From crystals to enzymes: Simple models of molecular recognition

Structural models resulting from the x-ray crystal structure determination of small molecules are shown to be closely mimicking some of the protein structure and function features. Crystals of inclusion compounds, a special type of solid molecular associates, lend special support to such a view. They are inherently suited for such modelling purpose due to easily visible association and molecular recognition effects. It is envisaged that combination of the analysis of high-resolution small molecule crystallographic models and those of protein structures will yield to further fruitful analogies.     

Hierarchical phylogenetic models for analyzing multipartite sequence data

Debate exists over how to incorporate information from multipartite sequence data in phylogenetic analyses. Strict combined-data approaches argue for concatenation of all partitions and estimation of one evolutionary history, maximizing the explanatory power of the data. Consensus/independence approaches endorse a two-step procedure where partitions are analyzed independently and then a consensus is determined from the multiple results. Mixtures across the model space of a strict combined-data approach and a priori independent parameters are popular methods to integrate these methods. We propose an alternative middle ground by constructing a Bayesian hierarchical phylogenetic model. Our hierarchical framework enables researchers to pool information across data partitions to improve estimate precision in individual partitions while permitting estimation and testing of tendencies in across-partition quantities. Such across-partition quantities include the distribution from which individual topologies relating the sequences within a partition are drawn. We propose standard hierarchical priors on continuous evolutionary parameters across partitions, while the structure on topologies varies depending on the research problem. We illustrate our model with three examples. We first explore the evolutionary history of the guinea pig (Cavia porcellus) using alignments of 13 mitochondrial genes. The hierarchical model returns substantially more precise continuous parameter estimates than an independent parameter approach without losing the salient features of the data. Second, we analyze the frequency of horizontal gene transfer using 50 prokaryotic genes. We assume an unknown species-level topology and allow individual gene topologies to differ from this with a small estimable probability. Simultaneously inferring the species and individual gene topologies returns a transfer frequency of 17%. We also examine HIV sequences longitudinally sampled from HIV+ patients. We ask whether posttreatment development of CCR5 coreceptor virus represents concerted evolution from middisease CXCR4 virus or reemergence of initial infecting CCR5 virus. The hierarchical model pools partitions from multiple unrelated patients by assuming that the topology for each patient is drawn from a multinomial distribution with unknown probabilities. Preliminary results suggest evolution and not reemergence.     

WinMGM: A fast CPK molecular graphics program for analyzing molecular structure

A molecular modeling program is presented which has been written for Microsoft windows 3.1 and Windows NT operating systems. The program permits interactive molecular manipulation and also provides analytical tools such as energy computations and solvent accessible surfaces. An extremely fast algorithm is used which generates realistic space-filling CPK images in addition to wire frame, ribbons, MIDAS, labels, and points. An important feature of this algorithm is a highly optimized Z-buffer, which is described.     

Impact of stents and flow diverters on hemodynamics in idealized aneurysm models

Cerebral aneurysms constitute a major medical challenge as treatment options are limited and often associated with high risks. Statistically, up to 3% of patients with a brain aneurysm may suffer from bleeding for each year of life. Eight percent of all strokes are caused by ruptured aneurysms. In order to prevent this rupture, endovascular stenting using so called flow diverters is increasingly being regarded as an alternative to the established coil occlusion method in minimally invasive treatment. Covering the neck of an aneurysm with a flow diverter has the potential to alter the hemodynamics in such a way as to induce thrombosis within the aneurysm sac, stopping its further growth, preventing its rupture and possibly leading to complete resorption. In the present study the influence of different flow diverters is quantified considering idealized patient configurations, with a spherical sidewall aneurysm placed on either a straight or a curved parent vessel. All important hemodynamic parameters (exchange flow rate, velocity, and wall shear stress) are determined in a quantitative and accurate manner using computational fluid dynamics when varying the key geometrical properties of the aneurysm. All simulations are carried out using an incompressible, Newtonian fluid with steady conditions. As a whole, 72 different cases have been considered in this systematic study. In this manner, it becomes possible to compare the efficiency of different stents and flow diverters as a function of wire density and thickness. The results show that the intra-aneurysmal flow velocity, wall shear stress, mean velocity, and vortex topology can be considerably modified thanks to insertion of a suitable implant. Intra-aneurysmal residence time is found to increase rapidly with decreasing stent porosity. Of the three different implants considered in this study, the one with the highest wire density shows the highest increase of intra-aneurysmal residence time for both the straight and the curved parent vessels. The best hemodynamic modifications are always obtained for a small aneurysm diameter.     

Developing a genetic system in Deinococcus radiodurans for analyzing mutations

We have applied a genetic system for analyzing mutations in Escherichia coli to Deinococcus radiodurans, an extremeophile with an astonishingly high resistance to UV- and ionizing-radiation-induced mutagenesis. Taking advantage of the conservation of the beta-subunit of RNA polymerase among most prokaryotes, we derived again in D. radiodurans the rpoB/Rif(r) system that we developed in E. coli to monitor base substitutions, defining 33 base change substitutions at 22 different base pairs. We sequenced >250 mutations leading to Rif(r) in D. radiodurans derived spontaneously in wild-type and uvrD (mismatch-repair-deficient) backgrounds and after treatment with N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and 5-azacytidine (5AZ). The specificities of NTG and 5AZ in D. radiodurans are the same as those found for E. coli and other organisms. There are prominent base substitution hotspots in rpoB in both D. radiodurans and E. coli. In several cases these are at different points in each organism, even though the DNA sequences surrounding the hotspots and their corresponding sites are very similar in both D. radiodurans and E. coli. In one case the hotspots occur at the same site in both organisms.     

Neural network models for promoter recognition

The problem of recognition of promoter sites in the DNA sequence has been treated with models of learning neural networks. The maximum network capacity admissible for this problem has been estimated on the basis of the total of experimental data available on the determined promoter sequences. The model of a block neural network has been constructed to satisfy this estimate and rules have been elaborated for its learning and testing. The learning process involves a small (of the order of 10%) part of the total set of promoter sequences. During this procedure the neural network develops a system of distinctive features (key words) to be used as a reference in identifying promoters against the background of random sequences. The learning quality is then tested with the whole set. The efficiency of promoter recognition has been found to amount to 94 to 99%. The probability of an arbitrary sequence being identified as a promoter is 2 to 6%.     

Alternative non-antibody scaffolds for molecular recognition

Originally proposed one decade ago, the idea of engineering proteins outside the immunoglobulin family for novel binding functions has evolved as a powerful technology. Several classes of protein scaffolds proved to yield reagents with specificities and affinities in a range that was previously considered unique to antibodies. Such engineered protein scaffolds are usually obtained by designing a random library with mutagenesis focused at a loop region or at an otherwise permissible surface area and by selection of variants against a given target via phage display or related techniques. Whereas a plethora of protein scaffolds has meanwhile been proposed, only few of them were actually demonstrated to yield specificities towards different kinds of targets and to offer practical benefits such as robustness, smaller size, and ease of expression that justify their use as a true alternative to conventional antibodies or their recombinant fragments. Currently, the most promising scaffolds with broader applicability are protein A, the lipocalins, a fibronectin domain, an ankyrin consensus repeat domain, and thioredoxin. Corresponding binding proteins are not only of interest as research reagents or for separation in biotechnology but also as potential biopharmaceuticals, especially in the areas of cancer, autoimmune and infectious diseases as well as for in vivo diagnostics. The medical prospects have boosted high commercial expectations, and many of the promising scaffolds are under development by biotech start-up companies. Although some issues still have to be addressed, for example immunogenicity, effector functions, and plasma half-life in the context of therapeutic use or low-cost high-throughput selection for applications in proteomics research, it has become clear that scaffold-derived binding proteins will play an increasing role in biotechnology and medicine.     

Low-cost image processing system for analyzing molecular RNA fingerprints.

A complete image digitizing and processing system is described for capturing, enhancing and analyzing molecular fingerprints. The low-cost, high-resolution system features a Motorola 68000 processor, multi-tasking, a separate video coprocessor, and color or gray scale processing. Thousands of manipulations are possible using functions which include histographic equalization, edge detection, filtering, overlays, false coloring, zoom, pan and print. All operations are initiated and controlled with a mouse. Techniques for enhancing, scaling and comparing molecular fingerprints are described. The techniques all involve using a graphical interface to select and manipulate the various processes. The system has been used successfully for about 1.5 years, and it has been ideal for our application which requires human judgment at many steps between processing and which probably would not lend itself to a completely automated analysis. Similar techniques could probably be used with this system on many other applications.     

Developing biochemical and molecular markers for cyanobacterial inoculants

Markers for evaluating the establishment of cyanobacteria based on their sensitivity or resistance to antibiotics, saccharide utilization patterns and PCR generated fingerprints were developed. Four selected strains (isolates from rhizosphere soils of diverse agro-ecosystems) have shown potential as diazotrophs and exhibited plant growth promoting abilities. Different responses were obtained on screening against 40 antibiotics, which aided in developing selectable antibiotic markers for each strain. Biochemical profiles generated using standardized chromogenic identification system (including saccharide utilization tests) revealed that 53 % of the saccharides tested were not utilized by any strain, while some strains exhibited unique ability for utilization of saccharides such as melibiose, cellobiose, maltose and glucosamine. PCR based amplification profiles developed using a number of primers based on repeat sequences revealed the utility of 3 primers in providing unique fingerprints for the strains.     

Macromolecular crowding and molecular recognition

The effects of macromolecular crowding upon macromolecular associations in solution, and upon binding of a macromolecular ligand to a surface site, ar ereviewed. It is demonstrated, by means of two examples, that crowding may lead to significant alterations of biochemical or biological recognition processes at the molecular level.     

DNA repair: models for damage and mismatch recognition

Maintaining the integrity of the genome is critical for the survival of any organism. To achieve this, many families of enzymatic repair systems which recognize and repair DNA damage have evolved. Perhaps most intriguing about the workings of these repair systems is the actual damage recognition process. What are the chemical characteristics which are common to sites of nucleic acid damage that DNA repair proteins may exploit in targeting sites? Importantly, thermodynamic and kinetic principles, as much as structural factors, make damage sites distinct from the native DNA bases, and indeed, in many cases, these are the features which are believed to be exploited by repair enzymes. Current proposals for damage recognition may not fulfill all of the demands required of enzymatic repair systems given the sheer size of many genomes, and the efficiency with which the genome is screened for damage. Here we discuss current models for how DNA damage recognition may occur and the chemical characteristics, shared by damaged DNA sites, of which repair proteins may take advantage. These include recognition based upon the thermodynamic and kinetic instabilities associated with aberrant sites. Additionally, we describe how small changes in base pair structure can alter also the unique electronic properties of the DNA base pair pi-stack. Further, we describe photophysical, electrochemical, and biochemical experiments in which mismatches and other local perturbations in structure are detected using DNA-mediated charge transport. Finally, we speculate as to how this DNA electron transfer chemistry might be exploited by repair enzymes in order to scan the genome for sites of damage.     

MANOSK: A graphics program for analyzing and modeling molecular structure and functions

A program written for the Evans and Sutherland PS300 graphic displays is described in this paper. Called MANOSK, it provides a flexible and powerful environment for displaying, manipulating and analyzing small molecules and macromolecules from atomic coordinates. Translations and rotations of up to four independent molecules are available from the dials, and screen-relative orientations of the molecules are used in all geometrical and energetical calculations. A gradual progression of functions complexity makes the program easy to use for occasional works and efficient for more sophisticated studies.