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T L James

Publications and source records attributed to T L James.

At least 37 records · Page 2Linked to original sources
PubMed1999

NOS inhibitors decrease hypoxia-induced ATP reductions in respiring cerebrocortical slices.

BACKGROUND: Excess neuronal nitric oxide (NO) production might cause adenosine triphosphate loss and cellular damage in hypoxic brain parenchyma. 31P nuclear magnetic resonance spectroscopy was used to study hypoxic intracellular responses in perfused respiring cerebrocortical slices, in which NO scavenging by hemoglobin is absent, during NO synthase blockade and NO augmentation. METHODS: Adenosine triphosphate concentrations were monitored at 4.7 Tesla in respiring slices before, during, and after 60 min of hypoxia (oxygen tension < 5 mmHg). Slices were not treated or were pretreated with 27 microM L-nitroarginine methyl ester (L-NAME), 27 microM 7-nitroindozole (7-NI), or 27 microM L-nitroarginine. Nitrotyrosine:tyrosine ratios of slice extracts were measured using high-performance liquid chromatography. Cresyl violet-stained sections (2 microm) from random slices were examined histologically. RESULTS: After 60 min of hypoxia, adenosine triphosphate decreased to < or = 3, < or = 3, 65 +/- 6, and 25 +/- 4% of control in slices that were untreated or treated with L-nitroarginine, L-NAME, and 7-NI, respectively. After 120 min of hyperoxic recovery, adenosine triphosphate levels returned to control values in slices pretreated with L-NAME and 7-NI, but to only 30% of control in untreated or L-nitroarginine-treated slices. Nitric oxide donors administered during posthypoxic recovery partially antagonized the adenosine triphosphate recovery found with L-NAME and 7-NI. Nitric oxide synthase activity in slice homogenates, assayed via conversion of L-arginine to citrulline, was < or = 2% of control after all inhibitory treatments. The nitrotyrosine:tyrosine ratio increased by 52% in slices treated with 7-NI and by 200-300% in all other groups. Pretreatment with L-NAME and 7-NI reduced histologic evidence of cell swelling. CONCLUSION: Neuronal NO is associated with rapid adenosine triphosphate reductions and peroxynitrite formation in acutely hypoxic cerebrocortical slices.

Adenosine Triphosphate↗
PubMed1999

Evaluation of individual and combined neurotoxicity of the immunosuppressants cyclosporine and sirolimus by in vitro multinuclear NMR spectroscopy.

Neurotoxicity, a crucial side effect of immunosuppressive therapy with cyclosporine, also has been demonstrated in vitro for sirolimus, a novel macrolide immunosuppressant, which is under clinical investigation in combination with cyclosporine. NMR spectroscopy was used to study the separate and combined effects of cyclosporine and sirolimus on cerebral metabolism, both in brain cells and in perfused rat brain slices. The high-energy phosphate metabolism was already affected significantly at cyclosporine concentrations as low as 100 micrograms/liter: phosphocreatine was reduced by 10 +/- 2% [half-maximal inhibition concentration (IC50) = 1850 +/- 600 micrograms/liter], and nucleoside triphosphate was reduced by 11 +/- 5% (IC50 = 1110 +/- 420 micrograms/liter; n = 4, P <.05). At 500 micrograms/liter cyclosporine, N-acetylaspartate and glutamate were decreased by 13 +/- 7% (IC50 = 1100 +/- 330 micrograms/liter) and 22 +/- 9% (IC50 = 360 +/- 220 micrograms/liter; n = 4, P <.05), respectively. As evaluated using an algorithm based on Loewe isobolograms, combination of cyclosporine and sirolimus resulted in a synergetic reduction of high-energy phosphate metabolites. Addition of sirolimus to the perfusion medium increased brain slice concentrations of cyclosporine. It is concluded that cyclosporine significantly reduced high-energy phosphate metabolism in brain tissue at in vivo relevant concentrations. Combination with sirolimus resulted in synergism, which, in part, is explained by a greater distribution of cyclosporine into the brain tissue in the presence of sirolimus.

Animals↗
PubMed1998

Small structural ensembles for a 17-nucleotide mimic of the tRNA T psi C-loop via fitting dipolar relaxation rates with the quadratic programming algorithm.

Solution structures are typically average structures determined with the help of nmr-derived distance and torsion angle information. However, when a biomolecule populates significantly different conformations, the average structure might be prone to artifacts, and other refinement strategies are necessary. For example, when experimental restraints are used in molecular dynamics simulations in a time-averaged fashion (MDtar), the experimental structural information does no longer need to be satisfied at each step of the simulation; instead, the whole trajectory must agree with the restraints. However, the resulting structural ensembles are large and not unique and it is not trivial to extract the essential dynamic features for a system. Here we demonstrate that large MDtar ensembles can be simplified substantially by reducing the number of members to just a few on the basis of adjusting the individual probabilities of the members with the PDQPRO program [N. B. Ulyanov et al. Biophysical Journal (1995), Vol. 68, p. 13]. This algorithm finds the global minimum for a search function that represents the best match of a given ensemble with the experimental dipolar interproton relaxation rates. We have applied this strategy to a 17-residue RNA hairpin, whose loop exhibited considerable flexibility evident from nmr data. This 17mer is a mimic of the T psi C-loop of tRNA, where nucleotide 54 is usually a ribosylthymidine. The methylation of U54, which is completely buried in transfer ribonucleic acid, is administered by tRNA (m5 U54)-methyltransferase (RUMT). Since the 17mer is a good substrate for RUMT, we previously concluded that the flexibility of the 17mer's loop is a key to how RUMT gains access to the methylation site [L. J. Yao et al. Journal of Biomolecular NMR (1996) Vol. 9. p. 229]. Application of the PDQPRO algorithm to the previously acquired MDtar trajectories allowed us to reduce the number of conformations from several hundred to one major and five or six minor conformations with individual populations from approximately 5% to approximately 50% without any deterioration in the match with the experimental data. The major conformation exhibits a continuation of A-form helicity through part of the loop, involving C60 and U59. In this and most other conformations the methylation site in U54 is no longer buried.

Algorithms↗
PubMed1998

A pseudosquare knot structure of DNA in solution.

We report a high-resolution NMR structure of a homodimer formed by a synthetic 25 residue DNA oligonucleotide GCTCCCATGGTTTTTGTGCACGAGC. This structure presents a novel structural motif for single-stranded nucleic acids, called a pseudosquare knot (PSQ). The oligonucleotide was originally designed to mimic a slipped-loop structure (SLS), another "unusual" DNA structure postulated as an alternative conformation for short direct repeats in double-stranded DNA. The design of the sequence is compatible with both SLS and PSQ structures, both of which possess identical sets of base-paired and unpaired nucleotides but different tertiary folds. We used deuteration of the H8 positions of purines to ascertain that the PSQ is actually formed under the conditions used. The PSQ structure was solved based on homonuclear proton nuclear Overhauser effect data using complete relaxation matrix methods. The structure essentially consists of two side-by-side helices connected by single-stranded loops. Each of the helices is well-defined; however, the relative orientation of the two remains undetermined by the NMR data. The sequences compatible with the PSQ formation are frequent in single-stranded genomes; this structure may play a role as a dimerization motif.

Base Composition↗
PubMed1998

Nuclear magnetic resonance structure of d(GCATATGATAG). d(CTATCATATGC): a consensus sequence for promoters recognized by sigma K RNA polymerase.

The three-dimensional structure of d(GCATATGATAG).d(CTATCATATGC), from the promoter region of a gene regulating sporulation in Bacillus subtilis mother cells, was determined utilizing two-dimensional nuclear Overhauser effect (2D NOE) and double-quantum-filtered COSY (2QF-COSY) spectra. To minimize the effect of methods used to obtain restraints and refine structure, several variables were studied. Interproton distance bounds were calculated very conservatively by running the complete relaxation matrix program MARDIGRAS hundreds of times using 2D NOE spectra for exchangeable and for nonexchangeable protons at different mixing times, assuming different overall correlation times and different starting structures. The 435 distance restraints were used with two different structural refinement methods: restrained molecular dynamics (rMD) and restrained Monte Carlo calculations (rMC). Refinement using different procedures and starting structures resulted in essentially the same structure (<0.8 A rmsd), indicating that the structure is defined by experimental restraints and not the refinement method or variables used. R factors indicate the structures fit the experimental NOE data very well. Some helical parameters, notably large negative X displacement, are characteristic of A-DNA, but others are characteristic of B-DNA. As with TG.CA steps in other duplex DNA sequences studied in our laboratory, the two TG.CA steps have a positive roll, with T6-G7 exhibiting the largest, and consequently a bent helix axis. The converged structure represents a time-averaged structure. However, multiple conformations, especially in deoxyriboses, were evident from vicinal coupling constants obtained from quantitative simulations of 2QF-COSY cross-peaks and from persistent inconsistencies in experimental distances due to nonlinear conformational averaging.

Base Sequence↗
PubMed1998

Insights into the dynamic nature of DNA duplex structure via analysis of nuclear Overhauser effect intensities.

Sequence-dependent structures of DNA duplexes in solution can be reliably determined using NMR data if care is taken to determine restraint bounds accurately. This entails use of complete relaxation matrix methods to analyze nuclear Overhauser effect (NOE) spectroscopic cross-peak intensities, yielding accurate distance restraints. NMR studies of various DNA duplexes have suggested that there may be some limited internal motions. First, it is typically not possible to reconcile all vicinal proton coupling constants in deoxyribose rings with a single conformer. In addition, with the increased accuracy of interproton distance measurements afforded by the complete relaxation matrix algorithm MARDIGRAS, we find inconsistencies in certain distances which can most readily be ascribed to limited conformational flexibility, since conformational averaging is nonlinear. As base-sugar interproton distances depend on both sugar pucker and glycosidic torsion angle chi, motion involving these structural variables should be reflected by experimental data. Possible motional models have been considered to account for all of the data for three DNA duplexes. Analysis of intraresidue base-sugar interproton NOE bounds patterns suggests a motional model with individual sugars in equilibrium between S (2'-endo) and N (3'-endo) conformations, with S being preferred. As sugar repuckering is correlated with changes in glycosidic torsion angle chi, different sugar conformers imply different values for chi, but this is insufficient to account for all data. A two-state jump between anti and syn glycosidic conformers was considered, but it was incapable of accounting for all data. However, a model with restricted diffusion (rocking) about the glycosidic bond in addition to sugar repuckering was capable of accommodating all experimental data. This motional model is in qualitative agreement with experimental 13C relaxation-derived order parameter values in a DNA duplex.

Base Composition↗
PubMed1998

NMR spectroscopy and MRI investigation of a potential bioartificial liver.

NMR feasibility was established for a coaxial hydrophobic-membrane bioreactor containing isolated rat hepatocytes with features designed to mimic the human liver. A novel triple-tuned NMR probe and a perfusion system controlling temperature, gas concentrations, flow-rate, and pH were used. We determined the optimum coaxial interfiber distance (i.e. diffusion distance) for maintaining hepatocyte viability in two bioreactor prototypes. Prototype no. 1 and no. 2 had diffusion distances of 500 microns and 200 microns, respectively. Cell viability was established by 31P NMR and trypan blue exclusion. Only prototype no. 2 maintained cell viability for more than 6 h, indicating the importance of diffusion distance. 31P spectra obtained over this 6 h time period were similar to in vivo spectra of rat liver. The 31P spectra were found to be more sensitive to subacute cell viability than trypan blue exclusion. In the 1H and 31P spectra, 1H2O and inorganic phosphate signals were split in two at all flow-rates, probably due to bulk magnetic susceptibility effects originating from the three bioreactor compartments. MRI was useful for quality control and determining flow dynamics, fiber integrity, and cell inoculate distribution. MRI revealed that the inner fibers were not centered in either prototype. Although an increased flow-rate did not influence spectral resolution or chemical shifts, significant degradation of MRI quality occurred above 50 mL/min. NMR spectroscopy and imaging provide valuable, real-time information on cell biochemistry and flow dynamics which can be used in development and monitoring of bioreactors designed as artificial livers.

Animals↗
PubMed1998

DNA duplex dynamics: NMR relaxation studies of a decamer with uniformly 13C-labeled purine nucleotides.

Dynamics in a DNA decamer duplex, d(CATTTGCATC). d(GATGCAAATG), were investigated via a detailed 13C NMR relaxation study. Every 2'-deoxyadenosine and 2'-deoxyguanidine was chemically enriched with 15% 13C and 98% 15N isotopes. Six nuclear relaxation parameters [R(13Cz), R(1Hz), R(2(1)Hz13Cz), R(13Cx), R(2(1)Hz13Cx) and steady-state 13C¿1H¿ NOE] were measured at 600 MHz and three were measured at 500 MHz (1H frequency) for the CH spin systems of sugar 1', 3', and 4' as well as base 8 and 2 positions. A dependence of relaxation parameter values on chemical position was clearly observed; however, no sequence-specific variation was readily evident within our experimental error of approximately 5-10%, except for 3' and 5' termini. It was demonstrated that the random 15% 13C enrichment effectively suppressed both scalar and dipolar contributions of the neighboring carbons and protons on the relaxation parameters. To analyze dynamics via all observed relaxation parameters, full spectral density mapping (1992, J. W. Peng and G. Wagner, J. Magn. Reson. 98, 308) and the "model-free" approach (1982, Lipari and Szabo, J. Am. Chem. Soc. 104, 4546) were applied complementarily. A linear correlation between three spectral density values, J(omegaC), J(omegaH - omegaC), and J(omegaH + omegaC) was observed in plots containing all measured values, but not for the other spectral density terms including J(0). These linear correlations reflect the effect of overall motion and similar internal motions for each CH vector in the decamer. The correlations yielded two correlation times, 3-4 ns and 10-200 ps. One value, 3-4 ns, corresponds to the value of 3.3 ns obtained for the overall isotropic tumbling correlation time determined from analysis of 13C T1/T2 ratios. The possibility of overall anisotropic tumbling was examined, but statistical analysis showed no advantage over the assumption of simple isotropic tumbling. Lack of correlations entailing J(0) implies that a relatively slow chemical exchange contributes to yielding of effective Jeff(0) values. Based on spectral density mapping and the T1/T2 ratio analysis, three basic assumptions were initially employed (and subsequently justified) for the model-free calculation: isotropic overall tumbling, one internal motion, and the presence of chemical exchange terms. Except for terminal residues, the order parameter S2 and the corresponding fast internal motion correlation time were determined to be about 0.8 +/- 0.1 and 20 +/- 20 ps, respectively, for the various CH vectors. Only a few differences were observed between or within sugars and bases. The internal motion is very fast (ps-ns time scale) and its amplitude restricted; e.g., assuming a simple wobble-in-a-cone model, the internal motion is restricted to an angular amplitude of +/-22. 5 degrees for each of the 1', 3', 4', 2, and 8 positions in the purine nucleotides in the entire duplex.

Adenosine↗
PubMed1998

Structure-based design of ligands for protein basic domains: application to the HIV-1 Tat protein.

A methodology has been developed for designing ligands to bind a flexible basic protein domain where the structure of the domain is essentially known. It is based on an empirical binding free energy function developed for highly charged complexes and on Monte Carlo simulations in internal coordinates with both the ligand and the receptor being flexible. HIV-1 encodes a transactivating regulatory protein called Tat. Binding of the basic domain of Tat to TAR RNA is required for efficient transcription of the viral genome. The structure of a biologically active peptide containing the Tat basic RNA-binding domain is available from NMR studies. The goal of the current project is to design a ligand which will bind to that basic domain and potentially inhibit the TAR-Tat interaction. The basic domain contains six arginine and two lysine residues. Our strategy was to design a ligand for arginine first and then a superligand for the basic domain by joining arginine ligands with a linker. Several possible arginine ligands were obtained by searching the Available Chemicals Directory with DOCK 3.5 software. Phytic acid, which can potentially bind multiple arginines, was chosen as a building block for the superligand. Calormetric binding studies of several compounds to methylguanidine and Arg-/Lys-containing peptides were performed. The data were used to develop an empirical binding free energy function for prediction of affinity of the ligands for the Tat basic domain. Modeling of the conformations of the complexes with both the superligand and the basic domain being flexible has been carried out via Biased Probability Monte Carlo (BPMC) simulations in internal coordinates (ICM 2.6 suite of programs). The simulations used parameters to ensure correct folding, i.e., consistent with the experimental NMR structure of a 25-residue Tat peptide, from a random starting conformation. Superligands for the basic domain were designed by joining together two molecules of phytic acid with peptidic and peptidomimetic linkers. The linkers were refined by varying the length and side chains of the linking residues, carrying out BPMC simulations, and evaluation of the binding free energy for the best energy conformation. The dissociation constant of the best ligand designed is estimated to be in the low- to mid-nanomolar range.

Amino Acid Sequence↗
PubMed1998

Structure of the dimer initiation complex of HIV-1 genomic RNA.

Retroviral genomes must dimerize to be fully infectious. Dimerization is directed by a unique RNA hairpin structure with a palindrome in its loop: hairpins of two strands first associate transiently through their loops, and then refold to a more stable, linear duplex. The structure of the initial, kissing-loop dimer from HIV-1, solved using 2D NMR, is bent and metastable, its interface being formed not only by standard basepairing between palindromes, but also by a distinctive pattern of interstrand stacking among bases at the stem-loop junctions. This creates mechanical distortions that partially melt both stems, which may facilitate spontaneous refolding of this RNA complex into linear form.

Base Composition↗
PubMed1998

Fructose-1,6-bisphosphate preserves adenosine triphosphate but not intracellular pH during hypoxia in respiring neonatal rat brain slices.

BACKGROUND: Fructose-1,6-bisphosphate (FBP) sometimes provides substantial cerebral protection during hypoxia or ischemia. 31P/1H nuclear magnetic resonance spectroscopy of cerebrocortical slices was used to study the effects of FBP on hypoxia-induced metabolic changes. In addition, 13C-labeled glucose was administered and 13C nuclear magnetic resonance spectroscopy was used to search for FBP-induced modulations in glycolysis and the pentose-phosphate pathway. METHODS: In each experiment, 80 slices (350 microm) obtained from ten 7-day-old Sprague-Dawley rat litter mates were placed together in a 20-mm nuclear magnetic resonance tube, perfused, and subjected to 30 min of hypoxia (PO2 < 3 mmHg). Nine experiments were performed, with n = 3 in each of three groups: (1) no treatment with FBP; (2) 60 min of prehypoxia treatment with FBP (2 mM); and (3) 60 min of posthypoxia treatment with FBP (2 mM). 31P/1H Interleaved nuclear magnetic resonance spectra at 4.7 T provided average adenosine triphosphate, intracellular pH, and lactate. Cresyl violet stains of random slices taken at predetermined time points were studied histologically. Some experiments had [2-13C]glucose in the perfusate. Slices from these studies were frozen for perchloric acid extraction of intracellular metabolites and studied with high-resolution 13C nuclear magnetic resonance spectroscopy at 11.75 T. RESULTS: With no pretreatment with FBP, hypoxia caused an approximately 50% loss of adenosine triphosphate, an approximately 700% increase in lactate, and a decrease in intracellular pH to approximately 6.4. Pretreatment with FBP resulted in no detectable loss of adenosine triphosphate, no increase in lactate, and minimal morphologic changes but did not alter decreases in intracellular pH. 13C Nuclear magnetic resonance spectra of extracted metabolites showed that pretreatment caused accumulation of [1-13C]fructose-6-phosphate, an early pentose-phosphate pathway metabolite. Posthypoxic treatment with FBP had no effects compared with no treatment. CONCLUSIONS: During severe hypoxia, pretreatment with FBP completely preserves adenosine triphosphate and almost completely preserves cell morphology but does not alter hypoxia-induced decreases in intracellular pH. Pretreatment also substantially augments the flux of glucose into the pentose-phosphate pathway.

Adenosine Triphosphate↗
PubMed1997

Structure of the recombinant full-length hamster prion protein PrP(29-231): the N terminus is highly flexible.

The prion diseases seem to be caused by a conformational change of the prion protein (PrP) from the benign cellular form PrPC to the infectious scrapie form PrPSc; thus, detailed information about PrP structure may provide essential insights into the mechanism by which these diseases develop. In this study, the secondary structure of the recombinant Syrian hamster PrP of residues 29-231 [PrP(29-231)] is investigated by multidimensional heteronuclear NMR. Chemical shift index analysis and nuclear Overhauser effect data show that PrP(29-231) contains three helices and possibly one short beta-strand. Most striking is the random-coil nature of chemical shifts for residues 30-124 in the full-length PrP. Although the secondary structure elements are similar to those found in mouse PrP fragment PrP(121-231), the secondary structure boundaries of PrP(29-231) are different from those in mouse PrP(121-231) but similar to those found in the structure of Syrian hamster PrP(90-231). Comparison of resonance assignments of PrP(29-231) and PrP(90-231) indicates that there may be transient interactions between the additional residues and the structured core. Backbone dynamics studies done by using the heteronuclear [1H]-15N nuclear Overhauser effect indicate that almost half of PrP(29-231), residues 29-124, is highly flexible. This plastic region could feature in the conversion of PrPC to PrPSc by template-assisted formation of beta-structure.

Amino Acid Sequence↗
PubMed1997

A conformational transition at the N terminus of the prion protein features in formation of the scrapie isoform.

The scrapie prion protein (PrPSc) is formed from the cellular isoform (PrPC) by a post-translational process that involves a profound conformational change. Linear epitopes for recombinant antibody Fab fragments (Fabs) on PrPC and on the protease-resistant core of PrPSc, designated PrP 27-30, were identified using ELISA and immunoprecipitation. An epitope region at the C terminus was accessible in both PrPC and PrP 27-30; in contrast, epitopes towards the N-terminal region (residues 90 to 120) were accessible in PrPC but largely cryptic in PrP 27-30. Denaturation of PrP 27-30 exposed the epitopes of the N-terminal domain. We argue from our findings that the major conformational change underlying PrPSc formation occurs within the N-terminal segment of PrP 27-30.

Animals↗
PubMed1997

Evidence for protein X binding to a discontinuous epitope on the cellular prion protein during scrapie prion propagation.

Studies on the transmission of human (Hu) prions to transgenic (Tg) mice suggested that another molecule provisionally designated protein X participates in the formation of nascent scrapie isoform of prion protein (PrPSc). We report the identification of the site at which protein X binds to the cellular isoform of PrP (PrPC) using scrapie-infected mouse (Mo) neuroblastoma cells transfected with chimeric Hu/MoPrP genes even though protein X has not yet been isolated. Substitution of a Hu residue at position 214 or 218 prevented PrPSc formation. The side chains of these residues protrude from the same surface of the C-terminal alpha-helix and form a discontinuous epitope with residues 167 and 171 in an adjacent loop. Substitution of a basic residue at positions 167, 171, or 218 also prevented PrPSc formation: at a mechanistic level, these mutant PrPs appear to act as "dominant negatives" by binding protein X and rendering it unavailable for prion propagation. Our findings seem to explain the protective effects of basic polymorphic residues in PrP of humans and sheep and suggest therapeutic and prophylactic approaches to prion diseases.

Animals↗
PubMed1997

Solution structure of a 142-residue recombinant prion protein corresponding to the infectious fragment of the scrapie isoform.

The scrapie prion protein (PrPSc) is the major, and possibly the only, component of the infectious prion; it is generated from the cellular isoform (PrPC) by a conformational change. N-terminal truncation of PrPSc by limited proteolysis produces a protein of approximately 142 residues designated PrP 27-30, which retains infectivity. A recombinant protein (rPrP) corresponding to Syrian hamster PrP 27-30 was expressed in Escherichia coli and purified. After refolding rPrP into an alpha-helical form resembling PrPC, the structure was solved by multidimensional heteronuclear NMR, revealing many structural features of rPrP that were not found in two shorter PrP fragments studied previously. Extensive side-chain interactions for residues 113-125 characterize a hydrophobic cluster, which packs against an irregular beta-sheet, whereas residues 90-112 exhibit little defined structure. Although identifiable secondary structure is largely lacking in the N terminus of rPrP, paradoxically this N terminus increases the amount of secondary structure in the remainder of rPrP. The surface of a long helix (residues 200-227) and a structured loop (residues 165-171) form a discontinuous epitope for binding of a protein that facilitates PrPSc formation. Polymorphic residues within this epitope seem to modulate susceptibility of sheep and humans to prion disease. Conformational heterogeneity of rPrP at the N terminus may be key to the transformation of PrPC into PrPSc, whereas the discontinuous epitope near the C terminus controls this transition.

Amino Acid Sequence↗
PubMed1997

Solution structure and backbone dynamics of the human alpha3-chain type VI collagen C-terminal Kunitz domain,.

The solution structure and backbone dynamics of the 58-residue C-terminal Kunitz domain fragment [alpha3(VI)] of human alpha3-chain type VI collagen has been studied by two-dimensional 1H-1H and 1H-15N nuclear magnetic resonance spectroscopy at 303 K. The solution structure is represented by an ensemble of 20 structures calculated with X-PLOR using 612 distance and 47 dihedral angle restraints. The distance restraints were obtained by a complete relaxation matrix analysis using MARDIGRAS. The root mean squared (rms) deviation is 0.91 A for the backbone atoms of the residues Thr2(8)-Gly12(18), Arg15(21)-Tyr35(41), and Gly40(46)-Pro57(63). The central beta-sheet [residues Ile18(24)-Tyr35(41)] and the C-terminal alpha-helix [residues Gln48(54)-Cys55(61)] are better defined with a backbone rms deviation of 0.46 A. The solution structure of alpha3(VI) is virtually identical to the crystal structure of alpha3(VI) and to the solution structure of bovine pancreatic trypsin inhibitor (BPTI). The 15N spin-lattice and spin-spin relaxation rates and the 1H-15N heteronuclear nuclear Overhauser enhancement (NOE) were analyzed using both the "model-free" formalism [Lipari, G., & Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546-4559 and 4559-4570] and the reduced spectral density mapping procedure [Farrow, N. A., Szabo, A., Torchia, D. A., & Kay, L. E. (1995) J. Biomol.NMR 6, 153-162]. The results obtained from the "model-free" analysis include an overall correlation time tauc of 3. 00 ns and backbone order parameters S2 in the range from 0.28 to 0. 93. The necessity of including an exchange term in the analysis of the relaxation data from 14 residues indicated that these residues are involved in motions on the micro- to millisecond time scale. The majority of the 14 residues are located in the vicinity of the Cys14(20)-Cys38(44) disulfide bond, suggesting the presence of a disulfide bond isomerization similar to the one observed in BPTI [Otting, G., Liepinsh, E., & Wüthrich, K. (1993) Biochemistry 32, 3571-3582]. It is suggested that this disulfide bond isomerization is the main reason for the surprisingly small effect on trypsin inhibition observed when Thr13(19) of alpha3(VI) is substituted with Pro.

Amino Acid Sequence↗
PubMed1997

Physical studies of conformational plasticity in a recombinant prion protein.

PrP(Sc) is known to be the major, if not the only, component of the infectious prion. Limited proteolysis of PrP(Sc) produces an N-terminally truncated polypeptide of about 142 residues, designated PrP 27-30. Recently, a recombinant protein (rPrP) of 142 residues corresponding to the Syrian hamster PrP 27-30 was expressed in Escherichia coli and purified (Mehlhorn et al., 1996). rPrP has been refolded into both alpha-helical and beta-sheet structures as well as various intermediates in aqueous buffers. The beta-sheet state and two pH-dependent alpha-helical states were characterized by CD and NMR. The alpha-helical conformation occurred only after the formation of an intramolecular disulfide bond, whereas the beta-sheet form was accessible either with or without the disulfide. Of the different alpha-helical forms studied, only those refolded in the pH range 5-8 were substantially soluble at physiological pH, exhibiting similar conformations and monomeric analytical sedimentation profiles throughout the above pH range. Furthermore, refolded alpha-rPrP showed NMR chemical shift dispersion typical of proteins with native conformations, although 2D NMR indicated large segments of conformational flexibility. It displayed a cooperative thermal denaturation transition; at elevated temperatures, it converted rapidly and irreversibly to the thermodynamically more stable beta-sheet form. Unfolding of alpha-rPrP by GdnHCl revealed a two-phase transition with a relatively stable folding intermediate at 2 M GdnHCl. The deltaG values were estimated to be 1.9 +/- 0.4 kcal/mol for the first phase and 6.5 +/- 1.2 kcal/mol for the second, consistent with a folding core surrounded by significant segments of flexible conformation. By NMR, alpha-rPrP(acid) isolated at pH 2 without refolding exhibited heterogeneous line widths, consistent with an acid-denatured molten globular state. We conclude that to the extent that rPrP constitutes a relevant folding domain of PrP(C), the various conformations exhibited by rPrP suggest that the PrP sequence may be intrinsically plastic in its conformations; indeed, portions of PrP(C) may possess a relatively open conformation which makes it susceptible to conversion into PrP(Sc) under appropriate conditions.

Animals↗