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.

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.

In May 2005 the University of Coimbra, Portugal hosted a Nobel Workshop in Chemistry entitled ‘Fundamentals of Biomolecular Function: Nucleic Acids, Proteins, and Membranes. This volume is based on presentations and discussions from this workshop.