Ice formation in aqueous solutions and suspensions involves a number of significant changes and processes in the residual liquid. The resulting effects were described concerning the redistribution of dissolved salts, the behaviour of gaseous solutes and bubble formation, the rejection and entrapment of secondphase particles. This set of conditions is also experienced by biological cells subjected to freezing. The influences of ice formation in that respect and their relevance for cryopreservation were considered as well.

A model of transient heat conduction and solute diffusion with a planar ice front, propagating through a system of finite length was found to be in good agreement with measured salt concentration profiles. The spacing of the subsequently developing columnar solidification pattern was of the same order of magnitude as the pertubation wavelengths predicted from the stability criterion. Non-planar solidification of binary salt solutions was described by a pure heat transfer model under the assumption of local thermodynamic equilibrium.

The rejection of gaseous solutes and the resulting gas concentration profile ahead of a planar ice front has been estimated by means of a test bubble method, yielding a distribution coefficient of 0·05 for oxygen. The nucleation of gas bubbles has been observed to occur at slightly less than 20-fold supersaturation. The subsequent radial growth of the bubbles obeys a square-root time dependence as expected from a diffusion controlled model until the still expanding bubbles become engulfed by the advancing ice-liquid interface. The maximum bubble radii decrease for increasing ice front velocities.

The transition between repulsion and entrapment of spherical latex particles by an advancing planar ice-front has been characterized by a critical value of the velocity of the solidification interface. The critical velocity is inversely proportional to the particle radius as suggested by models assuming an undisturbed ice front. The increase of the critical velocity for increasing thermal gradients shows good agreement with a theoretically predicted square-root type of dependence. Critical velocities have also been measured for yeast and red blood cells.

The effect of freezing on biological cells has been analyzed for human lymphocytes and erythrocytes. The reduction of cell volume observed during non-planar freezing agrees reasonably well with shrinkage curves calculated from a water transport model. The probability of intracellular ice formation has been characterized by threshold cooling rates above which the amount of water remaining within the cell is sufficient for crystallization. The cooling rate dependence of viability exhibits largely different maxima for lymphocytes and erythrocytes at about 30 and 4700 K/min, respectively. The decrease of viability above these values has been attributed to the damaging effect of intracellular ice formation.

It has been known for many years that there are two distinct ways of designing an electron microscope.

Protein folding and binding can be understood using energy landscape theory. When seeming deviations from the predictions of the funnel hypothesis are found, landscape theory helps us locate the cause. Sometimes the deviation reflects symmetry effects, allowing extra degeneracies to occur. Such effects seem to explain some kinetic anomalies in helical bundles. When binding processes were found to use apparently non-funneled landscapes this was traced to an inadequate understanding of biomolecular forces. The discrepancy allowed the discovery of new water-mediated forces – some of which act between hydrophilic residues. Introducing such forces into the algorithms greatly improves the quality of structure predictions.

The nicotinic acetylcholine (ACh) receptor, at the neuromuscular junction, is a neurotransmitter-gated ion channel that has been fine-tuned through evolution to transduce a chemical signal into an electrical signal with maximum efficiency and speed. It is composed from three similar and two identical polypeptide chains, arranged in a ring around a narrow membrane pore. Central to the design of this assembly is a hydrophobic gate in the pore, more than 50 Å away from sites in the extracellular domain where ACh binds. Although the molecular properties of the receptor have been explored intensively over the last few decades, only recently have structures emerged revealing its complex architecture and illuminating how ACh entering the binding sites opens the distant gate. Postsynaptic membranes isolated from the (muscle-derived) electric organ of the Torpedo ray have underpinned most of the structural studies: the membranes form tubular vesicles having receptors arranged on a regular surface lattice, which can be imaged directly in frozen physiological solutions. Advances in electron crystallographic techniques have also been important, enabling analysis of the closed- and open-channel forms of the receptor in unreacted tubes or tubes reacted briefly with ACh. The structural differences between these two forms show that all five subunits participate in a concerted conformational change communicating the effect of ACh binding to the gate, but that three of them (αγ, β and δ) play a dominant role. Flexing of oppositely facing pore-lining α-helices is the principal motion determining the closed/open state of the gate. These results together with the findings of biochemical, biophysical and other structural studies allow an integrated description of the receptor and of its mode of action at the synapse.

1. Introduction 258

2. Set-up of MD simulations 260

2.1 Constant-pressure dynamics 260

2.2 Grand-canonical dynamics 261

2.3 Boundary conditions 261

3. Force fields 262

3.1 Proteins 262

3.2 Nucleic acids 265

3.3 Carbohydrates 266

3.4 Phospholipids 266

3.5 Polarization 267

4. Electrostatics 267

4.1 Spherical truncation methods 268

4.2 Ewald summation methods 269

4.3 Fast multipole (FM) methods 271

4.4 Reaction-field methods 271

5. Implicit solvation models 271

6. Speeding-up the simulation 273

6.1 SHAKE and its relatives 273

6.2 Multiple time-step algorithms 274

6.3 Other algorithms 275

7. Conformational space sampling 275

7.1 Multiple copy simultaneous search (MCSS) and locally enhanced sampling (LES) 275

7.2 Steered or targeted MD 276

7.3 Self-guided MD 276

7.4 Leaving the standard 3D Cartesian coordinate system: 4D MD and internal coordinate MD 277

7.5 Temperature variations 277

8. Thermodynamic calculations 278

8.1 Lambda (λ) dynamics 278

8.2 Extracting thermodynamic information from simulations 279

8.3 Non-Boltzmann thermodynamic integration (NBTI) 279

8.4 Other methods 279

9. QM/MM calculations 282

10. MD simulations of protein folding and unfolding 283

10.1 High-temperature effects 284

10.2 Co-solvent and polarization effects 288

10.3 External force effects 288

11. On the horizon 291

12. Acknowledgements 292

13. References 292

Molecular dynamics simulations are widely used today to tackle problems in biochemistry and molecular biology. In the 25 years since the first simulation of a protein computers have become faster by many orders of magnitude, algorithms and force fields have been improved, and simulations can now be applied to very large systems, such as protein–nucleic acid complexes and multimeric proteins in aqueous solution. In this review we give a general background about molecular dynamics simulations, and then focus on some recent technical advances, with applications to biologically relevant problems.

The ability to design drugs (so-called ‘rational drug design’) has been one of the long-term objectives of chemistry for 50 years. It is an exceptionally difficult problem, and many of its parts lie outside the expertise of chemistry. The much more limited problem – how to design tight-binding ligands (rational ligand design) – would seem to be one that chemistry could solve, but has also proved remarkably recalcitrant. The question is ‘Why is it so difficult?’ and the answer is ‘We still don't entirely know’. This perspective discusses some of the technical issues – potential functions, protein plasticity, enthalpy/entropy compensation, and others – that contribute, and suggests areas where fundamental understanding of protein–ligand interactions falls short of what is needed. It surveys recent technological developments (in particular, isothermal titration calorimetry) that will, hopefully, make now the time for serious progress in this area. It concludes with the calorimetric examination of the association of a series of systematically varied ligands with a model protein. The counterintuitive thermodynamic results observed serve to illustrate that, even in relatively simple systems, understanding protein–ligand association is challenging.

This essay gives the autho's personal account on the development of concepts underlying single-particle reconstruction, a technique in electron microscopy of macromolecular assemblies with a remarkable record of achievements as of late. The ribosome proved to be an ideal testing ground for the development of specimen preparation methods, cryo-EM techniques, and algorithms, with discoveries along the way as a rich reward. Increasingly, cryo-EM and single-particle reconstruction, in combination with classification techniques, is revealing dynamic information on functional molecular machines uninhibited by molecular contacts.

1. Helicases as components of macromolecular machines 3

2. Helicases in replication 7

2.1 The loading of replicative helicases 7

2.1.1 Loading Rep helicase at the replication origin of bacteriophage ϕX174 7

2.1.2 How is a ssDNA strand passed through (and bound in?) the central channel of the hexameric replicative helicases? 8

2.1.3 Loading of E. coli DnaB helicase in the absence of an auxiliary protein-loading factor 8

2.1.4 The T7 gp4 primase-helicase is loaded by means of a facilitated ring-opening mechanism 10

2.1.5 Bacteriophage T4 gp61 primase can be viewed as a loading factor for the homologous gp41 helicase 11

2.1.6 DnaC serves as the loading factor for E. coli DnaB helicase 11

2.1.7 The role of bacteriophage T4 gp59 in loading the T4 gp41 helicase 12

2.1.8 Loading of helicases onto ssDNA covered by ssDNA-binding proteins (SSBPs) 15

2.2 DNA polymerase and ssDNA-binding proteins can serve as reporters for replicative helicases in their elongation mode 17

2.2.1 The DNA polymerase, the sliding clamp, and the clamp loader 17

2.2.2 The role of ssDNA-binding protein 18

2.2.3 Coupling is achieved by the DNA polymerase and the ssDNA-binding protein 18

2.3 Arrest of replicative helicases 18

2.3.1 The Ter sites and termination proteins 19

2.3.2 Models for orientation-specific fork arrest 20

3. Helicases in transcription 20

3.1 Assisted loading of E. coli RNAP by the sigma70 initiation factor 21

3.1.1 RNAP holoenzyme formation 23

3.1.2 Formation of closed promoter complexes RPc and RPi 24

3.1.3 Strand separation and the formation of the open complex 24

3.1.4 Promoter clearance 24

3.1.5 Conclusions 25

3.2 Transcript formation serves as a monitor (reporter) of RNAP helicase activity in the elongation phase of transcription 25

3.2.1 Structural aspects of transcription complex translocation 26

3.2.2 Transcript elongation is an approximately isoenergetic process 26

3.3 Termination of transcription 27

3.3.1 Intrinsic termination 27

3.3.2 Termination by transcription-termination helicase Rho 28

3.3.3 Conclusions 29

3.4 Loading of the Rho transcription-termination helicase 29

4. Helicases in nucleotide excision repair (NER) 30

4.1 The limited strand-separating activity of the UvrAB complex 31

4.2 UvrB is a DNA helicase adapted for NER 33

4.2.1 The ATP-binding site of UvrB is similar to that of other helicases 33

4.2.2 The putative DNA-binding site 33

4.3 UvrA as a UvrB loader 34

4.4 Assisted targeting of UvrAB to the transcribed strand of DNA sequences undergoing active transcription 34

4.4.1 Targeting of UvrAB to damaged DNA sites in the vicinity of promoters is assisted by RNAP 34

4.4.2 TRCF participates in the assisted targeting of UvrAB to a transcribing RNAP stalled by a DNA lesion 35

4.4.3 Conclusions 36

4.5 UvrC endonuclease is the reporter of UvrAB helicase activity in incision 36

4.6 Post-incision events 36

4.7 Mechanistic details of the helicase activity of UvrD 37

4.7.1 Structural organization and conformational changes 37

4.7.2 Translocation and unwinding activities 38

4.7.3 Step size of DNA unwinding 38

4.7.4 Oligomeric state 39

5. Helicases in recombination 39

5.1 Role of RecBCD and RecQ in the initiation of recombination 40

5.1.1 The RecBCD enzyme 40

5.1.1.1 Loading of RecBCD onto its DNA substrate does not require a separate loading protein 40

5.1.1.2 The endonuclease activity of RecD, and the binding of SSB protein, serve as reporters of RecBCD helicase activity 40

5.1.1.3 RecA can also serve as a reporter of RecBCD helicase activity 41

5.1.1.4 RecBCD step size and unwinding mechanism 41

5.1.1.5 RecBCD unwinding efficiency 42

5.1.2 The RecQ protein 43

5.2 Strand-exchange reaction catalyzed by RecA 43

5.2.1 The nucleoprotein filament 44

5.2.2 The strand-exchange reaction 46

5.2.2.1 A ‘minor-groove’ to ‘major-groove’ triple-helix transition 46

5.2.2.2 Role of the secondary DNA-binding site of RecA 46

5.2.2.3 SSB protein stimulates the strand-exchange reaction 46

5.2.2.4 Cost of the strand-exchange reaction 47

5.2.3 Conclusion: RecA is a ‘scaffolding’ protein that prepares DNA for a coupled unpairing–reannealing reaction 48

5.3 Role of the RuvAB helicase in processing recombination intermediates by a branch migration mechanism 48

5.3.1 A brief description of the RuvA and RuvB proteins 49

5.3.2 Crystal structures of RuvA and the RuvA–Holliday junction complex 50

5.3.3 RuvA as a scaffolding protein that prepares the homoduplex for strand separation 51

5.3.4 Branch migration mechanism 51

6. RNA unwindases in the spliceosome 52

6.1 RNA structural rearrangements within the spliceosome: an overview 52

6.2 The spliceosome consumes chemical free energy 54

6.3 RNA structural alterations require the concerted (or coupled) action of unwinding and reannealing proteins 54

6.4 The reannealing proteins of the spliceosome: contribution of the RNA recognition motifs (RRMs) 55

6.5 The RNA unwindases of the spliceosome 55

6.6 RNA targets of the RNA unwindases 56

7. Conclusions and overview 57

8. Acknowledgments 58

9. References 59

In Part I of this review [Delagoutte & von Hippel, Quarterly Reviews of Biophysics (2002) 35, 431–478] we summarized what is known about the properties, mechanisms, and structures of the various helicases that catalyze the unwinding of double-stranded nucleic acids. Here, in Part II, we consider these helicases as tightly integrated (or coupled) components of the various macromolecular machines within which they operate. The biological processes that are considered explicitly include DNA replication, recombination, and nucleotide excision repair, as well as RNA transcription and splicing. We discuss the activities of the constituent helicases (and their protein partners) in the assembly (or loading) of the relevant complex onto (and into) the specific nucleic acid sites at which the actions of the helicase-containing complexes are to be initiated, the mechanisms by which the helicases (and the complexes) translocate along the nucleic acids in discharging their functions, and the reactions that are used to terminate the translocation of the helicase-containing complexes at specific sites within the nucleic acid ‘substrate’. We emerge with several specific descriptions of how helicases function within the above processes of genetic expression which, we hope, can serve as paradigms for considering how helicases may also be coupled and function within other macromolecular machines.

The development of clustered regularly interspaced short-palindromic repeat (CRISPR)-Cas systems for genome editing has transformed the way life science research is conducted and holds enormous potential for the treatment of disease as well as for many aspects of biotechnology. Here, I provide a personal perspective on the development of CRISPR-Cas9 for genome editing within the broader context of the field and discuss our work to discover novel Cas effectors and develop them into additional molecular tools. The initial demonstration of Cas9-mediated genome editing launched the development of many other technologies, enabled new lines of biological inquiry, and motivated a deeper examination of natural CRISPR-Cas systems, including the discovery of new types of CRISPR-Cas systems. These new discoveries in turn spurred further technological developments. I review these exciting discoveries and technologies as well as provide an overview of the broad array of applications of these technologies in basic research and in the improvement of human health. It is clear that we are only just beginning to unravel the potential within microbial diversity, and it is quite likely that we will continue to discover other exciting phenomena, some of which it may be possible to repurpose as molecular technologies. The transformation of mysterious natural phenomena to powerful tools, however, takes a collective effort to discover, characterize, and engineer them, and it has been a privilege to join the numerous researchers who have contributed to this transformation of CRISPR-Cas systems.

During the last twenty-five years the development of quantum mechanical calculations and experimental measurements of chemical shifts of the different type of nuclei present in nucleic acids have run parallel in close relation to each other. The first calculations dealt with intramolecular effects on base proton shifts (Veillard, 1962) but the real breakthrough of the theory occurred with the advent of computations of intermolecular shielding due to the ring current effect of the nucleic acid bases (Giessner-Prettre & Pullman, 1970).

Some of the rate theories that are most useful for modeling biological processes are reviewed. By delving into some of the details and subtleties in the development of the theories, the review will hopefully help the reader gain a more than superficial perspective. Examples are presented to illustrate how rate theories can be used to generate insight at the microscopic level into biomolecular behaviors. An attempt is made to clear up a number of misconceptions in the literature regarding popular rate theories, including the appearance of Planck's constant in the transition-state theory and the Smoluchowski result as an upper limit for protein–protein and protein–DNA association rate constants. Future work in combining the implementation of rate theories through computer simulations with experimental probes of rate processes, and in modeling effects of intracellular environments so that theories can be used for generating rate constants for systems biology studies is particularly exciting.

Structure-based drug design has become an essential tool for rapid lead discovery and optimization. As available structural information has increased, researchers have become increasingly aware of the importance of protein flexibility for accurate description of the native state. Typical protein–ligand docking efforts still rely on a single rigid receptor, which is an incomplete representation of potential binding conformations of the protein. These rigid docking efforts typically show the best performance rates between 50 and 75%, while fully flexible docking methods can enhance pose prediction up to 80–95%. This review examines the current toolbox for flexible protein–ligand docking and receptor surface mapping. Present limitations and possibilities for future development are discussed.

The theory of van der Waals forces has now developed to a stage where it constitutes a powerful tool in theoretical investigations of many biological systems. In this review we shall consider both the theoretical and conceptual aspects of these forces with the emphasis on the way they may be involved in various biological processes.

This review is devoted to the presentation of recent developments in a field of theoretical molecular biophysics which seem to open large possibilities for progress in a hitherto relatively unexplored although very important direction. It is concerned with the establishment of a new methodology in the molecular orbital approach to the problem of the solvation of biological molecules. The methodology is still in an initial stage, susceptible of many refinements and its practical applications may also be considered as being in their beginnings. Nevertheless the successes obtained so far and the interest which they aroused in a number of laboratories incite us to present already now the general principles and the available results so as to offer a possibility of evaluating the advantages, present-day limitations and future potentialities of the procedure, which could then be explored by all those interested. This review may thus be said to be to a large extent oriented towards the future, with the acknowledged aim of producing an acceleration and widening of researches in this field. Possible progress in this respect is interesting from a double point of view.

It has been known for a number of years that calcium ions play a crucial role in excitation-contraction (e-c) coupling (Sandow, 1952). The majority of the calcium required for this process is derived, at least in vertebrate striated muscle fibres, from discrete intracellular stores located at sites within the cell: the terminal cysternae (tc)/junctional SR of the sarcoplasmic reticulum (SR) (Fig. 1 a). These storage sites not only form a compartment that is distinct from the sarcoplasm of the fibre, but they are also closely associated with the contractile elements, the myofibrils. The SR release sites are activated following the spread of electrical activity (Huxley and Taylor, 1958) along the transverse (T) tubular system (Eisenberg and Gage, 1967; Adrian et al. 1969a, b; Peachey, 1973) from the surface membrane (Bm).

The rotary ATPase family of membrane protein complexes may have only three members, but each one plays a fundamental role in biological energy conversion. The F1Fo-ATPase (F-ATPase) couples ATP synthesis to the electrochemical membrane potential in bacteria, mitochondria and chloroplasts, while the vacuolar H+-ATPase (V-ATPase) operates as an ATP-driven proton pump in eukaryotic membranes. In different species of archaea and bacteria, the A1Ao-ATPase (A-ATPase) can function as either an ATP synthase or an ion pump. All three of these multi-subunit complexes are rotary molecular motors, sharing a fundamentally similar mechanism in which rotational movement drives the energy conversion process. By analogy to macroscopic systems, individual subunits can be assigned to rotor, axle or stator functions. Recently, three-dimensional reconstructions from electron microscopy and single particle image processing have led to a significant step forward in understanding of the overall architecture of all three forms of these complexes and have allowed the organisation of subunits within the rotor and stator parts of the motors to be more clearly mapped out. This review describes the emerging consensus regarding the organisation of the rotor and stator components of V-, A- and F-ATPases, examining core similarities that point to a common evolutionary origin, and highlighting key differences. In particular, it discusses how newly revealed variation in the complexity of the inter-domain connections may impact on the mechanics and regulation of these molecular machines.

Mitosis is the process by which eukaryotic cells organize and segregate their chromosomes in preparation for cell division. It is accomplished by a cellular machine composed largely of microtubules (MTs) and their associated proteins. This article reviews literature on mitosis from a biophysical point of view, drawing attention to the assembly and motility processes required to do this complex job with precision. Work from both the recent and the older literature is integrated into a description of relevant biological events and the experiments that probe their mechanisms. Theoretical work on specific subprocesses is also reviewed. Our goal is to provide a document that will expose biophysicists to the fascination of this quite amazing process and provide them with a good background from which they can pursue their own research interests in the subject.

Molecules built up from a given set of atoms may differ in their three-dimensional structure. They may have one or more asymmetric centres that serve as reference points for the steric distribution of the atoms. Carbon atoms, common to all biomolecules, are often such centres. For example, the Cα atom between the carboxyl and amino groups in amino acids is an asymmetric centre: looking ON ward (i.e. from the carbOxyl to the amiNo group, with the Cα oriented so that it is above the carboxyl and amino groups) the radical characterizing the amino acid may be to the right (D-molecules) or to the left (L-molecules). Nineteen of the 20 amino acids occurring in proteins have such a structure (the exception is glycine, where the radical is a hydrogen atom). These pairs of molecules cannot be brought into coincidence with their own mirror image, as is the situation with our hands. The phenomenon has therefore been named handedness, or chirality, from the Greek word cheir, meaning hand. The two forms of the chiral molecules are called enantiomers or antipodes. They differ in rotating the plane of the polarized light either to the right or to the left. The sense of rotation depends on the wavelength of the measuring light, but at a given wavelength it is always opposite for a pair of enantiomers. Chirality may also occur when achiral molecules form chiral substances during crystallization (for example, quartz forms D-quartz or Lquartz). A detailed theoretical treatment of molecular chirality is given by Barron (1991).

For the cells of an organism to act in the coordinated fashion necessary for complex functioning, they must be able to receive and transmit information. Information transfer is mediated by molecules released by the cells and may be local, as in the case of neurotransmitters, or long range, as in the case of hormones. It is apparent, however, that irrespective of the range of interaction, a cell must be able to distinguish, with a high degree of precision, the signals relevant to it from an enormous flow of background noise.

Molecular recognition at the cell surface is mediated by receptors: cell surface glycoproteins that usually form an integral part of the plasma membrane (see, for example, Cuatrecasas & Greaves, 1978). Typically, receptors bind the ligands they are designed to recognize with affinities of the order of 108 M-1, and they translate that interaction into a sequence of signals that ultimately lead to biological activity.

Circular dichroism (CD) spectroscopy is a well-established technique for the study of proteins. Synchrotron radiation circular dichroism (SRCD) spectroscopy extends the utility of conventional CD spectroscopy (i.e. using laboratory-based instruments) because the high light flux from a synchrotron enables collection of data to lower wavelengths, detection of spectra with higher signal-to-noise levels and measurements in the presence of strongly absorbing non-chiral components such as salts, buffers, lipids and detergents. This review describes developments in instrumentation, methodologies and bioinformatics that have enabled new applications of the SRCD technique for the study of proteins. It includes examples of the use of SRCD spectroscopy for providing static and dynamic structural information on molecules, including determinations of secondary structures of intact proteins and domains, assessment of protein stability, detection of conformational changes associated with ligand and drug binding, monitoring of environmental effects, examination of the processes of protein folding and membrane insertion, comparisons of mutant and modified proteins, identification of intermolecular interactions and complex formation, determination of the dispositions of proteins in membranes, identification of natively disordered proteins and their binding partners and examination of the carbohydrate components of glycoproteins. It also discusses how SRCD can be used in conjunction with macromolecular crystallography and other biophysical techniques to provide a more complete picture of protein structures and functions, including how proteins interact with other macromolecules and ligands. This review also includes a discussion of potential new applications in structural and functional genomics using SRCD spectroscopy and future instrumentation and bioinformatics developments that will enable such studies. Finally, the appendix describes a number of computational/bioinformatics resources for secondary structure analyses that take advantage of the improved data quality available from SRCD. In summary, this review discusses how SRCD can be used for a wide range of structural and functional studies of proteins.