfortunately, Bruce did not have access to Michaels program; in fact, the present-day version, called ENCAD, is still is not generally available, although it has been used by some Levitts co-workers, perhaps most effectively in the recent simulations of protein unfolding by Valerie Daggett.19

THE BEGINNING OF MOLECULAR DYNAMICS OF BIOMOLECULES

Given a program that could calculate the forces on the atoms of a protein for minimization of the energy, the next step was to use these forces in Newtons equation to calculate the dynamics. This important development was introduced by Andy McCammon when he joined my group. An essential element that encouraged us in this attempt was the existence of molecular

dynamics simulation methods for simpler systems. Molecular dynamics had followed two pathways that come together in the study of biomolecule dynamics. One of these, usually referred to as trajectory calculations, has an ancient history that goes back to twobody scattering problems for which analytic solutions can be achieved. However, as is well known, even for only three particles with realistic interactions, difculties arise. An example is provided by the simplest H, for which a chemical reaction, H H2 3 H2 prototype calculation was attempted by Hirschfelder, Eyring, and Topley in 193620; they were able to calculate a few steps along one trajectory. It was nearly thirty years later that the availability of computers made it possible to complete the calculation.21 Much has been done since then in applying classical trajectory methods to a wide range of chemical reactions.22,23 These classical studies have been supplemented by semiclassical and quantum-mechanical calculations in areas where quantum effects can play an important role.24,25 The focus at present is on more complex molecules, the redistribution of their internal energy, and the effect of this on reactivity.26 The other pathway in molecular dynamics has been concerned with physical rather than chemical interactions and with the thermodynamics and dynamic properties of large numbers of particles, rather than detailed trajectories of a few particles. Although the basic ideas go back to van der Waals and Boltzmann, the modern era began with the work of Alder and Wainright on hardsphere liquids in the late 1950s.27 The paper by Rahman28 in 1964 on a molecular dynamics simulation of liquid argon with a soft sphere (LennardJones) potential represented an important next step. Simulations of complex uids followed; the original study of liquid water by Stillinger and Rahman was published in 1971.29 Since then there have been many studies on the equilibrium and nonequilibrium behavior of a wide range of homogeneous systems.30,31 The background I have outlined set the stage for the development of molecular dynamics of biomolecules.32 The size of an individual molecule, composed of 500 or more atoms for even a small protein, is such that its simulation in isolation can serve to obtain approximate equilibrium properties, as in the molecular dynamics of uids, although detailed aspects of the atomic motions are of considerable interest, as in trajectory calculations. A basic assumption in initiating such studies was that potential functions could be constructed that were sufciently accurate to give meaningful results for systems as complex as proteins or nucleic acids. In addition, it was necessary to assume that for these inhomogeneous systems, in contrast to the homogeneous character of even com-

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plex liquids such as water, simulations of an attainable time scale (approximately 10 100 ps in initial studies) could provide a useful sample of the phase space in the neighborhood of the native structure. Although for neither of these assumptions was there strong supporting evidence in 1975, the results obtained for BPTI32 have clearly stood the test of time, and perhaps more importantly, have served to open a new eld that has become the research focus of a large number of scientists. It is now twenty-ve years since the rst molecular dynamics simulation of a macromolecule of biological interest was published.32 The simulation concerned BPTI, which has served as the hydrogen molecule of protein dynamics because of its small size, high stability, and relatively accurate x-ray structure, available in 197533; interestingly, the physiological function of BPTI remains unknown. Although this simulation was done in vacuum with a crude molecular mechanics potential and lasted for only 9.2 ps, the results were instrumental in replacing our view of proteins as relatively rigid structures (In 1981, Sir D. L. Phillips commented: Brass models of DNA and a variety of proteins dominated the scene and much of the thinking.34) with the realization that they were dynamic systems, whose internal motions play a functional role. Of course, there were already experimental data, such as the hydrogen exchange experiments of LinderstromLang and his co-workers),35,36 pointing in this direction. It is now recognized that the x-ray structure of a protein provides the average atomic positions, but that the atoms exhibit uid-like motions of sizable amplitudes about these averages. The new understanding of protein dynamics subsumed the static picture in that the average positions are still useful for the discussion of many aspects of biomolecular function in the language of structural chemistry. However, the recognition of the importance of uctuations opened the way for more sophisticated and accurate interpretations of functional properties. The conceptual changes resulting from the early studies make one marvel at how much of great interest could be learned with so littlesuch poor potentials, such small systems, so little computer time. This is, of course, one of the great benets of taking the initial, somewhat faltering steps in a new eld where the questions are qualitative rather than quantitative, and any insights, even if crude, are better than none at all. Subsequent applications have been concerned with more detailed interpretations and predictions of phenomena that require the use of improved methods. Simulations based on more rened potentials, and the longer runs required for improved statistics, are be-

coming possible for increasingly complex systems as a result of the progress in the available computers, which continue to increase in speed by a factor of two or so every eighteen months, in accord with Moores Law. Molecular dynamics simulations of proteins, as of many other systems (e.g., liquids), can, in principle, provide the ultimate details of motional phenomena. The primary limitation of simulation methods is that they are approximate. It is here that experiment plays an essential role in validating the simulation methodsthat is, comparisons with experimental data serve to test the accuracy of the calculated results and provide criteria for improving the methodology. However, the experimental approaches to bimolecular dynamics are limited as to the information that can be obtained from them; e.g., if one is concerned with the time scale of motions, the frequency spectrum covered by experiments such as NMR is limited,37 so that motional models that are able to rationalize the data can be inaccurate. When experimental comparisons indicate that the simulations are meaningful, their capacity for providing detailed results often makes it possible to examine specic aspects of the atomic motions far more easily than by making measurements.

EARLY APPLICATIONS
Two years after the BPTI simulation, it was recognized38,39 that thermal (B) factors determined in x-ray crystallographic renement could be used to obtain information about the internal motions of proteins and plots of estimated mean-square uctuations vs residue number (introduced in Ref. 32) became a standard part of papers on high-resolution crystal structures, even though the contribution to the B factors of overall translation and rotation, as well as crystal disorder, continues to be a concern in their interpretation.40 During the subsequent ten years, a wide range of phenomena were investigated by molecular dynamics simulations of proteins and nucleic acids. Most of these studies focused on the physical aspects of the internal motions and the interpretation of experiments. They include the analysis of uorescence depolarization of tryptophan residues,41 the role of dynamics in measured NMR parameters,42 44 and inelastic neutron scattering,45,46 the effect of solvent and temperature on protein structure and dynamics,47 49 as well as the now widely used simulated annealing methods for x-ray structure renement50,51 and NMR structure determination.52,53 Simultaneously, there were a number of applications that

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demonstrated the importance of internal motions in biomolecular function, including the hinge bending modes for opening and closing active sites,54,55 the exibility of tRNA,56 the induced conformation change in the activation of trypsin,57 the uctuations required for ligand entrance and exit in heme proteins,16 and the role of congurational entropy in proteins and nucleic acids.58,59 Two attributes of molecular dynamics simulations have played an essential part in the explosive growth in the number of studies based on such simulations and the very wide range of applications that have been made. Simulations provide the ultimate detail concerning individual particle motions as a function of time, so they can be used to answer specic questions about the properties of a system, often more easily than experiments. For many aspects of biomolecule function, these are the details of interest (e.g., by what pathways does oxygen enter into and exit from the heme pocket in myoglobin). Of course, experiments play an essential role in validating the simulation methodsthat is, comparisons with experimental data can serve to test the accuracy of the calculated results and to provide criteria for improving the methodology. This is particularly important because theoretical estimates of the systematic errors inherent in the simulations are still lacking; i.e., the errors introduced by the use of empirical potentials are difcult to quantify. Another important aspect of simulations is that, although the potentials employed in simulations are approximate, they are completely under the users control, so that by removing or altering specic contributions their role in determining a given property can be examined. This is most graphically demonstrated by the use of computer alchemytransmuting the potential from that representing one system to another during a simulationin calculating free energy differences.60 62 There are three types of applications of simulation methods in the macromolecular area, as well as in other areas involving mesoscopic systems. The rst uses the simulation simply as a means of sampling conguration space. This is involved in the utilization of molecular dynamics, often with simulated annealing protocols, to determine or rene structures with data obtained from experiments. The second uses simulations to determine equilibrium averages, including structural and motional properties (e.g., atomic mean-square uctuation amplitudes) and the thermodynamics of the system. For such applications, it is necessary that the simulations adequately sample conguration space, as in the rst application, with the additional condition that each point be weighted by the appropriate Boltzmann factor. The third area

employs simulations to examine the actual dynamics. Here not only is adequate sampling of conguration space with appropriate Boltzmann weighting required, but it must be done so as to properly represent the time development of the system. For the rst two areas, Monte Carlo simulations can be utilized, as well as molecular dynamics. By contrast, in the third area where the motions and their time developments are of primary interest, only molecular dynamics can provide the necessary information. The three sets of applications make increasing demands on the simulation method in terms of the required accuracy.

CURRENT APPLICATIONS
Most of the motional phenomena examined during the rst ten years after the BPTI simulation paper was published32 continue to be studied both experimentally and theoretically. Every year more papers appear, stimulated in part by improvement in the methodology for the former and in the increase in computer power for the latter. Moreover, the increasing scope of molecular dynamics is making possible the study of systems of increasing complexity with more accurate methods (e.g., improved potentials) on everincreasing time scales. There are many recent examples of the use of molecular dynamics to obtain functionally important information that is difcult or impossible to obtain experimentally.

NMR-Related Studies
One area where there has been and continues to be a symbiotic relation between simulations and experiment is NMR, which in addition to its use for structure determinations51,52,63 is a powerful technique for the study of the dynamics and thermodynamics of macromolecules. Interpretations of relaxation rates (T1 and T2) and NOE measurement by molecular dynamics, initiated in the early 1980s,42 44 are now widespread.63 65 Yet, in a recent paper66 on the use of NMR to study motional properties, Lee and Wand state, Despite its importance, the precise nature of the internal motions of protein molecules remains a mystery. The papers by Lee and Wand66 and by Wand37 focus on internal protein motions (particularly side-chain librations) and the correlation of the measured exibility based on order parameters with the entropy. In fact, molecular dynamics simulations over the last 25 years have taught us much about these motions,67,68 and have been used successfully to calculate and interpret order parameters measured by NMR.42,44,63 65 It is interesting to note also that al-

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ready in 198358 (see also Ref. 69) normal mode calculations demonstrated that vibrational contributions to the internal entropy of BPTI are equal to about 600 kcal/mol (including quantum corrections, which so far have been neglected in NMR analyses) and that the value per residue of a protein is very similar to that for an isolated peptide.70 More recent simulations have shown the importance of the residual motion to the entropy of binding of internal waters, using BPTI as the model system,71 and of antiviral drugs to the capsid of rhinovirus72; for an experimental verication of the latter, see Ref. 73. A recent molecular dynamics analysis65 of the relation between NMR order parameters and protein entropy suggests that although there is a correlation, the NMR results correspond to only a fraction (25%) of the total entropy.

Table I

Average Mean Square Fluctuationsa Backbone 0.23 0.18 0.09 0.09 All Heavy Atoms 0.36 0.28 0.13 0.13

Temperature P300/S300b P180/S300 P300/S180 P180/S180

a Temperatures in degrees Kelvin and mean square uctuations in ngstroms. b P refers to the protein and S to solvent.

even if the solvent is at 300 K, because the internal motional barriers become dominant.

The Role of Solvent in Protein Dynamics

Molecular dynamics has been used to resolve the contentious question concerning the role of the solvent in determining the internal motions of proteins, particularly at temperatures below the so-called glass transition74,75; low-temperature studies have been very important in elucidating the fundamental role of internal motionsfor example, in bacteriorhodopsin.76 It has not been possible to determine experimentally whether or not the solvent uctuations drive the internal motion of a protein, but this was accomplished by employing an attribute of the simulation methodology to create a physical system not accessible in nature.77 Simulations can be performed with one part of the system (e.g., the protein) at one temperature and another part of the system (e.g., the surrounding solvent) at a different temperature. The amplitudes of the atomic uctuations (corresponding to the B factors) in carbonmonoxymyoglobin were calculated77 from simulations with the temperature of the protein and the solvent at either 300K or 180 K (i.e., above and below the protein glass transition around at 220 K). The results of the four possible temperature combinations are given in Table I. It is striking that the magnitude of the uctuations is only weakly dependent on the protein temperature; the uctuations are large when the solvent is at 300 K and small when the solvent is 180 K, independent of the protein temperature. This demonstrates unequivocally that the temperature of the solvent and its resultant mobility is the dominant factor in determining the functionally important protein uctuations in this temperature range. Additional simulations have shown that at still lower temperatures (around 80 K), the protein freezes (i.e., has only small uctuations),

Conformational Change in the Mechanism of GroEL

Chaperones are essential for the correct folding of many proteins in the highly concentrated milieu of the cell.78 One of the best characterized chaperonins is GroEL, which consists of two rings of seven identical subunits, stacked back-to-back.79 GroEL is required for growth of Escherchia coli; it has been estimated that 10% of its proteins are assisted in folding by GroEL.80 During the functional cycle, GroEL undergoes large conformational changes that are regulated by the binding and hydrolysis of ATP and involve the cochaperonin GroES, as demonstrated by x-ray crystallography.79 Each of the seven-membered rings has been shown to have a closed structure in the absence of ATP and GroES and an open structure in their presence. Molecular dynamics simulations81 have been used to nd the pathway between these two structures, which it is impossible to determine by experimental techniques. Since the actual transition between the open and closed structure is believed to be on the millisecond time scale (The full GroEL cycle takes about 15 s), a direct simulation of the opening and closing motion in the presence and absence of ATP by molecular dynamics is not possible as yet. However, a number of methods have been developed to follow the transition between two known states on the nanoseconds time scale accessible to simulations. One of these, targeted molecular dynamics,82 was used to determine a transition pathway of GroEL. It was shown that there is an intermediate form, which exists with ATP bound in the absence of GroEL, and it was demonstrated that the early downward motion of the small intermediate domain induced by nucleotide binding is the trigger for the larger movement of the apical and equatorial domains. Moreover, the results demonstrated that steric

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interactions and salt bridges between different subunits are the source of the measured positive cooperativity of ATP binding and hydrolysis within a ring and the negative cooperativity between the two rings. Recent cryo-electron microscopy results (Ref. 83; H. Saibil and A. L. Horwich, private communication) support the prediction from molecular dynamics of the mechanism by which the intermediate domain plays a key role in the allosteric transition of GroEL. There is still some uncertainty about the motion of the apical domain in this intermediate structure because the cryo electron microscopy model83 and the simulations81 appear to disagree.

A PERSPECTIVE ON FUTURE APPLICATIONS

One of the very exciting recent developments in molecular dynamics simulations is that the time scales becoming available with modern computers (100 ns to s) are making it possible to study phenomena of biological interest in real time. This is analogous, in an inverse sense, to the fact that while experiments on the picosecond time scale were an important development, it is when the resolution was extended to femtoseconds that the actual events involved in chemical reactions could be observed in real time.84,85 By running multiple simulations of 10 ns duration, real time visualization of water molecule migration through models of the aquaporin channel has been achieved.86,87 Although statistically signicant studies of the entire process of protein folding are still beyond the reach of present day simulations,88 (the experimental time scale is tens of microseconds or longer, and the potentials may not yet be good enough to identify the native state in many cases), the folding and unfolding of model peptides, such as a threestranded -sheet, with a simplied solvent model has been possible.89 Systems containing more than 100,000 atoms have been studied by molecular dynamics simulation since 1997.90 Current simulations include models of the nicotinic acetylcholine receptor91 and ATP synthase.92,93 The simulation of such complex systems for increasing periods of time is expected to provide unique insights into the specicity of binding in ligand-gated channels, the mechanisms of receptor activation and desensitization, and the action and regulation of molecular motors. It has always been my hope that when the methodology for molecular dynamics simulation had been codied in available programs, experimentalists (e.g., those doing x-ray crystallography, NMR spectros-

copy, and enzyme kinetics) would use them as a standard tool in their research. This is now beginning to happen. A beautiful example of such a study is the analysis of the role of the dynamic coupling between SH2 and SH3 domains in the control of their function.94 Another stage is the evolution of molecular dynamics simulations from the molecular to the supramolecular to the cellular scale. The transition to the supramolecular scale is already evident in the some of the studies mentioned above. These currently rely on crystallographic or other models of the molecular assemblages, and mainly consider structural changes of relatively modest amplitudes that are related to function. Studies of the formation of such assemblages will be more demanding. An interesting recent example of structure formation studied in real time by molecular dynamics concerns the formation of phospholipid bilayers.95,96 The simulation of such cellular activities as synaptic transmission,97 and how the nuclear membrane is dismantled on cell division by the motor protein cytoplasmic dynein,98 will eventually follow, building upon the detailed knowledge of the structure and dynamics of the channels, enzymes and other components. (A recent simulation that addressed a medical proteinthe destruction of brous caps and their relation to heart attacks is also indicative of what the future holds.99) More global simulations are likely to be initiated with less detailed models than the atomistic (Newtonian) ones considered in this review, but the ultimate descriptions, which will necessarily include such details as the possible effects of mechanical stress upon the channels and other components of a contracting neuromuscular synapse, will require studies by atomistic molecular dynamics simulations. Fortunately, the continued increase in computer power is (almost) keeping up with the demands made by such calculations.

SUMMARY
Molecular dynamics simulations of macromolecules have provided many insights concerning the internal motions of these systems since the rst protein was studied over 25 years ago. With continuing advances in the methodology and the speed of computers molecular dynamics studies are being extended to larger systems, greater conformational changes, and longer time scales. This is making possible the investigation of motions that have particular biological functions and to obtain information that is not accessible from experiment. The results available today make clear

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that the applications of molecular dynamics will play an even more important role in our understanding of biology in the future.
Much of the research described here was supported in part by grants from the National Science Foundation and the National Institutes of Health. As is evident from the references, many of my co-workers have contributed to the research. The opportunity of working with them has been one of the major rewards of my scientic career. Parts of this review are based on reference 100.

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