Automated NMR resonance assignments and structure determination using a minimal set of 4D spectra

Automated methods for NMR structure determination of proteins are continuously becoming more robust. However, current methods addressing larger, more complex targets rely on analyzing 6–10 complementary spectra, suggesting the need for alternative approaches. Here, we describe 4D-CHAINS/autoNOE-Rosetta, a complete pipeline for NOE-driven structure determination of medium- to larger-sized proteins. The 4D-CHAINS algorithm analyzes two 4D spectra recorded using a single, fully protonated protein sample in an iterative ansatz where common NOEs between different spin systems supplement conventional through-bond connectivities to establish assignments of sidechain and backbone resonances at high levels of completeness and with a minimum error rate. The 4D-CHAINS assignments are then used to guide automated assignment of long-range NOEs and structure refinement in autoNOE-Rosetta. Our results on four targets ranging in size from 15.5 to 27.3 kDa illustrate that the structures of proteins can be determined accurately and in an unsupervised manner in a matter of days

Refolding upon force quench and pathways of mechanical and thermal unfolding of ubiquitin

The refolding from stretched initial conformations of ubiquitin (PDB ID: 1ubq) under the quenched force is studied using the Go model and the Langevin dynamics. It is shown that the refolding decouples the collapse and folding kinetics. The force quench refolding times scale as tau_F ~ exp(f_q*x_F/k_B*T), where f_q is the quench force and x_F = 0.96 nm is the location of the average transition state along the reaction coordinate given by the end-to-end distance. This value is close to x_F = 0.8 nm obtained from the force-clamp experiments. The mechanical and thermal unfolding pathways are studied and compared with the experimental and all-atom simulation results in detail. The sequencing of thermal unfolding was found to be markedly different from the mechanical one. It is found that fixing the N-terminus of ubiquitin changes its mechanical unfolding pathways much more drastically compared to the case when the C-end is anchored. We obtained the distance between the native state and the transition state x_UF=0.24 nm which is in reasonable agreement with the experimental data.Comment: 35 pages, 15 figures, 1 tabl

A review of wildland fire spread modelling, 1990-present 2: Empirical and quasi-empirical models

In recent years, advances in computational power and spatial data analysis (GIS, remote sensing, etc) have led to an increase in attempts to model the spread and behaviour of wildland fires across the landscape. This series of review papers endeavours to critically and comprehensively review all types of surface fire spread models developed since 1990. This paper reviews models of an empirical or quasi-empirical nature. These models are based solely on the statistical analysis of experimentally obtained data with or without some physical framework for the basis of the relations. Other papers in the series review models of a physical or quasi-physical nature, and mathematical analogues and simulation models. The main relations of empirical models are that of wind speed and fuel moisture content with rate of forward spread. Comparisons are made of the different functional relationships selected by various authors for these variables.Comment: 22 pages + 7 pages references + 2 pages tables + 2 pages figures. Submitted to International Journal of Wildland Fir

A review of wildland fire spread modelling, 1990-present, 1: Physical and quasi-physical models

In recent years, advances in computational power and spatial data analysis (GIS, remote sensing, etc) have led to an increase in attempts to model the spread and behaviour of wildland fires across the landscape. This series of review papers endeavours to critically and comprehensively review all types of surface fire spread models developed since 1990. This paper reviews models of a physical or quasi-physical nature. These models are based on the fundamental chemistry and/or physics of combustion and fire spread. Other papers in the series review models of an empirical or quasi-empirical nature, and mathematical analogues and simulation models. Many models are extensions or refinements of models developed before 1990. Where this is the case, these models are also discussed but much less comprehensively.Comment: 31 pages + 8 pages references + 2 figures + 5 tables. Submitted to International Journal of Wildland Fir

Knowledge-based energy functions for computational studies of proteins

This chapter discusses theoretical framework and methods for developing knowledge-based potential functions essential for protein structure prediction, protein-protein interaction, and protein sequence design. We discuss in some details about the Miyazawa-Jernigan contact statistical potential, distance-dependent statistical potentials, as well as geometric statistical potentials. We also describe a geometric model for developing both linear and non-linear potential functions by optimization. Applications of knowledge-based potential functions in protein-decoy discrimination, in protein-protein interactions, and in protein design are then described. Several issues of knowledge-based potential functions are finally discussed.Comment: 57 pages, 6 figures. To be published in a book by Springe

Crystallization and preliminary X-ray diffraction analyses of the TIR domains of three TIR-NB-LRR proteins that are involved in disease resistance in Arabidopsis thaliana

The Toll/interleukin-1 receptor (TIR) domain is a protein-protein interaction domain that is found in both animal and plant immune receptors. The N-terminal TIR domain from the nucleotide-binding (NB)-leucine-rich repeat (LRR) class of plant disease-resistance (R) proteins has been shown to play an important role in defence signalling. Recently, the crystal structure of the TIR domain from flax R protein L6 was determined and this structure, combined with functional studies, demonstrated that TIR-domain homodimerization is a requirement for function of the R protein L6. To advance the molecular understanding of the function of TIR domains in R-protein signalling, the protein expression, purification, crystallization and X-ray diffraction analyses of the TIR domains of the Arabidopsis thaliana R proteins RPS4 (resistance to Pseudomonas syringae 4) and RRS1 (resistance to Ralstonia solanacearum 1) and the resistance-like protein SNC1 (suppressor of npr1-1, constitutive 1) are reported here. RPS4 and RRS1 function cooperatively as a dual resistance-protein system that prevents infection by three distinct pathogens. SNC1 is implicated in resistance pathways in Arabidopsis and is believed to be involved in transcriptional regulation through its interaction with the transcriptional corepressor TPR1 (Topless-related 1). The TIR domains of all three proteins have successfully been expressed and purified as soluble proteins in Escherichia coli. Plate-like crystals of the RPS4 TIR domain were obtained using PEG 3350 as a precipitant; they diffracted X-rays to 2.05 angstrom resolution, had the symmetry of space group P1 and analysis of the Matthews coefficient suggested that there were four molecules per asymmetric unit. Tetragonal crystals of the RRS1 TIR domain were obtained using ammonium sulfate as a precipitant; they diffracted X-rays to 1.75 angstrom resolution, had the symmetry of space group P4(1)2(1)2 or P4(3)2(1)2 and were most likely to contain one molecule per asymmetric unit. Crystals of the SNC1 TIR domain were obtained using PEG 3350 as a precipitant; they diffracted X-rays to 2.20 angstrom resolution and had the symmetry of space group P4(1)2(1)2 or P4(3)2(1)2, with two molecules predicted per asymmetric unit. These results provide a good foundation to advance the molecular and structural understanding of the function of the TIR domain in plant innate immunity

Optimal local propensities for model proteins

Lattice models of proteins were used to examine the role of local propensities in stabilizing the native state of a protein, using techniques drawn from spin-glass theory to characterize the free-energy landscapes. In the strong evolutionary limit, optimal conditions for folding are achieved when the contributions from local interactions to the stability of the native state is small. Further increasing the local interactions rapidly decreases the foldability. © 1995 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/38522/1/340220411_ftp.pd

Evaluation of fluoropyruvate as nucleophile in reactions catalysed by N-acetyl neuraminic acid lyase variants: Scope, limitations and stereoselectivity

The catalysis of reactions involving fluoropyruvate as donor by N-acetyl neuraminic acid lyase (NAL) variants was investigated. Under kinetic control, the wild-type enzyme catalysed the reaction between fluoropyruvate and N-acetyl mannosamine to give a 90:10 ratio of the (3R,4R)- and (3S,4R)-configured products; after extended reaction times, equilibration occurred to give a 30:70 mixture of these products. The efficiency and stereoselectivity of reactions of a range of substrates catalysed by the E192N, E192N/T167V/S208V and E192N/T167G NAL variants were also studied. Using fluoropyruvate and (2R,3S)- or (2S,3R)-2,3-dihydroxy-4-oxo-N,N-dipropylbutanamide as substrates, it was possible to obtain three of the four possible diastereomeric products; for each product, the ratio of anomeric and pyranose/furanose forms was determined. The crystal structure of S. aureus NAL in complex with fluoropyruvate was determined, assisting rationalisation of the stereochemical outcome of C-C bond formation