Guaifenesin stone matrix proteomics: a protocol for identifying proteins critical to stone formation.

Drug-related kidney stones are a diagnostic problem, since they contain a large matrix (protein) fraction and are frequently incorrectly identified as matrix stones. A urine proteomics study patient produced a guaifenesin stone during her participation, allowing us to both correctly diagnose her disease and identify proteins critical to this drug stone-forming process. The patient provided three random midday urine samples for proteomics studies; one of which contained stone-like sediment with two distinct fractions. These solids were characterized with optical microscopy and Fourier transform infrared spectroscopy. Immunoblotting and quantitative mass spectrometry were used to quantitatively identify the proteins in urine and stone matrix. Infrared spectroscopy showed that the sediment was 60 % protein and 40 % guaifenesin and its metabolite guaiacol. Of the 156 distinct proteins identified in the proteomic studies, 49 were identified in the two stone-components with approximately 50 % of those proteins also found in this patient's urine. Many proteins observed in this drug-related stone have also been reported in proteomic matrix studies of uric acid and calcium containing stones. More importantly, nine proteins were highly enriched and highly abundant in the stone matrix and 8 were reciprocally depleted in urine, suggesting a critical role for these proteins in guaifenesin stone formation. Accurate stone analysis is critical to proper diagnosis and treatment of kidney stones. Many matrix proteins were common to all stone types, but likely not related to disease mechanism. This protocol defined a small set of proteins that were likely critical to guaifenesin stone formation based on their high enrichment and high abundance in stone matrix, and it should be applied to all stone types.

Accuracy and precision of NMR relaxation experiments and MD simulations for characterizing protein dynamics.

Model-free parameters obtained from nuclear magnetic resonance (NMR) relaxation experiments and molecular dynamics (MD) simulations commonly are used to describe the intramolecular dynamical properties of proteins. To assess the relative accuracy and precision of experimental and simulated model-free parameters, three independent data sets derived from backbone 15N NMR relaxation experiments and two independent data sets derived from MD simulations of Escherichia-coli ribonuclease HI are compared. The widths of the distributions of the differences between the order parameters for pairs of NMR data sets are congruent with the uncertainties derived from statistical analyses of individual data sets; thus, current protocols for analyzing NMR data encapsulate random uncertainties appropriately. Large differences in order parameters for certain residues are attributed to systematic differences between samples for intralaboratory comparisons and unknown, possibly magnetic field-dependent, experimental effects for interlaboratory comparisons. The widths of distributions of the differences between the order parameters for two NMR sets are similar to widths of distributions for an NMR and an MD set or for two MD sets. The linear correlations between the order parameters for an MD set and an NMR set are within the range of correlations observed between pairs of NMR sets. These comparisons suggest that the NMR and MD generalized order parameters for the backbone amide N-H bond vectors are of comparable accuracy for residues exhibiting motions on a fast time scale (< 100 ps). Large discrepancies between NMR and MD order parameters for certain residues are attributed to the occurrence of "rare" motional events over the simulation trajectories, the disruption of an element of secondary structure in one of the simulations, and lack of consensus among the experimental data sets. Consequently, (easily detectable) severe distortions of local protein structure and infrequent motional events in MD simulations appear to be the most serious artifacts affecting the accuracy and precision, respectively, of MD order parameters relative to NMR values. In addition, MD order parameters for motions on a fast (< 100 ps) timescale are more precisely determined than their NMR counterparts, thereby permitting more detailed dynamic characterization of biologically important residues by MD simulation than is sometimes possible by experimental methods. Proteins 28:481-493, 1997.

Dynamics of ribonuclease H: temperature dependence of motions on multiple time scales.

The temperature dependence of the backbone motions in Escherichia coli ribonuclease HI was studied on multiple time scales by 15N nuclear magnetic spin relaxation. Laboratory frame relaxation data at 285, 300, and 310 K were analyzed using the model-free and reduced spectral density approaches. The temperature dependence of the order parameters was used to define a characteristic temperature for the motions of the backbone N-H bond vectors on picosecond to nanosecond time scales. The characteristic temperatures for secondary structure elements, loops, and the C-terminus are approximately 1000, approximately 300, and approximately 170 K, respectively. The observed variation in the characteristic temperature indicates that the energy landscape, and thus the configurational heat capacity, is markedly structure dependent in the folded protein. The effective correlation times for internal motions do not show significant temperature dependence. Conformational exchange was observed for a large number of residues forming a contiguous region of the protein that includes the coiled coil formed by helices alpha A and alpha D. Exchange broadening in the CPMG experiments decreased with increased temperature, directly demonstrating that the microscopic exchange rate is faster than the pulse repetition rate of 1.2 ms. The temperature dependence of the exchange contributions to the transverse relaxation rate constant shows approximately Arrhenius behavior over the studied temperature range with apparent activation enthalpies of approximately 20-50 kJ/mol. Numerical calculations suggest that these values underestimate the activation barriers by at most a factor of 2. The present results obtained at 300 K are compared to those reported previously [Mandel, A. M., Akke, M., & Palmer, A. G., III (1995) J. Mol. Biol. 246, 144-163] to establish the reproducibility of the experimental techniques.

Backbone dynamics of Escherichia coli ribonuclease HI: correlations with structure and function in an active enzyme.

Ribonuclease H is an endonuclease that hydrolyzes the RNA moiety of RNA-DNA duplex molecules. Escherichia coli ribonuclease H is involved in DNA replication, and retroviral ribonuclease H is essential for reverse transcription of the viral genome. To characterize the intramolecular dynamical properties of E. coli ribonuclease H, spin-lattice relaxation rate constants, spin-spin relaxation rate constants and steady state nuclear Overhauser effects for the 15N nuclear spins were measured by using proton-detected heteronuclear NMR spectroscopy. The relaxation data were analyzed by using a series of dynamical models in conjunction with a statistical model selection protocol. Ribonuclease H exhibits a complex array of dynamical features, most notably in the parallel beta-strands of the principal five-stranded beta-sheet, the coiled-coil helical interface, the active site, and the loop regions surrounding the active site. The dynamical properties are correlated with local structural environments of the 15N spins and suggest possible relationships to the functional properties of ribonuclease H. Results for E. coli ribonuclease H are compared to previously reported results for the human immunodeficiency virus type 1 ribonuclease H domain of reverse transcriptase.

The electrostatic basis for the interfacial binding of secretory phospholipases A2.

Biochemical and structural data suggest that electrostatic forces play a critical role in the binding of secretory phospholipases A2 to substrate aggregates (micelles, vesicles, monolayers, and membranes). This initial binding (adsorption) of the enzyme to the interface is kinetically distinct from the subsequent binding of substrate to the buried active site. Thus, in the absence of specific active-site interactions, electrostatic forces operating at the molecular surface may orient and hold the enzyme at the interface. We have calculated the electrostatic potentials for 10 species of secretory phospholipases A2 whose atomic coordinates have been determined by x-ray crystallography. Most of these enzymes show a marked electrostatic sidedness that is accentuated to a variable degree by the presence of the essential cofactor calcium ion. This asymmetry suggests a discrete interfacial binding region on the protein's surface, the location of which is in general agreement with proposals derived from the results of chemical modification, mutational, and crystallographic experiments.

Complete cDNA sequence of a South American isolate of potato virus X.

The complete cDNA sequence corresponding to the genomic RNA of a South American strain of potato virus X (PVXc) is reported. The sequence (6432 nucleotides) contains five open reading frames coding for polypeptides with molecular weights of 165.3, 24.3, 12.3, 7.6 and 25.0 and displays an overall homology of 77.4% with those previously reported for two European isolates. Comparison of amino acid sequences shows an average homology of 87%. Two major domains of variability, located between amino acids 476-615 of ORF 1 and 64-100 of ORF 5, are identified. Sequence similarities between RNA stretches lying upstream of ORFs 2, 4 and 5, and at the 3'-non coding regions of PVX and other plus-strand RNA viruses are described.