1. Introduction 98

2. Historical background of two-photon effects 99

2.1 2PE 100

2.2 Harmonic generation 100

2.3 Fluorescence correlation spectroscopy 100

3. Basic principles of two-photon excitation of fluorescent molecules and implications for microscopy and spectroscopy 101

3.1 General considerations 101

3.2 Fluorescence intensity under the 2PE condition 103

3.3 Optical consequences of 2PE 104

3.4 Saturation effects in 2PE 108

3.5 Fluorescence correlation spectroscopy 109

3.5.1 Autocorrelation analysis 110

3.5.2 Photon-counting histogram analysis 112

4. Two-photon-excited probes 115

5. Design considerations for a 2PE fluorescence microscope 119

5.1 General aspects 119

5.2 Descanned and non-descanned 2PE imaging 121

5.3 Lens objectives and pulse broadening 122

5.4 Laser sources 125

5.5 Example of a practical realization 127

6. Applications 134

6.1 Biological applications of 2PE 134

6.1.1 Brain images 134

6.1.2 Applications on the kidney 139

6.1.3 Mammalian embryos 139

6.1.4 Applications to immuno-response 141

6.1.5 Myocytes 141

6.1.6 Retina 142

6.1.7 DNA imaging 143

6.1.8 FISH applications 144

6.2 2PE imaging of single molecules 144

6.3 FCS applications 148

6.4 Signals from nonlinear interactions 151

7. Conclusions 153

8. Acknowledgements 154

9. References 155

This review is concerned with two-photon excited fluorescence microscopy (2PE) and related techniques, which are probably the most important advance in optical microscopy of biological specimens since the introduction of confocal imaging. The advent of 2PE on the scene allowed the design and performance of many unimaginable biological studies from the single cell to the tissue level, and even to whole animals, at a resolution ranging from the classical hundreds of nanometres to the single molecule size. Moreover, 2PE enabled long-term imaging of in vivo biological specimens, image generation from deeper tissue depth, and higher signal-to-noise images compared to wide-field and confocal schemes. However, due to the fact that up to this time 2PE can only be considered to be in its infancy, the advantages over other techniques are still being evaluated. Here, after a brief historical introduction, we focus on the basic principles of 2PE including fluorescence correlation spectroscopy. The major advantages and drawbacks of 2PE-based experimental approaches are discussed and compared to the conventional single-photon excitation cases. In particular we deal with the fluorescence brightness of most used dyes and proteins under 2PE conditions, on the optical consequences of 2PE, and the saturation effects in 2PE that mostly limit the fluorescence output. A complete section is devoted to the discussion of 2PE of fluorescent probes. We then offer a description of the central experimental issues, namely: choice of microscope objectives, two-photon excitable dyes and fluorescent proteins, choice of laser sources, and effect of the optics on 2PE sensitivity. An inevitably partial, but vast, overview of the applications and a large and up-to-date bibliography terminate the review. As a conclusive comment, we believe that 2PE and related techniques can be considered as a mainstay of the modern biophysical research milieu and a bright perspective in optical microscopy.

This review focuses on the so-called SNARE (soluble N-ethyl maleimide sensitive factor attachment protein receptor) proteins that are involved in exocytosis at the pre-synpatic plasma membrane. SNAREs play a role in docking and fusion of synaptic vesicles to the active zone, as well as in the Ca2+-triggering step itself, most likely in combination with the Ca2+ sensor synaptotagmin. Different SNARE domains are involved in different processes, such as regulation, docking, and fusion. SNAREs exhibit multiple configurational, conformational, and oliogomeric states. These different states allow SNAREs to interact with their matching SNARE partners, auxiliary proteins, or with other SNARE domains, often in a mutually exclusive fashion. SNARE core domains undergo progressive disorder to order transitions upon interactions with other proteins, culminating with the fully folded post-fusion (cis) SNARE complex. Physiological concentrations of neuronal SNAREs can juxtapose membranes, and promote fusion in vitro under certain conditions. However, significantly more work will be required to reconstitute an in vitro system that faithfully mimics the Ca2+-triggered fusion of a synaptic vesicle at the active zone.

1. Introduction 222

2. What is an RNA motif? 222

2.1 Sequence vs. structural motifs 222

2.2 RNA structural motifs 223

2.3 RNA structural elements vs. motifs 223

2.4 Specific recognition motifs 224

2.5 Tools for identifying and classifying elements and motifs 226

3. Types of RNA structural motifs 228

3.1 Helices 228

3.2 Hairpin loops 228

3.3 Internal loops 230

3.4 Junction loops/multiloops 230

3.5 Binding motifs 232

3.5.1 Metal binding 232

3.5.2 Natural and selected aptamers 234

3.6 Tertiary interactions 234

4. Future directions 236

5. Acknowledgments 239

6. References 239

RNAs are modular biomolecules, composed largely of conserved structural subunits, or motifs. These structural motifs comprise the secondary structure of RNA and are knit together via tertiary interactions into a compact, functional, three-dimensional structure and are to be distinguished from motifs defined by sequence or function. A relatively small number of structural motifs are found repeatedly in RNA hairpin and internal loops, and are observed to be composed of a limited number of common ‘structural elements’. In addition to secondary and tertiary structure motifs, there are functional motifs specific for certain biological roles and binding motifs that serve to complex metals or other ligands. Research is continuing into the identification and classification of RNA structural motifs and is being initiated to predict motifs from sequence, to trace their phylogenetic relationships and to use them as building blocks in RNA engineering.

The Nobel Workshop ‘Fundamentals of Biomolecular Function: Nucleic Acids, Proteins, and Membranes’, was hosted by the city of Coimbra, Portugal, and the University of Coimbra in May 2005. The workshop focused on three subjects: (i) the functions of biological macromolecules, (ii) the interactions underlying these functions, and (iii) new techniques for studying these interactions. A brief summary of the discussions follows.

1. Introduction 2

2. The macroscopic and microscopic views of protein folding 2

2.1 The macroscopic view: the experimental folding free-energy profile 2

2.2 The microscopic view: an underlying energy landscape 3

3. The micro to macro projection: from an energy landscape to a free-energy profile 6

4. Global features of the protein folding transition-state ensemble 12

4.1 Overall transition state location β[Dagger]: a measure of compactness 12

4.2 What makes folding so robust ? 13

5. Structural characterization of the transition-state ensemble 16

5.1 Insights from ϕ-value analysis 16

6. Deviations from ideality 20

6.1 β[Dagger] shifts along seemingly robust trajectories 21

6.2 Anomalous ϕ values, frustration and inhomogeneities 25

7. Intermediates 28

8. Detours, traps and frustration 29

8.1 Premature collapse and non-native trapping 29

9. Diffusion on the energy landscape and the elementary events of protein folding 30

10. Malleability of folding routes: changes of the dominant collective coordinates for folding 33

11. The evolution of the shape of the energy landscape 35

11.1 Negative design: the hidden dimension of the folding code 35

12. Mechanistic multiplicity and evolutionary choice 36

13. Acknowledgements 37

14. References 38

We review what has been learned about the protein-folding problem from experimental kinetic studies. These studies reveal patterns of both great richness and surprising simplicity. The patterns can be interpreted in terms of proteins possessing an energy landscape which is largely, but not completely, funnel-like. Issues such as speed limitations of folding, the robustness of folding, the origin of barriers and cooperativity and the ensemble nature of transition states, intermediate and traps are assessed using the results from several experimental groups highlighting energy-landscape ideas as an interpretive framework.

Homing endonucleases are encoded by open reading frames that are embedded within group I, group II and archael introns, as well as inteins (intervening sequences that are spliced and excised post-translationally). These enzymes initiate transfer of those elements (and themselves) by generating strand breaks in cognate alleles that lack the intervening sequence, as well as in additional ectopic sites that broaden the range of intron and intein mobility. Homing endonucleases can be divided into several unique families that are remarkable in several respects: they display extremely high DNA-binding specificities which arise from long DNA target sites (14–40 bp), they are tolerant of a variety of sequence variations in these sites, and they display disparate DNA cleavage mechanisms. A significant number of homing endonucleases also act as maturases (highly specific cofactors for the RNA splicing reactions of their cognate introns). Of the known homing group I endonuclease families, two (HNH and His-Cys box enzymes) appear to be diverged from a common ancestral nuclease. While crystal structures of several representatives of the LAGLIDADG endonuclease family have been determined, only structures of single members of the HNH (I-HmuI), His-Cys box (I-PpoI) and GIY-YIG (I-TevI) families have been elucidated. These studies provide an important source of information for structure–function relationships in those families, and are the centerpiece of this review. Finally, homing endonucleases are significant targets for redesign and selection experiments, in hopes of generating novel DNA binding and cutting reagents for a variety of genomic applications.

1. Introduction 168

1.1 Genomic annotation of metalloproteins 168

1.2 Why NMR structures? 168

1.3 Why paramagnetic metalloproteins? 169

2. General theory 170

2.1 Nuclear and electron spins 170

2.2 Hyperfine coupling 171

2.3 The effect of the hyperfine coupling on the NMR shift: the hyperfine shift 173

2.4 The effect of the hyperfine coupling on nuclear relaxation 174

2.5 Interplay between electron spin properties and features of the NMR spectra 178

3. Paramagnetism-based structural restraints 180

3.1 Contact shifts and relaxation rates as restraints 181

3.2 Locating the metal ion within the protein frame: pseudocontact shifts 184

3.3 Cross-correlation rates 186

3.4 Residual dipolar couplings 188

3.5 Interplay between different restraints 190

4. NMR without1H detection 191

4.1 The protocol for 13C-detected protonless assignment of backbone and side-chains 194

4.2 Protonless heteronuclear NMR experiments tailored to paramagnetic systems 196

5. The use of lanthanides as paramagnetic probes 198

6. The case of Cu(II) proteins 202

7. Perspectives 208

8. Acknowledgments 209

9. References 209

Metalloproteins represent a large share of the proteome and many of them contain paramagnetic metal ions. The knowledge, at atomic resolution, of their structure in solution is important to understand processes in which they are involved, such as electron transfer mechanisms, enzymatic reactions, metal homeostasis and metal trafficking, as well as interactions with their partners. Formerly considered as unfeasible, the first structure in solution by nuclear magnetic resonance (NMR) of a paramagnetic protein was obtained in 1994. Methodological and instrumental advancements pursued over the last decade are such that NMR structure of paramagnetic proteins may be now routinely obtained. We focus here on approaches and problems related to the structure determination of paramagnetic proteins in solution through NMR spectroscopy. After a survey of the background theory, we show how the effects produced by the presence of a paramagnetic metal ion on the NMR parameters, which are in many cases deleterious for the detection of NMR spectra, can be overcome and turned into an additional source of structural restraints. We also briefly address features and perspectives given by the use of 13C-detected protonless NMR spectroscopy for proteins in solution. The structural information obtained through the exploitation of a paramagnetic center are discussed for some Cu2+-binding proteins and for Ca2+-binding proteins, where the replacement of a diamagnetic metal ion with suitable paramagnetic metal ions suggests novel approaches to the structural characterization of proteins containing diamagnetic and NMR-silent metal ions.

During the last 15 years, scientists have developed methods that permit the direct mechanical manipulation of individual molecules. Using this approach, they have begun to investigate the effect of force and torque in chemical and biochemical reactions. These studies span from the study of the mechanical properties of macromolecules, to the characterization of molecular motors, to the mechanical unfolding of individual proteins and RNA. Here I present a review of some of our most recent results using mechanical force to unfold individual molecules of RNA. These studies make it possible to follow in real time the trajectory of each molecule as it unfolds and characterize the various intermediates of the reaction. Moreover, if the process takes place reversibly it is possible to extract both kinetic and thermodynamic information from these experiments at the same time that we characterize the forces that maintain the three-dimensional structure of the molecule in solution. These studies bring us closer to the biological unfolding processes in the cell as they simulate in vitro, the mechanical unfolding of RNAs carried out in the cell by helicases. If the unfolding process occurs irreversibly, I show here that single-molecule experiments can still provide equilibrium, thermodynamic information from non-equilibrium data by using recently discovered fluctuation theorems. Such theorems represent a bridge between equilibrium and non-equilibrium statistical mechanics. In fact, first derived in 1997, the first experimental demonstration of the validity of fluctuation theorems was obtained by unfolding mechanically a single molecule of RNA. It is perhaps a sign of the times that important physical results are these days used to extract information about biological systems and that biological systems are being used to test and confirm fundamental new laws in physics.

1. Viruses and other informational parasites are a ‘fact of life’ 304

2. Evolution drives diversity in viral strategies and host interactions 304

3. Cellular defense often involves recognizing nucleic acid structures that are indicative of unwanted information duplication 305

3.1 Double-stranded RNA (dsRNA) 305

3.2 Single-stranded DNA (ssDNA) 305

3.3 Unspliced mRNAs 306

3.4 Polycistronic mRNAs 306

3.5 Multi-copy DNAs 306

3.6 Other structures 306

4. dsRNA as a case study: building a multilevel response to foreign structure 306

5. Getting there is half the fun: disseminated immunity in RNAi 308

6. Viruses strike back … so why do we still get sick? 308

Cells face a constant struggle against unwanted instructions that arrive in the form of viruses and transposons. At the core of this battle are two issues: how can cellular machinery recognize certain informational molecules as ‘unwanted’ and how can the cell use this recognition to effectively silence malicious genetic activity. While defenses against some specific parasites may be triggered by individual nucleic acid or protein sequences, such sequence-specific mechanisms have the limitation of allowing the parasite to evade following relatively minor evolutionary change. A more general set of defense mechanisms is based on recognition of structural features that are intrinsic aspects of one or more parasitic lifestyle. Recognition of extended regions of double-stranded RNA (dsRNA) provides cells with one such defensive modality. Essentially absent during ‘normal’ gene expression, long stretches of dsRNA within a cell serve as a dramatic warning that a segment of information may be replicating as RNA. In addition to exemplifying many of the mechanistic issues in genome defense, the cellular response to dsRNA provides several examples of the logic by which organisms attempt to focus their limited immunity resources on the most immediate and dangerous targets.

Introduction 312

Triple helices in DNA 312

Chemically modified TFOs 313

Further development 316

Recognition of GC base pairs 316

Recognition of TA base pairs 316

Recognition of AT base pairs 317

Recognition of CG base pairs 317

RNA triplexes 317

Kinetics of triplex formation 318

Practical applications of triplexes 318

Conclusions 319

References 319

Watson–Crick base pairing is a natural molecular recognition process that has been exploited in molecular biology and universally adopted in many fields. An additional mode of nucleic acid sequence recognition that could be used in combination with normal base pairing would add an exta dimension to nucleic acid interactions and open up many new applications. In principle the triplex approach could provide this if developed to recognize any DNA sequence. To this end modified nucleosides have been incorporated into triple-helix-forming oligonucleotides (TFOs) and used to recognize mixed sequence DNA with high selectivity and affinity at neutral pH. Continuing developments are directed towards improving TFO affinity at high pH and increasing triplex association kinetics. A number of applications of triplexes are currently being explored.

1. Introduction 321

2. Tyrosine kinase receptors 322

3. Histidine kinase sensors 325

4. G-protein coupled receptors 327

5. Principles 328

6. Acknowledgments 329

7. References 330

Biological cells need to be responsive to various stimuli, primarily chemical ligands from their environments. Specific receptor molecules embedded in the plasma membrane detect the different biochemical signals that impact the cell, and these receptors are the conduits for transmission of this information to the cell interior for action. There are several classes of signal transduction receptors and many specific receptors within each of the major classes. This review emphasizes the structural biology of three major classes of transmembrane receptors – tyrosine kinase receptors, histidine kinase sensors, and G-protein coupled receptors. Biophysical principles that govern the processes of signal transduction across cell membranes are also discussed.

Biological interactions are ‘processes’ 331

Intermolecular forces involved 332

Synergy between different forces occurring at different locations 333

Non-equilibrium, rate and time-dependent interactions 335

Reversible and irreversible interactions 337

The interaction forces between biological molecules and surfaces are much more complex than those between non-biological molecules or surfaces, such as colloidal particle surfaces. This complexity is due to a number of factors: (i) the simultaneous involvement of many different molecules and different non-covalent forces – van der Waals, electrostatic, solvation (hydration, hydrophobic), steric, entropic and ‘specific’, and (ii) the flexibility of biological macromolecules and fluidity of membranes. Biological interactions are better thought of as ‘processes’ that evolve in space and time and, under physiological conditions, involve a continuous input of energy. Such systems are, therefore, not at thermodynamic equilibrium, or even tending towards equilibrium. Recent surface forces apparatus (SFA) and atomic force microscopy (AFM) measurements on supported model membrane systems (protein-containing lipid bilayers) illustrate these effects. It is suggested that the major theoretical challenge is to establish manageable theories or models that can describe the spatial and time evolution of systems consisting of different molecules subject to certain starting conditions or energy inputs.

It has become clear that there is no simple ‘code’ for protein–DNA recognition and that selecting an optimal binding sequence along the DNA double helix corresponds to more than simply forming a set of specific hydrogen bonds or steric interactions. However, it has been difficult to characterize the so-called indirect components of recognition. While DNA deformation certainly underlies indirect recognition, it is not easy to determine how local fine structure and deformability depend on base sequence or exactly what percentage of recognition should be attributed to such factors. Molecular modelling can help to develop these ideas into a quantitative model, provided the calculations can be carried out fast enough to enable a comprehensive survey of base-sequence effects. I present here some recent results from our group and their consequences for improving our understanding of protein–DNA binding, and their potential for predicting, and eventually modulating, protein–DNA binding.

1. Introduction 345

2. Peptide nucleic acid (PNA) 346

3. ‘Cell penetrating peptides’ (CPPs) 346

4. Endosomal escape 347

5. Cellular delivery of PNA 347

6.In vivobioavailability of PNA 349

7. References 350

Recent results on the cellular delivery of antisense peptide nucleic acids (PNA) via peptide conjugation is briefly discussed, in particular in the context of endosomal entrapment and escape.

Introduction 351

Part I. A succession of concepts 352

1. Cooperativity 352

2. Cosolvent interaction 352

3. Linearity 354

4. Solvent exchange 354

5. Excluded volume 356

6. Summation 357

Part II

1. The Kirkwood–Buff approach 357

Acknowledgments 360

References 360

In Part I the history of progress in the stabilization and destabilization of protein conformations by means of cosolvents is outlined in terms of distinct conceptual steps. In Part II it is shown that a straightforward application of the Kirkwood–Buff theory of solutions leads to formulas for the preferential interaction and the free energy of unfolding, which confirm and generalize the results of Part I.

1. Introduction 363

2. Motif and sequence design 364

3. Structural and topological constructions 366

4. Nanomechanical devices 367

5. Conclusions, applications and challenges 370

6. Acknowledgments 371

7. References 371

Structural DNA nanotechnology consists of constructing objects, lattices and devices from branched DNA molecules. Branched DNA molecules open the way for the construction of a variety of N-connected motifs. These motifs can be joined by cohesive interactions to produce larger constructs in a bottom-up approach to nanoconstruction. The first objects produced by this approach were stick polyhedra and topological targets, such as knots and Borromean rings. These were followed by periodic arrays with programmable patterns. It is possible to exploit DNA structural transitions and sequence-specific binding to produce a variety of DNA nanomechanical devices, which include a bipedal walker and a machine that emulates the translational capabilities of the ribosome. Much of the promise of this methodology involves the use of DNA to scaffold other materials, such as biological macromolecules, nanoelectronic components, and polymers. These systems are designed to lead to improvements in crystallography, computation and the production of diverse and exotic materials.

1. Introduction 373

2. Are rafts probable? 374

3. Micro-, nano- or ephemeral domains? 375

4. How can we reliably assess ‘raft’ composition? 376

5. Are rafts plausible? 379

6. What more can model systems contribute to ‘raft’ studies? 381

7. References 382

The concept of ‘lipid rafts’ and related liquid-ordered membrane microdomains has attracted great attention in the field of membrane biology, both as a novel paradigm in models of membrane organization and for the potential importance of such domains in phenomena such as membrane signaling and the differential trafficking of various membrane components. Studies of biological and of model membranes have gone hand in hand in shaping our current picture of the possible organization and functions of liquid-ordered lipid microdomains in membranes. This essay discusses some important current questions concerning the existence and functional importance of lipid microdomains in mammalian cell membranes, and the potential as well as the limitations of using model systems to help to address such questions.

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.

The assembly of ribosomes requires a significant fraction of the energy expenditure for rapidly growing bacteria. The ribosome is composed of three large RNA molecules and over 50 small proteins that must be rapidly and efficiently assembled into the molecular machine responsible for protein synthesis. For over 30 years, the 30S ribosome has been a key model system for understanding the process of ribosome biogenesis through in vitro assembly experiments. We have recently developed an isotope pulse-chase experiment using quantitative mass spectrometry that permits assembly kinetics to be measured in real time. Kinetic studies have revealed an assembly energy landscape that ensures efficient assembly by a flexible and robust pathway.

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.