A computer program for the design of optimal synthetic oligonucleotide probes for protein coding genes

A computer program has been written in FORTRAN 77 to locateon a protein sequence a region with optimum length and limiteddegeneracy in order to design artificial oligonucleotide probesfor use in molecular cloning. In addition the program checksfor regions of homology between this probe and any other basesequence found in nucleotide sequence data banks. There areoptions in the program to eliminate rare codons or to make preferentialchoices of bases in order to minimize the degeneracy of probes. Received on February 17, 1987; accepted on June 26, 1987     

A generic protein purification method for protein complex characterization and proteome exploration.

We have developed a generic procedure to purify proteins expressed at their natural level under native conditions using a novel tandem affinity purification (TAP) tag. The TAP tag allows the rapid purification of complexes from a relatively small number of cells without prior knowledge of the complex composition, activity, or function. Combined with mass spectrometry, the TAP strategy allows for the identification of proteins interacting with a given target protein. The TAP method has been tested in yeast but should be applicable to other cells or organisms.     

A multiple alignment program for protein sequences

A program for the multiple alignment of protein sequences ispresented. The program is an extension of the fast alignmentprogram by Wilbur et al. (1984) into higher dimensions. Theuse of hash procedures on fragments of the protein sequencesincreases the speed of calculation. Thereby we also take intoaccount fragments which are present in some, but not in all,sequences considered. The results of some multiple alignmentsare given. Received on September 11, 1986; accepted on March 18, 1987     

Improving the accuracy of protein stability predictions with multistate design using a variety of backbone ensembles

Multistate computational protein design (MSD) with backbone ensembles approximating conformational flexibility can predict higher quality sequences than single‐state design with a single fixed backbone. However, it is currently unclear what characteristics of backbone ensembles are required for the accurate prediction of protein sequence stability. In this study, we aimed to improve the accuracy of protein stability predictions made with MSD by using a variety of backbone ensembles to recapitulate the experimentally measured stability of 85 Streptococcal protein G domain β1 sequences. Ensembles tested here include an NMR ensemble as well as those generated by molecular dynamics (MD) simulations, by Backrub motions, and by PertMin, a new method that we developed involving the perturbation of atomic coordinates followed by energy minimization. MSD with the PertMin ensembles resulted in the most accurate predictions by providing the highest number of stable sequences in the top 25, and by correctly binning sequences as stable or unstable with the highest success rate (≈90%) and the lowest number of false positives. The performance of PertMin ensembles is due to the fact that their members closely resemble the input crystal structure and have low potential energy. Conversely, the NMR ensemble as well as those generated by MD simulations at 500 or 1000 K reduced prediction accuracy due to their low structural similarity to the crystal structure. The ensembles tested herein thus represent on‐ or off‐target models of the native protein fold and could be used in future studies to design for desired properties other than stability. Proteins 2014; 82:771–784. © 2013 Wiley Periodicals, Inc.     

Alpha-synuclein multistate folding thermodynamics: implications for protein misfolding and aggregation

Alpha-synuclein aggregation has been tightly linked with the pathogenesis of Parkinson's disease and other neurodegenerative disorders. Despite the protein's putative function in presynaptic vesicle regulation, the roles of lipid binding in modulating alpha-synuclein conformations and the aggregation process remain to be fully understood. This study focuses on a detailed thermodynamic characterization of monomeric alpha-synuclein folding in the presence of SDS, a well-studied lipid mimetic. Far-UV CD spectroscopy was employed for detection of conformational transitions induced by SDS, temperature, and pH. The data we present here clearly demonstrate the multistate nature of alpha-synuclein folding, which involves two predominantly alpha-helical partially folded thermodynamic intermediates that we designate as F (most folded) and I (intermediately folded) states. Likely structures of these alpha-synuclein conformational states are also discussed. These partially folded forms can exist in the presence of either monomeric or micellar forms of SDS, which suggests that alpha-synuclein has an intrinsic propensity for adopting multiple alpha-helical structures even in the absence of micelle or membrane binding, a feature that may have implications for its biological activity and toxicity. Additionally, we discuss the relation between alpha-synuclein three-state folding and its aggregation, within the context of isothermal titration calorimetry and transmission electron microscopy measurements of SDS-initiated oligomer formation.     

A multistate model for early decision-making in oncology

The development of oncology drugs progresses through multiple phases, where after each phase, a decision is made about whether to move a molecule forward. Early phase efficacy decisions are often made on the basis of single-arm studies based on a set of rules to define whether the tumor improves (“responds”), remains stable, or progresses (response evaluation criteria in solid tumors [RECIST]). These decision rules are implicitly assuming some form of surrogacy between tumor response and long-term endpoints like progression-free survival (PFS) or overall survival (OS). With the emergence of new therapies, for which the link between RECIST tumor response and long-term endpoints is either not accessible yet, or the link is weaker than with classical chemotherapies, tumor response-based rules may not be optimal. In this paper, we explore the use of a multistate model for decision-making based on single-arm early phase trials. The multistate model allows to account for more information than the simple RECIST response status, namely, the time to get to response, the duration of response, the PFS time, and time to death. We propose to base the decision on efficacy on the OS hazard ratio (HR) comparing historical control to data from the experimental treatment, with the latter predicted from a multistate model based on early phase data with limited survival follow-up. Using two case studies, we illustrate feasibility of the estimation of such an OS HR. We argue that, in the presence of limited follow-up and small sample size, and making realistic assumptions within the multistate model, the OS prediction is acceptable and may lead to better early decisions within the development of a drug.     

A program for template matching of protein sequences

The matching of a template to a protein sequence is simplifiedby treating it as a special case of sequence alignment. Restrictionof the distances between motifs in the template controls againstspurious matches within very long sequences. The program usingthis algorithm is fast enough to be used in scanning large databasesfor sequences matching a complex template. Received on August 17, 1987; accepted on January 11, 1988     

A fast and efficient program for modeling protein loops

We developed an efficient Monte Carlo Simulated Annealing (MCSA) program for modeling protein loops with high speed. The total conformational energy in each step of MCSA simulation consists of two parts: the nonbonded atomic interaction represented by a simple soft-sphere potential and the harmonic distance constraint to ensure the smooth connection of the loop segment to the rest of the protein structure. The soft-sphere potential was a simplified potential that has been successfully used by the authors in modeling the carbohydrate part of glycoprotein systems [H. Zhang, Y. Yang, L. Lai, and Y. Tang (1996), Carbohydrate Research, Vol. 284, pp. 25–34]. It only considers the purely repulsive steric interactions to avoid artificial attractive forces between atoms in the absence of solvent molecules. The N-terminal of the loop segment was connected to the bulk protein part, and two dummy main-chain atoms N and Cα immediately following the C-terminal of the loop segment were constrained to their real positions in the protein structure, which not only assures the correct geometry of loop-protein connection but also is more rigorous than the previous work. To improve the speed, two strategies, the local region method and grid-mapping method, were devised to accelerate the computation of environmental interaction that is responsible for the major part of the computing consumption. The grid-mapping method can reduce computational time dramatically. Conformations with rational steric packing and smooth connection to the rest of the protein structure were generated by the MCSA program, and then were refined by the empirical force field CHARMm [B. R. Brook, R. E. Braccoleri, B. D. Olafson, D. J. States, S. Swaminathan, and M. Karplus (1983), Journal of Computational Chemistry, Vol. 4, pp. 187–217]. Bovine pancreatic trypsin inhibitor (BPTI) was used as an example to test the ability of loop modeling of the method, and five loops in BPTI were calculated. Conformations close to the crystal structure were generated for all of them. With the criteria of CHARMm energy, near-native conformations can be selected, for example, the backbone rms deviation 0.93 A from the crystal structure was gotten for the longest 9-residue loop. © 1997 John Wiley & Sons, Inc.     

A multistate model for bivariate interval-censored failure time data

SUMMARY: Interval-censored life-history data arise when the events of interest are only detectable at periodic assessments. When interest lies in the occurrence of two such events, bivariate-interval censored event time data are obtained. We describe how to fit a four-state Markov model useful for characterizing the association between two interval-censored event times when the assessment times for the two events may be generated by different inspection processes. The approach treats the two events symmetrically and enables one to fit multiplicative intensity models that give estimates of covariate effects as well as relative risks characterizing the association between the two events. An expectation-maximization (EM) algorithm is described for estimation in which the maximization step can be carried out with standard software. The method is illustrated by application to data from a trial of HIV patients where the events are the onset of viral shedding in the blood and urine among individuals infected with cytomegalovirus.     

A computer graphics program system for protein structure representation

We have developed a computer graphics program system for the schematic representation of several protein secondary structure analysis algorithms. The programs calculate the probability of occurrence of alpha-helix, beta-sheet and beta-turns by the method of Chou and Fasman and assign unique predicted structure to each residue using a novel conflict resolution algorithm based on maximum likelihood. A detailed structure map containing secondary structure, hydrophobicity, sequence identity, sequence numbering and the location of putative N-linked glycosylation sites is then produced. In addition, helical wheel diagrams and hydrophobic moment calculations can be performed to further analyze the properties of selected regions of the sequence. As they require only structure specification as input, the graphics programs can easily be adapted for use with other secondary structure prediction schemes. The use of these programs to analyze protein structure-function relationships is described and evaluated.     

A maximum likelihood framework for protein design

Background  

The aim of protein design is to predict amino-acid sequences compatible with a given target structure. Traditionally envisioned as a purely thermodynamic question, this problem can also be understood in a wider context, where additional constraints are captured by learning the sequence patterns displayed by natural proteins of known conformation. In this latter perspective, however, we still need a theoretical formalization of the question, leading to general and efficient learning methods, and allowing for the selection of fast and accurate objective functions quantifying sequence/structure compatibility.     

A generic high-throughput screening assay for kinases: protein kinase a as an example

A generic high-throughput screening assay based on the scintillation proximity assay technology has been developed for protein kinases. In this assay, the biotinylated (33)P-peptide product is captured onto polylysine Ysi bead via avidin. The scintillation signal measuring the product formation increases linearly with avidin concentration due to effective capture of the product on the bead surface via strong coulombic interactions. This novel assay has been optimized and validated in 384-well microplates. In a pilot screen, a signal-to-noise ratio of 5- to 9-fold and a Z' factor ranging from 0.6 to 0.8 were observed, demonstrating the suitability of this assay for high-throughput screening of random chemical libraries for kinase inhibitors.     

A multistate Markov chain model for evaluating a sterilization policy

"A multistate Markov chain model corresponding to varying fertility and mortality rates at different levels of surviving children of a couple was developed. Asymptotic probabilities of having a fixed number of children have been worked out." The implied geographical focus is on India.     

A general multistate model for the analysis of hydrogen-exchange kinetics

In proton nmr, the chemical exchange rates of slowly exchanging labile hydrogens (with lifetimes in the range ～ 10 msec – ～ 1 sec) of peptides, proteins, and nucleic acids can be measured in H₂O by a combination of two separate experiments: (1) the transfer of solvent saturation and (2) saturation-recovery experiments. When these molecules exist in a dynamic equilibrium among different conformations, the experiments cannot be analyzed in a straightforward manner to derive the intrinsic exchange rates. In the present study we have derived analytical expressions for the above two experiments on a biomolecule under certain limiting conditions: (1) the extreme low-motility limit, where each of the conformational transitions is much slower than the corresponding hydrogen exchange rate with the solvent; (2) the high-motility limit (EX₂ mechanism), which is the opposite extreme of the previous limit; and (3) the low-motility limit (EX₁ mechanism), which is a mixture of limits (1) and (2), i.e., for some of the conformations, the exchange rate with the solvent is much faster than their conformational transition rates, while for the remaining conformations the reverse situation is realized. The results may be considered as a generalization to an arbitrary number of states of the two-state model treated by Hvidt. Equations have also been derived that are applicable to the iostope exchange method of measuring very slow exchange rates (with life-times of the order of minutes and longer) in biomolecules. The saturation recovery experiments performed in H₂O on the active pentapeptide fragment of thymopoietin serve to illustrate the high-motility limit. The theoretical formulation presented in this study can be easily adapted to other double-resonance techniques and also to situations where the kinetics of an arbitrary system existing in a multistate equilibrium are of interest.     

A computer program for translating DNA sequences into protein.

This paper describes a comprehensive program for translating one or two DNA sequences into amino acid sequences. Written in FORTRAN, it was designed for maximum flexibility of use and easy maintenance, modification and portability. It has full comments throughout.     

MATRAS: A program for protein 3D structure comparison

The recent accumulation of large amounts of 3D structural data warrants a sensitive and automatic method to compare and classify these structures. We developed a web server for comparing protein 3D structures using the program Matras (http://biunit.aist-nara.ac.jp/matras). An advantage of Matras is its structure similarity score, which is defined as the log-odds of the probabilities, similar to Dayhoff's substitution model of amino acids. This score is designed to detect evolutionarily related (homologous) structural similarities. Our web server has three main services. The first one is a pairwise 3D alignment, which is simply align two structures. A user can assign structures by either inputting PDB codes or by uploading PDB format files in the local machine. The second service is a multiple 3D alignment, which compares several protein structures. This program employs the progressive alignment algorithm, in which pairwise 3D alignments are assembled in the proper order. The third service is a 3D library search, which compares one query structure against a large number of library structures. We hope this server provides useful tools for insights into protein 3D structures.     

Combinatorial peptidomics: a generic approach for protein expression profiling

Traditional approaches to protein profiling were built around the concept of investigating one protein at a time and have long since reached their limits of throughput. Here we present a completely new approach for comprehensive compositional analysis of complex protein mixtures, capable of overcoming the deficiencies of current proteomics techniques. The Combinatorial methodology utilises the peptidomics approach, in which protein samples are proteolytically digested using one or a combination of proteases prior to any assay being carried out. The second fundamental principle is the combinatorial depletion of the crude protein digest (i.e. of the peptide pool) by chemical crosslinking through amino acid side chains. Our approach relies on the chemical reactivities of the amino acids and therefore the amino acid content of the peptides (i.e. their information content) rather than their physical properties. Combinatorial peptidomics does not use affinity reagents and relies on neither chromatography nor electrophoretic separation techniques. It is the first generic methodology applicable to protein expression profiling, that is independent of the physical properties of proteins and does not require any prior knowledge of the proteins. Alternatively, a specific combinatorial strategy may be designed to analyse a particular known protein on the basis of that protein sequence alone or, in the absence of reliable protein sequence, even the predicted amino acid translation of an EST sequence. Combinatorial peptidomics is especially suitable for use with high throughput micro- and nano-fluidic platforms capable of running multiple depletion reactions in a single disposable chip.     

A semiparametric transition model with latent traits for longitudinal multistate data

SUMMARY: We propose a general multistate transition model. The model is developed for the analysis of repeated episodes of multiple states representing different health status. Transitions among multiple states are modeled jointly using multivariate latent traits with factor loadings. Different types of state transition are described by flexible transition-specific nonparametric baseline intensities. A state-specific latent trait is used to capture individual tendency of the sojourn in the state that cannot be explained by covariates and to account for correlation among repeated sojourns in the same state within an individual. Correlation among sojourns across different states within an individual is accounted for by the correlation between the different latent traits. The factor loadings for a latent trait accommodate the dependence of the transitions to different competing states from a same state. We obtain the semiparametric maximum likelihood estimates through an expectation-maximization (EM) algorithm. The method is illustrated by studying repeated transitions between independence and disability states of activities of daily living (ADL) with death as an absorbing state in a longitudinal aging study. The performance of the estimation procedure is assessed by simulation studies.     

First-aid: A design philosophy and a program for on-line symptom processing

A major problem for computer-aided diagnosis systems, particularly those designed for on-line use in clinical settings, is that their operation may be obtrusive or unacceptable within established clinical routine. Criteria for a general approach to the design of on-line aids in clinical decision-making are presented. An interpreter is described that is designed to be broadly applicable within the area of statistical diagnosis, but which is also designed to encourage the development of congenial clinical systems. The use of the program and its features are illustrated by an application in the diagnosis of ‘dyspepsia’.     

Algorithms for protein design

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