Why twenty amino acid residue types suffice(d) to support all living systems.

It is well known that proteins are built up from an alphabet of 20 different amino acid types. These suffice to enable the protein to fold into its operative form relevant to its required functional roles. For carrying out these allotted functions, there may in some cases be a need for post-translational modifications and it has been established that an additional three types of amino acid have at some point been recruited into this process. But it still remains the case that the 20 residue types referred to are the major building blocks in all terrestrial proteins, and probably "universally". Given this fact, it is surprising that no satisfactory answer has been given to the two questions: "why 20?" and "why just these 20?". Furthermore, a suggestion is made as to how these 20 map to the codon repertoire which in principle has the capacity to cater for 64 different residue types. Attempts are made in this paper to answer these questions by employing a combination of quantum chemical and chemoinformatic tools which are applied to the standard 20 amino acid types as well as 3 "non-standard" types found in nature, a set of fictitious but feasible analog structures designed to test the need for greater coverage of function space and the collection of candidate alternative structures found either on meteorites or in experiments designed to reconstruct pre-life scenarios.

A tensegrity model for hydrogen bond networks in proteins.

Hydrogen-bonding networks in proteins considered as structural tensile elements are in balance separately from any other stabilising interactions that may be in operation. The hydrogen bond arrangement in the network is reminiscent of tensegrity structures in architecture and sculpture. Tensegrity has been discussed before in cells and tissues and in proteins. In contrast to previous work only hydrogen bonds are studied here. The other interactions within proteins are either much stronger - covalent bonds connecting the atoms in the molecular skeleton or weaker forces like the so-called hydrophobic interactions. It has been demonstrated that the latter operate independently from hydrogen bonds. Each category of interaction must, if the protein is to have a stable structure, balance out. The hypothesis here is that the entire hydrogen bond network is in balance without any compensating contributions from other types of interaction. For sidechain-sidechain, sidechain-backbone and backbone-backbone hydrogen bonds in proteins, tensegrity balance ("closure") is required over the entire length of the polypeptide chain that defines individually folding units in globular proteins ("domains") as well as within the repeating elements in fibrous proteins that consist of extended chain structures. There is no closure to be found in extended structures that do not have repeating elements. This suggests an explanation as to why globular domains, as well as the repeat units in fibrous proteins, have to have a defined number of residues. Apart from networks of sidechain-sidechain hydrogen bonds there are certain key points at which this closure is achieved in the sidechain-backbone hydrogen bonds and these are associated with demarcation points at the start or end of stretches of secondary structure. Together, these three categories of hydrogen bond achieve the closure that is necessary for the stability of globular protein domains as well as repeating elements in fibrous proteins.

The fate of proteins in outer space.

It is well established that any properly conducted biophysical studies of proteins must take appropriate account of solvent. For water-soluble proteins it has been an article of faith that water is largely responsible for stabilizing the fold, a notion that has recently come under increasing scrutiny. Further, there are some instances when proteins are studied experimentally in the absence of solvent, as in matrix-assisted laser desorption/ionization or electrospray mass spectrometry, for example, or in organic solvents for protein engineering purposes. Apart from these considerations, there is considerable speculation as to whether there is life on planets other than Earth, where conditions including the presence of water (both in liquid or vapor form and indeed ice), temperature and pressure may be vastly different from those prevailing on Earth. Mars, for example, has only 0.6% of Earth's mean atmospheric pressure which presents profound problems to protein structures, as this paper and a large corpus of experimental work demonstrate. Similar objections will most likely apply in the case of most exoplanets and other bodies such as comets whose chemistry and climate are still largely unknown. This poses the question, how do proteins survive in these different environments? In order to cast some light on these issues we have conducted a series of molecular dynamics simulations on protein dehydration under a variety of conditions. We find that, while proteins undergoing dehydration can retain their integrity for a short duration they ultimately become disordered, and we further show that the disordering can be retarded if superficial water is kept in place on the surface. These findings are compared with other published results on protein solvation in an astrobiological and astrochemical setting. Inter alia, our results suggest that there are limits as to what to expect in terms of the existence of possible extraterrestrial forms as well to what can be achieved in experimental investigations on living systems despatched from Earth. This finding may appear to undermine currently held hopes that life will be found on nearby planets, but it is important to be aware that the presence of ice and water are by themselves not sufficient; there has to be an atmosphere which includes water vapor at a sufficiently high partial pressure for proteins to be active. A possible scenario in which there has been a history of adequate water vapor pressure which allowed organisms to prepare for a future dessicated state by forming suitable protective capsules cannot of course be ruled out.

Melody discrimination and protein fold classification.

One of the greatest challenges in theoretical biophysics and bioinformatics is the identification of protein folds from sequence data. This can be regarded as a pattern recognition problem. In this paper we report the use of a melody generation software where the inputs are derived from calculations of evolutionary information, secondary structure, flexibility, hydropathy and solvent accessibility from multiple sequence alignment data. The melodies so generated are derived from the sequence, and by inference, of the fold, in ways that give each fold a sound representation that may facilitate analysis, recognition, or comparison with other sequences.

Comparison of Algorithms for Prediction of Protein Structural Features from Evolutionary Data.

Proteins have many functions and predicting these is still one of the major challenges in theoretical biophysics and bioinformatics. Foremost amongst these functions is the need to fold correctly thereby allowing the other genetically dictated tasks that the protein has to carry out to proceed efficiently. In this work, some earlier algorithms for predicting protein domain folds are revisited and they are compared with more recently developed methods. In dealing with intractable problems such as fold prediction, when different algorithms show convergence onto the same result there is every reason to take all algorithms into account such that a consensus result can be arrived at. In this work it is shown that the application of different algorithms in protein structure prediction leads to results that do not converge as such but rather they collude in a striking and useful way that has never been considered before.

The dipeptide conformations of all twenty amino acid types in the context of biosynthesis.

There have been many studies of dipeptide structure at a high level of accuracy using quantum chemical methods. Such calculations are resource-consuming (in terms of memory, CPU and other computational imperatives) which is the reason why most previous studies were restricted to the two simplest amino-acid residue types, glycine and alanine. We improve on this by extending the scope of residue types to include all 20 naturally occurring residue types. Our results reveal differences in secondary structure preferences for the all residue types. There are in most cases very deep energy troughs corresponding either to the polyproline II (collagen) helix and the α-helix or both. The ß-strand was not strongly favoured energetically although the extent of this depression in the energy surface is, while not "deeper" (energetically), has a wider extent than the other two types of secondary structure. There is currently great interest in the question of cotranslational folding, the extent to which the nascent polypeptide begins to fold prior to emerging from the ribosome exit tunnel. Accordingly, while most previous quantum studies of dipeptides were carried out in the (simulated) gas or aqueous phase, we wished to consider the first step in polypeptide biosynthesis on the ribosome where neither gas nor aqueous conditions apply. We used a dielectric constant that would be compatible with the water-poor macromolecular (ribosome) environment.

The preferred conformation of dipeptides in the context of biosynthesis.

Globular proteins are folded polypeptide structures comprising stretches of secondary structures (helical (α- or 310 helix type), polyproline helix or ß-strands) interspersed by regions of less well-ordered structure ("random coil"). Protein fold prediction is a very active field impacting inte alia on protein engineering and misfolding studies. Apart from the many studies of protein refolding from the denatured state, there has been considerable interest in studying the initial formation of peptides during biosynthesis, when there are at the outset only a few residues in the emerging polypeptide. Although there have been many studies employing quantum chemical methods of the conformation of dipeptides, these have mostly been carried out in the gas phase or simulated water. None of these conditions really apply in the interior confines of the ribosome. In the present work, we are concerned with the conformation of dipeptides in this low dielectric environment. Furthermore, only the residue types glycine and alanine have been studied by previous authors, but we extend this repertoire to include leucine and isoleucine, position isomers which have very different structural propensities.

Current progress in Structure-Based Rational Drug Design marks a new mindset in drug discovery.

The past decade has witnessed a paradigm shift in preclinical drug discovery with structure-based drug design (SBDD) making a comeback while high-throughput screening (HTS) methods have continued to generate disappointing results. There is a deficit of information between identified hits and the many criteria that must be fulfilled in parallel to convert them into preclinical candidates that have a real chance to become a drug. This gap can be bridged by investigating the interactions between the ligands and their receptors. Accurate calculations of the free energy of binding are still elusive; however progresses were made with respect to how one may deal with the versatile role of water. A corpus of knowledge combining X-ray structures, bioinformatics and molecular modeling techniques now allows drug designers to routinely produce receptor homology models of increasing quality. These models serve as a basis to establish and validate efficient rationales used to tailor and/or screen virtual libraries with enhanced chances of obtaining hits. Many case reports of successful SBDD show how synergy can be gained from the combined use of several techniques. The role of SBDD with respect to two different classes of widely investigated pharmaceutical targets: (a) protein kinases (PK) and (b) G-protein coupled receptors (GPCR) is discussed. Throughout these examples prototypical situations covering the current possibilities and limitations of SBDD are presented.

Accelerated simulation of unfolding and refolding of a large single chain globular protein.

We have developed novel strategies for contracting simulation times in protein dynamics that enable us to study a complex protein with molecular weight in excess of 34 kDa. Starting from a crystal structure, we produce unfolded and then refolded states for the protein. We then compare these quantitatively using both established and new metrics for protein structure and quality checking. These include use of the programs Concoord and Darvols. Simulation of protein-folded structure well beyond the molten globule state and then recovery back to the folded state is itself new, and our results throw new light on the protein-folding process. We accomplish this using a novel cooling protocol developed for this work.

Inactivation and reactivation of ribonuclease A studied by computer simulation.

The year 2011 marked the half-centenary of the publication of what came to be known as the Anfinsen postulate, that the tertiary structure of a folded protein is prescribed fully by the sequence of its constituent amino acid residues. This postulate has become established as a credo, and, indeed, no contradictions seem to have been found to date. However, the experiments that led to this postulate were conducted on only a single protein, bovine ribonuclease A (RNAse). We conduct molecular dynamics (MD) simulations on this protein with the aim of mimicking this experiment as well as making the methodology available for use with basically any protein. There have been many attempts to model denaturation and refolding processes of globular proteins in silico using MD, but only a few examples where disulphide-bond containing proteins were studied. We took the view that if the reductive deactivation and oxidative reactivation processes of RNAse could be modelled in silico, this would provide valuable insights into the workings of the classical Anfinsen experiment.

G-protein-coupled receptor dynamics: dimerization and activation models compared with experiment.

Our previously derived models of the active state of the ß2-adrenergic receptor are compared with recently published X-ray crystallographic structures of activated GPCRs (G-protein-coupled receptors). These molecular dynamics-based models using experimental data derived from biophysical experiments on activation were used to restrain the receptor to an active state that gave high enrichment for agonists in virtual screening. The ß2-adrenergic receptor active model and X-ray structures are in good agreement over both the transmembrane region and the orthosteric binding site, although in some regions the active model is more similar to the active rhodopsin X-ray structures. The general features of the microswitches were well reproduced, but with minor differences, partly because of the unexpected X-ray results for the rotamer toggle switch. In addition, most of the interacting residues between the receptor and the G-protein were identified. This analysis of the modelling has also given important additional insight into GPCR dimerization: re-analysis of results on photoaffinity analogues of rhodopsin provided additional evidence that TM4 (transmembrane helix 4) resides at the dimer interface and that ligands such as bivalent ligands may pass between the mobile helices. A comparison, and discussion, is also carried out between the use of implicit and explicit solvent for active-state modelling.

On dating stages in prebiotic chemical evolution.

The notion that RNA must have had a unique and decisive role in the development of life needs hardly be questioned. However, the chemical complexity and other properties of RNA, such as high solubility in water and vulnerability to degradation, make it improbable that RNA could have had an early presence in the development of life on Earth or on any comparable telluric planet. Rather, the task of origin of life research must surely be to identify those chemical processes which could have taken place on Earth that could accumulate the complexity and rich molecular information content needed to sustain primitive life, and ultimately give rise to RNA. A collection of likely chemical precursors to modern biomolecules is listed here together with calculations of their molecular complexity. These complexity scores are then used to propose an ordering, on a timescale, of when they might have appeared on Earth. These pre-RNA living systems would have flourished during the first ~0.3 Gyrs after the start of the Archaean era (~4.2 Gyr ago). If there ever was an "RNA-world" it could have started after that initial period (~3.9 Gyrs ago), later to be complemented with the appearance of duplex DNA at about ~3.6 Gyrs ago, some time before the earliest known stromatolites (~3.4 Gyr).

Membrane-spanning peptides and the origin of life.

An explanation is given as to why membrane-spanning peptides must have been the first "information-rich" molecules in the development of life. These peptides are stabilised in a lipid bilayer membrane environment and they are preferentially made from the simplest, and likewise oldest, of the amino acids that survive today. Transmembrane peptides can exercise functions that are essential for biological systems such as signal transduction and material transport across membranes. More complex peptides possessing catalytic properties could later develop on either side of the membrane as independently folding functional units formed by extension of the protruding ends of the transmembrane peptides within an aqueous environment and thereby give rise to more of the functions that are necessary for life. But the membrane was the cradle for the development of the first information-rich biomolecules.

Residues from transmembrane helices 3 and 5 participate in leukotriene B4 binding to BLT1.

Leukotrienes are inflammatory mediators that bind to seven transmembrane, G-protein-coupled receptors (GPCRs). Here we examine residues from transmembrane helices 3 and 5 of the leukotriene B4 (LTB4) receptor BLT1 to elucidate how these residues are involved in ligand binding. We have selected these residues on the basis of (1) amino acid sequence analysis, (2) receptor binding and activation studies with a variety of leukotriene-like ligands and recombinant BLT1 receptors, (3) previously published recombinant BLT1 mutants, and (4) a computed model of the active structure of the BLT1 receptor. We propose that LTB4 binds with the polar carboxylate group of LTB4 near the extracellular surface of BLT1 and with the hydrophobic LTB4 tail pointing into the transmembrane regions of the receptor protein. The carboxylate group and the two hydroxyls of LTB4 interact with Arg178 and Glu185 in transmembrane helix 5. Residues from transmembrane helix 3, Val105 and Ile108, also line the pocket deeper inside the receptor. LTB4 is becoming increasingly important as an immunomodulator during a number of pathologies, including atherosclerosis. Detailed information about the LTB4 binding mechanism, and the receptor residues involved, will hopefully aid in the design of new immunomodulatory drugs.

Did life begin on the beach?

Water is one of the prerequisites of life. Further requirements are the existence of a system of interacting organic molecules capable of capturing and converting the supply of external energy and elaborating the replicating function that is needed for propagation. None of this would be possible without the existence of some means of concentrating, selecting, and then containing these mutually interacting substances in proximity to one another, i.e., a primitive cell. Starting from this hypothesis we propose a model for the development of life on Earth. Our model embodies the following new features: (1) rapid cycles of catalysis and transport of material, (2) desegregation (separation by tidal action and degradation by catalysis) as well as segregation (by chromatography on tidal beaches), (3) cross-catalysis instead of auto-catalysis, as well as (4) compartmentalization, although the latter idea is of course not new. But our "lipid first" model, in contrast to earlier "peptide first" or "RNA first" models, provides for the compartments needed to act as a cradle for the subsequent development of information- rich molecules like peptides and RNA. If anything, the earliest information-rich molecules were probably membrane-spanning peptides/proteins.

Location and nature of the residues important for ligand recognition in G-protein coupled receptors.

The overall structure of the biogenic amine subclass of the G-protein-coupled receptors, and of their ligand binding sites, is discussed with the aim of highlighting the major structural features of these receptors that are responsible for ligand recognition. A comparison is made between biogenic amine receptors, peptide receptors of the rhodopsin class, and the secretin receptors which all have peptide ligands. The question of where the peptide ligands bind, whether at extracellular sites or within the transmembrane helix bundle, is discussed. The suitability of the rhodopsin crystal structure as a template for construction of homology models is discussed and it is concluded that there are many reasons why a caution should be issued against using it uncritically.

Toward the active conformations of rhodopsin and the beta2-adrenergic receptor.

Using sets of experimental distance restraints, which characterize active or inactive receptor conformations, and the X-ray crystal structure of the inactive form of bovine rhodopsin as a starting point, we have constructed models of both the active and inactive forms of rhodopsin and the beta2-adrenergic G-protein coupled receptors (GPCRs). The distance restraints were obtained from published data for site-directed crosslinking, engineered zinc binding, site-directed spin-labeling, IR spectroscopy, and cysteine accessibility studies conducted on class A GPCRs. Molecular dynamics simulations in the presence of either "active" or "inactive" restraints were used to generate two distinguishable receptor models. The process for generating the inactive and active models was validated by the hit rates, yields, and enrichment factors determined for the selection of antagonists in the inactive model and for the selection of agonists in the active model from a set of nonadrenergic GPCR drug-like ligands in a virtual screen using ligand docking software. The simulation results provide new insights into the relationships observed between selected biochemical data, the crystal structure of rhodopsin, and the structural rearrangements that occur during activation.

De novo generation of molecular structures using optimization to select graphs on a given lattice.

A recurrent problem in organic chemistry is the generation of new molecular structures that conform to some predetermined set of structural constraints that are imposed in an endeavor to build certain required properties into the newly generated structure. An example of this is the pharmacophore model, used in medicinal chemistry to guide de novo design or selection of suitable structures from compound databases. We propose here a method that efficiently links up a selected number of required atom positions while at the same time directing the emergent molecular skeleton to avoid forbidden positions. The linkage process takes place on a lattice whose unit step length and overall geometry is designed to match typical architectures of organic molecules. We use an optimization method to select from the many different graphs possible. The approach is demonstrated in an example where crystal structures of the same (in this case rigid) ligand complexed with different proteins are available.

Recognition of privileged structures by G-protein coupled receptors.

Privileged structures are ligand substructures that are widely used to generate high-affinity ligands for more than one type of receptor. To explain this, we surmised that there must be some common feature in the target proteins. For a set of class A GPCRs, we found a good correlation between conservation patterns of residues in the ligand binding pocket and the privileged structure fragments in class A GPCR ligands. A major part of interior surface of the common ligand binding pocket of class A receptors, identified in many GPCRs, is lined with variable residues that are responsible for selectivity in ligand recognition, while other regions, typically located deeper into the binding pocket, are more conserved and retain a predominantly hydrophobic and aromatic character. The latter is reflected in the chemical nature of most GPCR privileged structures and is proposed to be the common feature that is recognized by the privileged structures. Further, we find that this subpocket is conserved even in distant orthologs within the class A family. Three pairs of ligands recognizing widely different receptor types were docked into receptor models of their target receptors utilizing available structure- activity relationships and mutagenesis data. For each pair of ligands, the ligand-receptor complexes reveal that the nature of the privileged structure binding pocket is conserved between the two complexes, in support of our hypothesis. Only part of the privileged structures can be accommodated within the conserved subpocket. Some contacts are established between the privileged structure and the nonconserved parts of the binding pocket. This implies that any one particular privileged structure can target only a subset of receptors, those complementary to the full privileged structure. Our hypothesis leads to a valuable novelty in that ligand libraries can be designed without any foreknowledge of the structure of the endogenous ligand, which in turn means that even orphan receptors can in principle now be addressed as potential drug targets.