Gram-negative bacteria such as Escherichia coli (E. coli) and Salmonella typhimurium (S. typhimurium) have two layers of membranes in the cellular envelope – the cytoplasmic membrane and the outer membrane (Fig. I). Between these membranes is a periplasmic space in which there is a peptidoglycan layer that provides the cells with mechanical rigidity. In this periplasmic space, there are also a variety of hydrolases and binding proteins. The composition of the outer membrane is somewhat unusual. This membrane bilayer is asymmetric, having an inner (periplasmic) leaflet composed of phospholipids and an outer (extracellular) leaflet formed by lipopolysaccharide (LPS). Unlike phospholipids having two acyl chains, LPS has six or seven saturated fatty acid chains (see reviews, Lugtenberg & Van Alphen, 1983; Nikaido & Vaara, 1985; Nakae, 1986). The head groups of LPS have a strong affinity for divalent cations such as Ca2+, and given a sufficient concentration of these ions the outer membrane can form quite a formidable permeability barrier through this head group/salt bridge network (Nikaido & Vaara, 1985). The function of the outer membrane is to serve as a protective envelope against hostile environments such as those in the intestinal tract of animals where harmful and toxic substances - for example, bile salts and various enzymes - are often found. The outer membrane itself would be impermeable to most hydrophilic solutes were it not for the presence of membrane channels. The presence of a large number of pore-forming proteins provides both specific and nonspecific diffusion pathways across the outer membrane for solutes such as nutrients and waste products to diffuse into or out of the cell.

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.

1. INTRODUCTION 399

2. DEFINITIONS 401

3. CIS BASEPAIRS 410

3.1 Cis Watson–Crick/Watson–Crick 410

3.2 Wobble pairings 411

3.3 Cis Watson–Crick/Hoogsteen pairings 416

3.4 Bifurcated pairings 417

3.5 Cis open and water-inserted 421

4. TRANS BASEPAIRS 423

4.1 Trans Watson–Crick/Watson–Crick 423

4.2 Trans wobble pairs 424

4.3 Trans Watson–Crick/Hoogsteen pairs 424

4.4 Trans Hoogsteen/Hoogsteen pairs 430

4.5 Trans bifurcated pairings 432

5. SHALLOW-GROOVE PAIRINGS 432

5.1 Hoogsteen/Shallow-groove pairs 433

5.2 Watson–Crick/Shallow-groove pairings 438

5.3 Shallow-groove/Shallow-groove pairings 440

6. SIDE-BY-SIDE BASES 446

7. DEFINING A LIBRARY OF ISOSTERIC PAIRINGS 446

8. CONCLUSIONS 451

9. ACKNOWLEDGEMENTS 452

10. REFERENCES 452

RNA molecules fold into a bewildering variety of complex 3D structures. Almost every new RNA structure obtained at high resolution reveals new, unanticipated structural motifs, which we are rarely able to predict at the current stage of our theoretical understanding. Even at the most basic level of specific RNA interactions – base-to-base pairing – new interactions continue to be uncovered as new structures appear. Compilations of possible non-canonical base-pairing geometries have been presented in previous reviews and monographs (Saenger, 1984; Tinoco, 1993). In these compilations, the guiding principle applied was the optimization of hydrogen-bonding. All possible pairs with two standard H-bonds were presented and these were organized according to symmetry or base type. However, many of the features of RNA base-pairing interactions that have been revealed by high-resolution crystallographic analysis could not have been anticipated and, therefore were not incorporated into these compilations. These will be described and classified in the present review. A recently presented approach for inferring basepair geometry from patterns of sequence variation (Gautheret & Gutell, 1997) relied on the 1984 compilation of basepairs (Saenger, 1984), and was extended to include all possible single H-bond combinations not subject to steric clashes. Another recent review may be consulted for a discussion of the NMR spectroscopy and thermodynamic effects of non-canonical (‘mismatched’) RNA basepairs on duplex stability (Limmer, 1997).

In a recent review of the transport of salts and water across multicellular secretory tissues in animals (Keynes, 1969), a summary was given of the various types of active transport of ions necessary to explain the experimental observations in a very wide range of tissues, and five basic types of ion pump were discussed. The question of whether plants and animals have any common mechanisms for the transport of salts and water was specifically excluded. The original aim of the present review was to survey the types of ion pump found in plant cells and tissues, and to compare these with those found in animals. Its aims narrowed very considerably in writing. It now reviews ion transport processes in giant algal cells, and tries to assess progress towards understanding the mechanisms involved. It indicates the existence of similar ion transports in higher plant cells, but it does not present a complete review of the experimental work on higher plants.

All higher animals, whether they live in water or on dry land, are faced with the necessity of regulating rather closely their intake and excretion of salts and water in order to maintain the constancy of their internal ionic environment. The kidney is in general the most important organ of the body as far as the excretion of sodium, potassium, chloride and water is concerned, but there are other tissues which also play a part in controlling the ionic balance between the internal and external environments, such as the intestinal mucosa, the skin and urinary bladder in amphibia, the gill epithelium in fishes, the salt gland in marine birds, and the epithelium of the rumen in ruminants. In addition to excretory and absorptive organs of this type, there are others which are secretory and whose function involves the production of fluids differing in ionic composition from the blood plasma. Examples include the glands which secrete saliva and sweat, the oxyntic acid-producing cells of the gastric mucosa, and the epithelium of the stria vascularis which generates the potassium-rich endolymph of the mammalian cochlea. The purpose of this article is to consider briefly what is known about the active transport of salts and water across some typical multicellular secretory tissues, and to attempt in the process to discern what properties they have in common and in what respects they are specialized.

In all organisms, fatty acid synthesis is achieved in variations of a common cyclic reaction pathway by stepwise, iterative elongation of precursors with two-carbon extender units. In bacteria, all individual reaction steps are carried out by monofunctional dissociated enzymes, whereas in eukaryotes the fatty acid synthases (FASs) have evolved into large multifunctional enzymes that integrate the whole process of fatty acid synthesis. During the last few years, important advances in understanding the structural and functional organization of eukaryotic FASs have been made through a combination of biochemical, electron microscopic and X-ray crystallographic approaches. They have revealed the strikingly different architectures of the two distinct types of eukaryotic FASs, the fungal and the animal enzyme system. Fungal FAS is a 2·6 MDa α6β6 heterododecamer with a barrel shape enclosing two large chambers, each containing three sets of active sites separated by a central wheel-like structure. It represents a highly specialized micro-compartment strictly optimized for the production of saturated fatty acids. In contrast, the animal FAS is a 540 kDa X-shaped homodimer with two lateral reaction clefts characterized by a modular domain architecture and large extent of conformational flexibility that appears to contribute to catalytic efficiency.

It has long been recognized that measurements of the permeability characteristics of membranes (and of epithelia) may be strongly influenced by the presence of unstirred, or diffusion, layers adjacent to the membranes: see Dainty (1963) for the first systematic discussion of the subject in a biological context and Barry & Diamond (1983) for an important recent review. Consider a homogeneous, plane, uncharged membrane separating two solutions of the same solute at different bulk concentrations Cb+ and Cb- (Fig. 1), but at the same hydrostatic pressure and electrostatic potential (note that in this paper subscripts + and − refer to the two sides of the membrane, not to cation and anion parameters; electroneutrality is assumed throughout).

The ribosome is a complex macromolecular machine that translates the message encoded in the messenger RNA and synthesizes polypeptides by linking the individual amino acids carried by the cognate transfer RNAs (tRNAs). The protein elongation cycle, during which the tRNAs traverse the ribosome in a coordinated manner along a path of more than 100 Å, is facilitated by large-scale rearrangements of the ribosome. These rearrangements go hand in hand with conformational changes of tRNA as well as elongation factors EF-Tu and EF-G – GTPases that catalyze tRNA delivery and translocation, respectively. This review focuses on the structural data related to the dynamics of the ribosomal machinery, which are the basis, in conjunction with existing biochemical, kinetic, and fluorescence resonance energy transfer data, of our knowledge of the decoding and translocation steps of protein elongation.

Recent progress in membrane biology has brought us to a stage where it is possible to associate complex biological processes to identifiable membrane proteins. Technical advances in the biochemical characterization and purification of membrane proteins have contributed a wealth of structural information. The reconstitution approach has proved to be valuable in our efforts to understand the molecular mechanisms of membrane transport and energy transduction.

This review of the peripheral auditory apparatus represents an attempt to analyse critically recent developments in the field. The coverage is not exhaustive, the emphasis is on functional aspects and no attempt is made to review the anatomy of the ear. Particular emphasis is placed on three broad sections: the physiology of the middle ear, basilar membrane mechanics and the electrophysiology of the cochlea. It is in these areas that recent technical advances have led to experiments which throw doubt on traditionally held concepts. Such an advance is the application of the Mössbauer technique to the problem of middle- and inner-ear mechanics. Because of its novelty, this technique is discussed in detail. Other new methods such as laser holography are just starting to be used, as are the ion selective microelectrodes for measuring dynamic changes in endolymph concentration. After many years of slow progress there is a sudden spark of enthusiasm for peripheral auditory research, and the coming decade promises to be most exciting indeed.

Biological neural networks are large systems of complex elements interacting through a complex array of connexions. Individual neurons express a large number of active conductances (Connors et al. 1982; Adams & Gavin, 1986; Llinás, 1988; McCormick, 1990; Hille, 1992) and exhibit a wide variety of dynamic behaviours on time scales ranging from milliseconds to many minutes (Llinás, 1988; Harris-Warrick & Marder, 1991; Churchland & Sejnowski, 1992; Turrigiano et al. 1994).

1. INTRODUCTION 358

1.1 Summary 358

1.2 Overview 359

1.3 Four classes of pore-forming K+channel subunits – necessary and (sometimes) sufficient 361

1.4 Soluble and peripheral membrane proteins that interact with P loop subunits to alter function 362

1.5 Integral membrane proteins that interact with P loop subunits to alter function 363

2. MinK DETERMINES THE FUNCTION OF MIXED CHANNEL COMPLEXES 363

2.1 The KCNE1 gene product (MinK) gives rise to K+-selective currents and controversy 363

2.2 MinK assembles with a P loop protein, KvLQT1, to form K+channels with unique function 364

2.2.1 Single-channel conductance of KvLQT1 and MinK/KvLQT1 channels 366

2.2.2 Other differences between KvLQT1 and MinK/KvLQT1 channels 367

2.3 MinK assembles with HERG, another P loop subunit, to regulate channel activity 368

2.4 MinK does not form chloride-selective ion channels 368

3. EXPERIMENTAL AND NATURAL MinK MUTATIONS 369

3.1 Site-directed mutations 369

3.1.1 MinK mutation alters basic channel attributes and identifies key residues 369

3.1.2 MinK is a Type I transmembrane peptide 370

3.1.3 MinK is intimately associated with the IKspore 370

3.1.4 The number of MinK subunits in IKschannel complexes 372

3.2 KCNE1 mutations associated with arrhythmia and deafness alter IKschannel function 373

3.3 Summary of MinK sites critical to IKschannel function 374

4. MinK-RELATED PEPTIDES: AN EMERGING SUPERFAMILY 374

4.1 KCNE2, 3 and 4 encode MinK-related peptides 1, 2 and 3 (MiRPs) 374

4.2 MiRP1 assembles with a P loop protein, HERG, to form K+channels with unique function 375

4.2.1 MiRP1 alters activation, deactivation and single-channel conductance 376

4.2.2 MiRP1 alters regulation by K+ion and confers biphasic kinetics to channel blockade 378

4.2.3 Stable association of MiRP1 and HERG subunits 380

4.3 KCNE2 mutations are associated with arrhythmia and decreased K+flux 383

4.4 Summary of the evidence that cardiac IKrchannels are MiRP1/HERG complexes 385

5. MinK-RELATED PEPTIDES: COMMONALTIES AND IMPLICATIONS 386

5.1 Genetics and structure 386

5.2 Cell biology and function 387

6. ANSWERS, SOME OUTSTANDING ISSUES, CONCLUSIONS 387

7. ACKNOWLEDGEMENTS 389

8. REFERENCES 389

MinK and MinK-related peptide 1 (MiRP1) are integral membrane peptides with a single transmembrane span. These peptides are active only when co-assembled with pore-forming K+ channel subunits and yet their role in normal ion channel behaviour is obligatory. In the resultant complex the peptides establish key functional attributes: gating kinetics, single-channel conductance, ion selectivity, regulation and pharmacology. Co-assembly is required to reconstitute channel behaviours like those observed in native cells. Thus, MinK/KvLQT1 and MiRP1/HERG complexes reproduce the cardiac currents called IKs and IKr, respectively. Inherited mutations in KCNE1 (encoding MinK) and KCNE2 (encoding MiRP1) are associated with lethal cardiac arrhythmias. How these mutations change ion channel behaviour has shed light on peptide structure and function. Recently, KCNE3 and KCNE4 were isolated. In this review, we consider what is known and what remains controversial about this emerging superfamily.

The passage of molecules and information across cell membranes is mediated largely by membrane-spanning proteins acting as channels, pumps, receptors and enzymes. These proteins perform many tasks: they control electrochemical gradients across the membrane, receive signals from the environment or from other cells, convert light energy into chemical signals, transport small molecules into and out of cells, and harness proton gradients to generate the energy consumed in metabolism. Indeed, of the estimated 50000–100000 genes in the human genome, fully 20–40 % are thought to encode integral membrane proteins. If one also includes membrane-associated proteins, which are attached to the membrane surface through fatty acyl chains or electrostatic interactions, this percentage is likely to be much higher.

Deciphering the folding pathways and predicting the structures of complex three-dimensional biomolecules is central to elucidating biological function. RNA is single-stranded, which gives it the freedom to fold into complex secondary and tertiary structures. These structures endow RNA with the ability to perform complex chemistries and functions ranging from enzymatic activity to gene regulation. Given that RNA is involved in many essential cellular processes, it is critical to understand how it folds and functions in vivo. Within the last few years, methods have been developed to probe RNA structures in vivo and genome-wide. These studies reveal that RNA often adopts very different structures in vivo and in vitro, and provide profound insights into RNA biology. Nonetheless, both in vitro and in vivo approaches have limitations: studies in the complex and uncontrolled cellular environment make it difficult to obtain insight into RNA folding pathways and thermodynamics, and studies in vitro often lack direct cellular relevance, leaving a gap in our knowledge of RNA folding in vivo. This gap is being bridged by biophysical and mechanistic studies of RNA structure and function under conditions that mimic the cellular environment. To date, most artificial cytoplasms have used various polymers as molecular crowding agents and a series of small molecules as cosolutes. Studies under such in vivo-like conditions are yielding fresh insights, such as cooperative folding of functional RNAs and increased activity of ribozymes. These observations are accounted for in part by molecular crowding effects and interactions with other molecules. In this review, we report milestones in RNA folding in vitro and in vivo and discuss ongoing experimental and computational efforts to bridge the gap between these two conditions in order to understand how RNA folds in the cell.

Metals are commonly found as natural constituents of proteins. Since many such metals can interact specifically with their corresponding unfolded proteins in vitro, cofactor-binding prior to polypeptide folding may be a biological path to active metalloproteins. By interacting with the unfolded polypeptide, the metal may create local structure that initiates and directs the polypeptide-folding process. Here, we review recent literature that addresses the involvement of metals in protein-folding reactions in vitro. To date, the best characterized systems are simple one such as blue-copper proteins, heme-binding proteins, iron-sulfur-cluster proteins and synthetic metallopeptides. Taken together, the available data demonstrates that metals can play diverse roles: it is clear that many cofactors bind before polypeptide folding and influence the reaction; yet, some do not bind until a well-structured active site is formed. The significance of characterizing the effects of metals on protein conformational changes is underscored by the many human diseases that are directly linked to anomalous protein–metal interactions.

Like DNA and the various forms of RNA, a protein molecule is an information storage system. It contains in fact one or more polypeptide chains which may be regarded as linear sequences of twenty different types of monomer units. It is now clearly established that the chemical information corresponding to a given sequence of a polypeptide chain, containing n amino acid residues, is stored in a segment of one of the two strands of DNA containing 3n nucleotides. The transfer of such information from a gene to a polypeptide chain takes place according to the well-known process involving a transcription and a chemical translation step. This last step leads to a polymer which, in appropriate conditions, takes a three-dimensional conformation or tertiary structure which should correspond to a free-energy minimum of the molecule and its surrounding water solution.

Spectroscopic probes have become powerful tools in analysing the correlation between structure and function of biological macromolecules. Though these spectral methods cannot give as circumstantial information about the anatomy of a biological structure as, for example, X-ray diffraction they can provide information on physical properties at defined loci in a macromolecule which are not accessible by other techniques. Most important, spectroscopic studies have provided means to study the dynamics of structural changes and interactions in time domains spanning from nanoseconds and less up to infinite time.

Writing a review gives authors a splendid opportunity to view developments in a particular area of science from a very personal angle. They are at liberty to select material, emphasize aspects of direct interest to their own work and air speculations which, wisely or not, referees have caused to have removed from their publications. Such personal accounts often make good reading, but may be somewhat misleading especially for readers seeking an introduction to the field. One remedy is to aim at a comprehensive review with equal weight given to all publications but boredom, if not bias, is then likely to creep in.

In order to study proteins that do not occur in two- or three-dimensionally ordered form, one may take two different approaches: either search for conditions that induce the formation of crystals, and proceed with the established methods of X-ray or electron crystallography, or attempt to study the molecules in the form of single particles with the EM. Although many proteins have been successfully crystallized, and some general recipes for inducing ordered arrangement have been found (Mannella, 1984; Uzgiris & Kornberg, 1983), there exists a large number of proteins and protein assemblies that have resisted such attempts for a long time. Furthermore, there are macromolecular assemblies, associated with membranes and engaged in switching or gating, whose function is tied to their occurrence in isolated form, and hence are best studied without extraction from the membrane. For these reasons, the single-particle approach to the study of macromolecular structure (Frank et al. 1978, 1981; Radermacher et al. 1987a, b; for recent reviews, see Frank et al. 1985, 1988e; Frank, 1989) has found numerous applications after initial technical and conceptual hurdles were overcome. Although atomic resolution cannot be achieved with this approach for a variety of reasons, a quantitative description of architecture on the quaternary level is nevertheless possible, as exemplified in the 3D studies of ribosomal particles (overviews, see Frank et al. 1988a) and, most recently, the junctional channel complex (Wagenknecht et al. 1989a).

The last decade has witnessed a renewed interest in the coarse-grained (CG) models for biopolymers, also stimulated by the needs of modern molecular biology, dealing with nano- to micro-sized bio-molecular systems and larger than microsecond timescale. This combination of size and timescale is, in fact, hard to access by atomic-based simulations. Coarse graining the system is a route to be followed to overcome these limits, but the ways of practically implementing it are many and different, making the landscape of CG models very vast and complex.

In this paper, the CG models are reviewed and their features, applications and performances compared. This analysis, restricted to proteins, focuses on the minimalist models, namely those reducing at minimum the number of degrees of freedom without losing the possibility of explicitly describing the secondary structures. This class includes models using a single or a few interacting centers (beads) for each amino acid.

From this analysis several issues emerge. The difficulty in building these models resides in the need for combining transferability/predictive power with the capability of accurately reproducing the structures. It is shown that these aspects could be optimized by accurately choosing the force field (FF) terms and functional forms, and combining different parameterization procedures. In addition, in spite of the variety of the minimalist models, regularities can be found in the parameters values and in FF terms. These are outlined and schematically presented with the aid of a generic phase diagram of the polypeptide in the parameter space and, hopefully, could serve as guidelines for the development of minimalist models incorporating the maximum possible level of predictive power and structural accuracy.