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.

Exposure to various chemicals, either due to occupation or lifestyle, is considered to be a major contributing factor to tumour formation in man (Higginson, 1969; Doll & Peto, 1981). An important and prevalent class of potent carcinogenic compounds present in the environment is polycyclic aromatic hydrocarbons (PAHs), which are found in various petroleum and combustion products derived from heat and power generation and motor vehicle exhausts (Baum, 1978). Furthermore, since PAHs are generally formed by pyrolysis of organic matters such as tobacco smoking and certain procedures of food preparation, the PAH exposure to humans is extensive

The form of multicellular animals and their organs is mainly defined by the curvature of cell layers. They are boundaries for solid tissues; and some organs and organisms consist mainly of distinct cell layers (Fig. I a). The form of adult organisms results from a complex interplay of tissue evagination, growth patterns, production of and interaction with extra-cellular material, and other effects; but the rudiments and basic features of the forms produced can often be traced back to processes of evagination or invagination of nearly flat cell sheets at defined locations in the course of embryogenesis.

In the original Hodgkin & Huxley analysis of the membrane currents in squid giant axon, the time dependence of the currents was attributed to voltage-dependent changes in the permeability of the membrane to the ions involved. According to this theoretical framework, the time constants for the rate of change of current should be unique functions of the membrane potential, and should be independent of the previous history of the voltage and current flowing. In recent years, however, there has been increasing awareness of the fact that, for many physiological preparations, the space just outside the cell membrane is not in good diffusive contact with the bulk extra-cellular fluid perfusing the preparation. Consequently, when current flows across the cell membrane, there are changes in the ion concentrations in this so-called ‘restricted extra-cellular space’ (RECS).

Exciting new technological developments have pushed the boundaries of structural biology, and have enabled studies of biological macromolecules and assemblies that would have been unthinkable not long ago. Yet, the enhanced capabilities of structural biologists to pry into the complex molecular world have also placed new demands on the abilities of protein engineers to reproduce this complexity into the test tube. With this challenge in mind, we review the contents of the modern molecular engineering toolbox that allow the manipulation of proteins in a site-specific and chemically well-defined fashion. Thus, we cover concepts related to the modification of cysteines and other natural amino acids, native chemical ligation, intein and sortase-based approaches, amber suppression, as well as chemical and enzymatic bio-conjugation strategies. We also describe how these tools can be used to aid methodology development in X-ray crystallography, nuclear magnetic resonance, cryo-electron microscopy and in the studies of dynamic interactions. It is our hope that this monograph will inspire structural biologists and protein engineers alike to apply these tools to novel systems, and to enhance and broaden their scope to meet the outstanding challenges in understanding the molecular basis of cellular processes and disease.

An exciting aspect of NMR spectroscopy is its ability to monitor, non-invasively, a variety of small molecules in cells and tissues. This leads to the possibility of investigating details of cellular biochemistry previously obscured by separation and purification procedures.

Biological systems must have evolved in an interplay between a great many organic and inorganic compounds. As a result a considerable number of elements - estimates range between 25 and 30 - are essential for higher life forms such as animals and man (Underwood, 1977; Williams, 1983, 1984).

This review takes the regulation of breathing in man as an example of a highly developed and much studied control system in higher mammals.

1. Introduction 68

2. Ferredoxin reduction by photosystem I 72

3. Ferredoxins 73

4. Ferredoxin[ratio ]thioredoxin reductase 73

4.1 Spectroscopic investigations of FTR 76

4.2 The three-dimensional structure of FTR from the cyanobacterium Synechocystis sp. PCC6803 77

4.2.1 The variable subunit 77

4.2.2 The catalytic subunit 81

4.2.3 The iron–sulfur center and active site disulfide bridge 82

4.2.4 The dimer 84

4.3 Thioredoxin f and m 85

4.4 Ferredoxin and thioredoxin interactions 86

4.5 Mechanism of action 88

4.6 Comparison with other chloroplast FTRs 92

5. Target enzymes 95

5.1 NADP-dependent malate dehydrogenase 95

5.1.1 Regulatory role of the N-terminal extension 97

5.1.2 Regulatory role of the C-terminal extension 99

5.1.3 Thioredoxin interactions 101

5.2 Fructose-1,6-bisphosphatase 101

5.3 Redox regulation of chloroplast target enzymes 103

6. Conclusion 103

7. Acknowledgements 104

8. References 104

A pre-requisite for life on earth is the conversion of solar energy into chemical energy by photosynthetic organisms. Plants and photosynthetic oxygenic microorganisms trap the energy from sunlight with their photosynthetic machinery and use it to produce reducing equivalents, NADPH, and ATP, both necessary for the reduction of carbon dioxide to carbohydrates, which are then further used in the cellular metabolism as building blocks and energy source. Thus, plants can satisfy their energy needs directly via the light reactions of photosynthesis during light periods. The situation is quite different in the dark, when these organisms must use normal catabolic processes like non-photosynthetic organisms to obtain the necessary energy by degrading carbohydrates, like starch, accumulated in the chloroplasts during daylight. The chloroplast stroma contains both assimilatory enzymes of the Calvin cycle and dissimilatory enzymes of the pentose phosphate cycle and glycolysis. This necessitates a strict, light-sensitive control that switches between assimilatory and dissimilatory pathways to avoid futile cycling (Buchanan, 1980, 1991; Buchanan et al. 1994; Jacquot et al. 1997; Schürmann & Buchanan, 2000).

A distinction has been made between ‘metalloproteins’ and ‘metal-protein complexes’. The former exhibit high metal-ligand stability constants (the metal is not removed during the isolation of the protein), while the latter bind metal ions only loosely (Vallee, 1955). Actually, all proteins can be considered as existing as metal-protein complexes. Selectivity, however, among proteins in their tendency to combine with inorganic ions (both with regard to the type of ion as well as number of ions) suggests that particular configurations produce specific reactive sites. In turn, binding of ions to these sites alters the electronic and consequently the steric conformation of the protein. Thus, effects on macromolecular conformation may not only be due to structural changes of the solvent induced by ions, which modifies solvent-macromole interaction, but also to specific ligation of the ions to the protien which alters local molecular arrangement.

The major elements of membranes, such as proteins, lipids and polysaccharides, are in dynamic interaction with each other (Alberts et al. 1983). Protein diffusion in the lipid matrix of the membrane, the lipid diffusion and dynamic domain formation below and above their transition temperature from gel to fluid state, have many functional implications. This type of behaviour of membranes is often summarized in one frequently used word membrane fluidity (coined by Shinitzky & Henkart, 1979). The dynamic behaviour of the cell membrane includes rotational, translational and segmental movements of membrane elements (or their domain-like associations) in the plane of, and perpendicular to the membrane. The ever changing proximity relationships form a dynamic pattern of lipids, proteins and saccharide moieties and are usually described as ‘cell-surface dynamics’ (Damjanovich et al. 1981). The knowledge about the above defined behaviour originates from experiments performed mostly on cytoplasmic membranes of eukaryotic cells. Nevertheless numerous data are available also on the mitochondrial and nuclear membranes, as well as endo (sarco-)plasmic reticulum (Martonosi, 1982; Slater, 1981; Siekevitz, 1981).

The two best-known photophysiological processes are photosynthesis and vision. In photosynthesis light energy is absorbed and transformed into ‘life energy’, in vision the information content of light is utilized by organsims. In photosynthesis chlorophyll and other pigments involved in light-gathering are not chemically changed (except that some of the chlorophyll or phaeophytin molecules are oxidized and immediately reduced again). In human vision the rhodopsin and other visual pigments undergo a reaction cycle induced by photon absorption. The pigment is brought back to its original state only after a long chain of chemical reactions.

Kinetics of acid-induced chlorophyll demetallation was recorded in microdroplets by fusing a stream of microdroplets containing 40 µM chlorophyll a or b dissolved in methanol with a stream of aqueous microdroplets containing 35 mM hydrochloric acid (pH = 1·46). The kinetics of the demetallation of chlorophyll in the fused microdroplets (14 ± 6 µm diameter; 84 ± 18 m s−1 velocity) was recorded by controlling the traveling distance of the fused microdroplets between the fusion region and the inlet of a mass spectrometer. The rate of acid-induced chlorophyll demetallation was about 960 ± 120 times faster in the charged microdroplets compared with that reported in bulk solution. If no voltage was applied to the sprayed microdroplets, then the acceleration factor was about 580 ± 90, suggesting that the applied voltage is not a major factor determining the acceleration. Chlorophyll a was more rapidly demetallated than chlorophyll b by a factor of ~26 in bulk solution and ~5 in charged microdroplets. The demetallation kinetics was second order in the H+ concentration, but the acceleration factor of microdroplets compared with bulk solution appeared to be unchanged in going from pH = 1·3 to 7·0. The water:methanol ratio of the fused microdroplets was varied from 7:3 to 3:7 causing an increase in the reaction rate of chlorophyll a demetallation by 20%. This observation demonstrates that the solvent composition, which has different evaporation rates, does not significantly affect the acceleration. We believe that a major portion of the acceleration can be attributed to confinement effects involving surface reactions rather than either to evaporation of solvents or to the introduction of charges to the microdroplets.

The discoveries of myriad non-coding RNA molecules, each transiting through multiple flexible states in cells or virions, present major challenges for structure determination. Advances in high-throughput chemical mapping give new routes for characterizing entire transcriptomes in vivo, but the resulting one-dimensional data generally remain too information-poor to allow accurate de novo structure determination. Multidimensional chemical mapping (MCM) methods seek to address this challenge. Mutate-and-map (M2), RNA interaction groups by mutational profiling (RING-MaP and MaP-2D analysis) and multiplexed •OH cleavage analysis (MOHCA) measure how the chemical reactivities of every nucleotide in an RNA molecule change in response to modifications at every other nucleotide. A growing body of in vitro blind tests and compensatory mutation/rescue experiments indicate that MCM methods give consistently accurate secondary structures and global tertiary structures for ribozymes, ribosomal domains and ligand-bound riboswitch aptamers up to 200 nucleotides in length. Importantly, MCM analyses provide detailed information on structurally heterogeneous RNA states, such as ligand-free riboswitches that are functionally important but difficult to resolve with other approaches. The sequencing requirements of currently available MCM protocols scale at least quadratically with RNA length, precluding general application to transcriptomes or viral genomes at present. We propose a modify-cross-link-map (MXM) expansion to overcome this and other current limitations to resolving the in vivo ‘RNA structurome’.

Fluorescence spectroscopy offers particularly sensitive tools for the investigation of physical properties of macromolecules in solution. Differences in molecular structure under varying experimental conditions are often reflected in quantum yield and lifetime of natural or synthetically inserted fluorescent probes. inserted fluorescent probes. Energy transfer between fluorescent groups at known places of a primary sequence can be used to measure distances between the label sites (Eisinger, 1976) as well as to investigate their relative motion, e.g. measurements of the kinetics of variations in the end to end distance of a polymer (E. Haas & I. Steinberg, personal communication).

Theoretical as well as experimental studies of translational accuracy have most often been concerned with the selection of aminoacyl-tRNA by codon-programmed ribosomes. The selection of the successive codons on the mRNA has received much less attention, probably because it represents both conceptually and experimentally, a much more demanding physical problem. Nevertheless, it would seem that errors in the selection of the codon are potentially much more destructive than errors in selection of aminoacyl-tRNA species. This can be appreciated from the following.

Until recently there has been very limited information concerning the molecular geometry and conformation of biogenic monoamines and this lack of knowledge has seriously hampered efforts to unravel the structure—function relationships at the molecular level. Nevertheless, several theories have been proposed regarding interaction between the monoamines and their receptor sites. This is especially true for the neurohumoral transmitters dopamine, noradrenaline (norepinephrine) and serotonin (5-hydroxytryptamine), the target areas of which seem to be specific not only in the sense of chemical structure but also from the steric point of view. The well-known differences in biological activity due to chiralty illustrate the importance of the three-dimensional architecture of the molecules and indicate also the presence of stereospecific receptor sites. A detailed knowledge of the conformation of the biogenic monoamines would not only elucidate the requirements upon a natural or synthetic monoamine for proper function but also give indirect information about the geometry of the receptor sites.

DNA dynamics can only be understood by taking into account its complex mechanical behavior at different length scales. At the micrometer level, the mechanical properties of single DNA molecules have been well-characterized by polymer models and are commonly quantified by a persistence length of 50 nm (~150 bp). However, at the base pair level (~3.4 Å), the dynamics of DNA involves complex molecular mechanisms that are still being deciphered. Here, we review recent single-molecule experiments and molecular dynamics simulations that are providing novel insights into DNA mechanics from such a molecular perspective. We first discuss recent findings on sequence-dependent DNA mechanical properties, including sequences that resist mechanical stress and sequences that can accommodate strong deformations. We then comment on the intricate effects of cytosine methylation and DNA mismatches on DNA mechanics. Finally, we review recently reported differences in the mechanical properties of DNA and double-stranded RNA, the other double-helical carrier of genetic information. A thorough examination of the recent single-molecule literature permits establishing a set of general ‘rules’ that reasonably explain the mechanics of nucleic acids at the base pair level. These simple rules offer an improved description of certain biological systems and might serve as valuable guidelines for future design of DNA and RNA nanostructures.