The phenomenon of electro-optic orientation was discovered by John Kerr in 1875 and has been used extensively for determining the optical polarizability anisotropy of small molecules and for high-speed transmission of optical signals. Measurements on biopolymers have been made at least since 1950, but only in the last decade have these yielded definitive structural and physical information. In the course of this review, it should become obvious that among the reasons for this late development is the inherent difficulty of analysing optical data that depend simultaneously on intrinsic optical-structural properties of the molecules, and on their degree of orientation under the conditions of the experiment. The problem has been particularly difficult far biopolymers such as the nucleic acids, whose polarization in an electric field is dependent on their special polyelectrolyte properties. These unique electrostatic properties are an important feature in the interpretation of the experimental observations.

Branched DNA molecules provide a challenging set of structural problems. Operationally we define branched DNA species as molecules in which double helical segments are interrupted by abrupt discontinuities, and we draw together a number of different kinds of structure in the class, including helical junctions of different orders, and base bulges (Fig. 1).

Many cells release preformed material contained in secretory granules by exocytosis. Exocytosis is a specialized means of secretion in which the granules fuse with the plasma membrane and thereby discharge their contents through the fusion pores. This mechanism mediates, for example, the formation of the fertilization envelope in eggs, the release of neurotransmitters and neuropeptides by neurons, the release of a variety of enzymes and mediators by mast cells and granulocytes or the secretion of hormones by endocrine cells. Classical methods for investigating exocytosis usually measure release of secreted material.

The preceding paper by Douglas Magde has recounted the basic principles of Fluorescence Correlation Spectroscopy (FCS) as originally described (see Magde, Elson & Webb, 1972; Elson & Magde, 1974; Magde, Elson & Webb, 1974 Elson & Webb, 1975; referred to collectively as MEW), and has described the first application to chemical kinetics. In this paper I shall first illustrate the same principles of FCS with a simple graphical demonstration model based on the scheme for application to lateral diffusion in membranes as it was developed in our laboratory by Dr T. J. Herbert; I shall then proceed to discuss some current research in our group organized jointly with Professor E. L. Elson at Cornell.

The strong, long-range electrostatic forces described by Coulomb's law disappear for ions in water, and the behavior of these ions is instead controlled by their water affinity – a weak, short-range force which arises from their charge density. This was established experimentally in the mid-1980s by size-exclusion chromatography on carefully calibrated Sephadex® G-10 (which measures the effective volume and thus the water affinity of an ion) and by neutron diffraction with isotopic substitution (which measures the density and orientation of water molecules near the diffracting ion and thus its water affinity). These conclusions have been confirmed more recently by molecular dynamics simulations, which explicitly model each individual water molecule. This surprising change in force regime occurs because the oppositely charged ions in aqueous salt solutions exist functionally as ion pairs (separated by 0, 1 or 2 water molecules) as has now been shown by dielectric relaxation spectroscopy; this cancels out the strong long-range electrostatic forces and allows the weak, short-range water affinity effects to come to the fore. This microscopic structure of aqueous salt solutions is not captured by models utilizing a macroscopic dielectric constant. Additionally, the Law of Matching Water Affinity, first described in 1997 and 2004, establishes that contact ion pair formation is controlled by water affinity and is a major determinant of the solubility of charged species since only a net neutral species can change phases.

Biological electron transfers often occur between metal-containing cofactors that are separated by very large molecular distances. Employing photosensitizer-modified iron and copper proteins, we have shown that single-step electron tunneling can occur on nanosecond to microsecond timescales at distances between 15 and 20 Å. We also have shown that charge transport can occur over even longer distances by hole hopping (multistep tunneling) through intervening tyrosines and tryptophans. In this perspective, we advance the hypothesis that such hole hopping through Tyr/Trp chains could protect oxygenase, dioxygenase, and peroxidase enzymes from oxidative damage. In support of this view, by examining the structures of P450 (CYP102A) and 2OG-Fe (TauD) enzymes, we have identified candidate Tyr/Trp chains that could transfer holes from uncoupled high-potential intermediates to reductants in contact with protein surface sites.

There has been a surge of recent interest, reflected by a sharp increase in the number of publications, in the area of high-resolution nuclear magnetic resonance (NMR) studies of DNA. The goal of many of these studies is to monitor the structure of biologically important DNA sequences directly in solution; the impetus for such studies was the realization, from early single-crystal X-ray structures, that nearest-neighbor context effects are a major determinant of local structure in short double-helical DNAs (Dickerson & Drew, 1981; Dickerson, 1983). Thus, instead of the previously assumed regular averaged structure of the double helix derived from fibre diffraction analysis, the more interesting concept emerged that specific sequence-dependent distortions from ‘classical’ DNA structure might be responsible for the recognition of such sequences by a variety of ligands such as repressors, polymerases, drugs, etc.

The glmS ribozyme is the first known example of a natural ribozyme that has evolved to require binding of an exogenous small molecule for activity. In Gram-positive bacteria, this RNA domain is part of the messenger RNA (mRNA) encoding the essential enzyme that synthesizes glucosamine-6-phosphate (GlcN6P). When present at physiologic concentration, this small molecule binds to the glmS ribozyme and uncovers a latent self-cleavage activity that ultimately leads to degradation of the mRNA. Biochemical and structural studies reveal that the RNA adopts a rigid fold stabilized by three pseudoknots and the packing of a peripheral domain against the ribozyme core. GlcN6P binding to this pre-organized RNA does not induce conformational changes; rather, the small molecule functions as a coenzyme, providing a catalytically essential amine group to the active site. The ribozyme is not a passive player, however. Active site functional groups are essential for catalysis, even in the presence of GlcN6P. In addition to being a superb experimental system with which to analyze how RNA catalysts can exploit small molecule coenzymes to broaden their chemical versatility, the presence of the glmS ribozyme in numerous pathogenic bacteria make this RNA an attractive target for the development of new antibiotics and antibacterial strategies.

Few proteins have come under such intense scrutiny as superoxide dismutase-1 (SOD1). For almost a century, scientists have dissected its form, function and then later its malfunction in the neurodegenerative disease amyotrophic lateral sclerosis (ALS). We now know SOD1 is a zinc and copper metalloenzyme that clears superoxide as part of our antioxidant defence and respiratory regulation systems. The possibility of reduced structural integrity was suggested by the first crystal structures of human SOD1 even before deleterious mutations in the sod1 gene were linked to the ALS. This concept evolved in the intervening years as an impressive array of biophysical studies examined the characteristics of mutant SOD1 in great detail. We now recognise how ALS-related mutations perturb the SOD1 maturation processes, reduce its ability to fold and reduce its thermal stability and half-life. Mutant SOD1 is therefore predisposed to monomerisation, non-canonical self-interactions, the formation of small misfolded oligomers and ultimately accumulation in the tell-tale insoluble inclusions found within the neurons of ALS patients. We have also seen that several post-translational modifications could push wild-type SOD1 down this toxic pathway. Recently we have come to view ALS as a prion-like disease where both the symptoms, and indeed SOD1 misfolding itself, are transmitted to neighbouring cells. This raises the possibility of intervention after the initial disease presentation. Several small-molecule and biologic-based strategies have been devised which directly target the SOD1 molecule to change the behaviour thought to be responsible for ALS. Here we provide a comprehensive review of the many biophysical advances that sculpted our view of SOD1 biology and the recent work that aims to apply this knowledge for therapeutic outcomes in ALS.

The problem of how the brain stores and retrieves information is ultimately an experimental one, and its solution will doubtless call for the combined resources of psychology, physiology and molecular biology. But it is also a problem of great theoretical sophistication; and one of the major tasks confronting the brain scientist is the construction of theoretical models which are worthy of, and open to, experimental test. In this review we shall be concerned with the latter aspect of the problem of memory, which has attracted quite a lot of attention in the last few years. It is early yet to judge the relative merits of the various models in any detail; but as we shall see, most of those which have been developed beyond their initial hypotheses have a certain family resemblance, and it seems as if we may now be in possession of the basic ideas which will be needed for the understanding of one of the central problems of memory, namely the mechanism of associative recall.

It is more than 80 years since Gratia first described ‘a remarkable antagonism between two strains of Escherichia coli’. Shown subsequently to be due to the action of proteins (or peptides) produced by one bacterium to kill closely related species with which it might be cohabiting, such bacteriocins have since been shown to be commonplace in the internecine warfare between bacteria. Bacteriocins have been studied primarily from the twin perspectives of how they shape microbial communities and how they penetrate bacteria to kill them. Here, we review the modes of action of a family of bacteriocins that cleave nucleic acid substrates in E. coli, known collectively as nuclease colicins, and the specific immunity (inhibitor) proteins that colicin-producing organisms make in order to avoid committing suicide. In a process akin to targeting in mitochondria, nuclease colicins engage in a variety of cellular associations in order to translocate their cytotoxic domains through the cell envelope to the cytoplasm. As well as informing on the process itself, the study of nuclease colicin import has also illuminated functional aspects of the host proteins they parasitize. We also review recent studies where nuclease colicins and their immunity proteins have been used as model systems for addressing fundamental problems in protein folding and protein–protein interactions, areas of biophysics that are intimately linked to the role of colicins in bacterial competition and to the import process itself.

All living cells require a supply of energy from appropriate metabolic pathways in order in fulfill their physiological functions. A special function common to peripheral nerve fibres and the electroplates of the electric organ is the generation of electrical potentials and a consequent flow of current. A large fraction of their metabolism is therefore devoted basically to maintenance of the unequal distribution of sodium and potassium ions across the cell membranes on which their electrical excitability depends, and involves a consumption of ATP by the membrane-bound Na, K—ATPase system known as the sodium pump.

The discovery of mode-locked laser operation now nearly two decades ago has started a development which enables researchers to probe the dynamics of ultrafast physical and chemical processes at the molecular level on shorter and shorter time scales. Naturally the first applications were in the fields of photophysics and photochemistry where it was then possible for the first time to probe electronic and vibrational relaxation processes on a sub-nanosecond timescale. The development went from lasers producing pulses of many picoseconds to the shortest pulses which are at present just a few femtoseconds long. Soon after their discovery ultrashort pulses were applied also to biological systems which has revealed a wealth of information contributing to our understanding of a broadrange of biological processes on the molecular level.It is the aim of this review to discuss the recent advances and point out some future trends in the study of ultrafast processes in biological systems using laser techniques. The emphasis will be mainly on new results obtained during the last 5 or 6 years. The term ultrafast means that I shall restrict myself to sub-nanosecond processes with a few exceptions.

The ability of metal surfaces and nanostructures to localize and enhance optical fields is the primary reason for their application in biosensing and imaging. Local field enhancement boosts the signal-to-noise ratio in measurements and provides the possibility of imaging with resolutions significantly better than the diffraction limit. In fluorescence imaging, local field enhancement leads to improved brightness of molecular emission and to higher detection sensitivity and better discrimination. We review the principles of plasmonic fluorescence enhancement and discuss applications ranging from biosensing to bioimaging.

1. Summary 288

2. Introduction 288

3. Relationship between neuroenergetics and neurotransmitter flux 294

4. A model of coupling between neuroenergetics and neurotransmission 296

5. Relationship between neuroenergetics and neural spiking frequency 297

6. Comparison with previous electrophysiological and fMRI measurements 298

7. Contributions of non-oxidative energetics to a primarily oxidative brain 299

8. Possible explanation for non-oxidative energetics contributions 300

9. A model of total neuronal activity to support cerebral function 302

10. Implications for interpretation of fMRI studies 305

11. The restless brain 306

12. Acknowledgements 310

13. Appendix A. CMRO2by13C-MRS 310

14. Appendix B.Vcycand test of model 313

15. Appendix C. CMRO2by calibrated BOLD 316

16. Appendix D. Comparison of spiking activity of a neuronal ensemble with CMRO2318

17. References 320

In vivo13C magnetic resonance spectroscopy (MRS) studies of the brain have quantitatively assessed rates of glutamate–glutamine cycle (Vcyc) and glucose oxidation (CMRGlc(ox)) by detecting 13C label turnover from glucose to glutamate and glutamine. Contrary to expectations from in vitro and ex vivo studies, the in vivo13C-MRS results demonstrate that glutamate recycling is a major metabolic pathway, inseparable from its actions of neurotransmission. Furthermore, both in the awake human and in the anesthetized rat brain, Vcyc and CMRGlc(ox) are stoichiometrically related, where more than two thirds of the energy from glucose oxidation supports events associated with glutamate neurotransmission. The high energy consumption of the brain measured at rest and its quantitative relation to neurotransmission reflects a sizeable activity level for the resting brain. The high activity of the non-stimulated brain, as measured by cerebral metabolic rate of oxygen use (CMRO2), establishes a new neurophysiological basis of cerebral function that leads to reinterpreting functional imaging data because the large baseline signal is commonly discarded in cognitive neuroscience paradigms. Changes in energy consumption (ΔCMRO2%) can also be obtained from magnetic resonance imaging (MRI) experiments, using the blood oxygen level- dependent (BOLD) image contrast, provided that all the separate parameters contributing to the functional MRI (fMRI) signal are measured. The BOLD-derived ΔCMRO2% when compared with alterations in neuronal spiking rate (Δν%) during sensory stimulation in the rat reveals a stoichiometric relationship, in good agreement with 13C-MRS results. Hence fMRI when calibrated so as to provide ΔCMRO2% can provide high spatial resolution evaluation of neuronal activity. Our studies of quantitative measurements of changes in neuroenergetics and neurotransmission reveal that a stimulus does not provoke an arbitrary amount of activity in a localized region, rather a total level of activity is required where the increment is inversely related to the level of activity in the non-stimulated condition. These biophysical experiments have established relationships between energy consumption and neuronal activity that provide novel insights into the nature of brain function and the interpretation of fMRI data.

Except when radiation participates, all biological activities involve contact interactions between constituent reactants. At the molecular level, if one of the participants is smaller than its complementary partner, the former is usually designated the ‘ligand’ and the latter the ‘receptor’. Thus in an enzyme–substrate complex, the substrate is the ligand, the enzyme is the receptor. In immunological interactions, the ligand is the hapten or antigen, the receptor is the immunoglobulin. Neurotransmitters or hormones are effector ligands when they are bound to receptor sites at synapses or on cell membranes.

Helicases are a class of nucleic acid (NA) motors that catalyze NTP-dependent unwinding of NA duplexes into single strands, a reaction essential to all areas of NA metabolism. In the last decade, single-molecule (sm) technology has proven to be highly useful in revealing mechanistic insight into helicase activity that is not always detectable via ensemble assays. A combination of methods based on fluorescence, optical and magnetic tweezers, and flow-induced DNA stretching has enabled the study of helicase conformational dynamics, force generation, step size, pausing, reversal and repetitive behaviors during translocation and unwinding by helicases working alone and as part of multiprotein complexes. The contributions of these sm investigations to our understanding of helicase mechanism and function will be discussed.

Thermally-induced protein unfolding is commonly described with the two-state model. This model assumes only two types of protein molecules in solution, the native (N) and the denatured, unfolded (U) protein. In reality, protein unfolding is a multistep process, even if intermediate states are only sparsely populated. As an alternative approach we explore the Zimm–Bragg theory, originally developed for the α-helix-to-random coil transition of synthetic polypeptides. The theory includes intermediate structures with concentrations determined by the cooperativity of the unfolding reaction. We illustrate the differences between the two-state model and the Zimm–Bragg theory with measurements of apolipoprotein A-1 and lysozyme by differential scanning calorimetry (DSC) and CD spectroscopy. Nine further protein examples are taken from the literature. The Zimm–Bragg theory provides a perfect fit of the calorimetric unfolding transitions for all proteins investigated. In contrast, the transition curves and enthalpies predicted by the two-state model differ considerably from the experimental results. Apolipoprotein A-1 is ~50% α-helical at ambient temperature and its unfolding follows the classical α-helix-to-random coil equilibrium. The unfolding of proteins with little α-helix content, such as lysozyme, can also be analyzed with the Zimm–Bragg theory by introducing the concept of ‘folded’ and ‘unfolded’ peptide units assuming an average unfolding enthalpy per peptide unit. DSC is the method of choice to measure the unfolding enthalpy, $\Delta H_{\rm exp} ^0 $, but CD spectroscopy in combination with the two-state model is often used to deduce the unfolding enthalpy. This can lead to erroneous result. Not only are different enthalpies required to describe the CD and DSC transition curves but these values deviate distinctly from the experimental result. In contrast, the Zimm–Bragg theory predicts the DSC and CD unfolding transitions with the same set of parameters.

One of the important problems of biophysics is to understand in detail the mechanisms by which animals produce acoustic signals, and the ways in which other animals, of the same or different species, detect and process these signals, through the masking effects of ambient noise, to extract their information content. At a primitive level this information relates simply to the presence of the other animal and perhaps to its proximity and direction. At the next level we find some sort of coding in the acoustic signal which identifies the species and perhaps the sex, and finally there is the whole range of information content and emotional overtone contained in a complex sound like human speech or music.

DNA polymerases play a central role in biology by transferring genetic information from one generation to the next during cell division. Harnessing the power of these enzymes in the laboratory has fueled an increase in biomedical applications that involve the synthesis, amplification, and sequencing of DNA. However, the high substrate specificity exhibited by most naturally occurring DNA polymerases often precludes their use in practical applications that require modified substrates. Moving beyond natural genetic polymers requires sophisticated enzyme-engineering technologies that can be used to direct the evolution of engineered polymerases that function with tailor-made activities. Such efforts are expected to uniquely drive emerging applications in synthetic biology by enabling the synthesis, replication, and evolution of synthetic genetic polymers with new physicochemical properties.