Biomedical subjects

E Liepinsh

Publications and source records attributed to E Liepinsh.

30 records · Page 2Linked to original sources

PubMed1996

Proton exchange rates from amino acid side chains--implications for image contrast.

The proton exchange rates between water and the hydroxyl protons of threonine, serine, tyrosine, the amino protons of lysine, and the guanidinium protons of arginine were measured in the pH range 0.5 to 8.5 and for the temperatures 4 degrees C, 10 degrees C, 20 degrees C, 30 degrees C, and 36 degrees C. The intrinsic exchange rates of the hydroxyl and amino protons at pH 7.0 degrees C and 36 degrees C were found to be in the range 700 to about 10,000 s-1. In addition, the exchange catalysis by phosphate, carbonate, carboxyl-, and amino-groups was investigated. The presence of these exchange catalysts at physiological concentrations increased the proton exchange rates from hydroxyl and amino groups several fold. The proton exchange rates are sufficiently fast that the total magnetization transfer between biomolecules and free bulk water is not rate limited by the proton exchange rate, but by the intramolecular cross-relaxation rates between the exchangeable and nonexchangeable protons of the biomolecules. Since the cross-relaxation rates between surface hydration water molecules and biomolecules are usually vanishingly small because of too rapid exchange with the free bulk water, it is proposed that the contrast in MR images is a fingerprint of the number of the exchangeable protons from OH and NH groups of the tissue, as far as the contrast depends on the magnetization transfer between biomolecules and water.

Amino Acids↗

PubMed1995

The mechanism of the anaerobic Escherichia coli ribonucleotide reductase investigated with nuclear magnetic resonance spectroscopy.

During the reduction of ribonucleotides with [3H]formate by the class III anaerobic ribonucleotide reductase from Escherichia coli tritium appears in water and not in the product deoxyribonucleotide. In D2O, deuterium replaces the OH-group at carbon-2' with retention of configuration. In addition we find 1-2% deuterium in the 3'-position demonstrating a small exchange of this hydrogen with the protons of water during catalysis. Class I and II enzymes catalyze identical reactions. Members of the three classes of reductases apparently use the same chemical mechanism in spite of having completely different protein structures.

Deoxycytidine↗

PubMed1994

Hydration of DNA in aqueous solution: NMR evidence for a kinetic destabilization of the minor groove hydration of d-(TTAA)2 versus d-(AATT)2 segments.

The residence times of the hydration water molecules near the base protons of d-(GTGGAATTCCAC)2 and d-(GTGGTTAACCAC)2 were investigated by nuclear magnetic resonance (NMR) spectroscopy. Nuclear Overhauser effects (NOE) were observed between base protons of the DNA and hydration water in NOESY and ROESY experiments. Large positive NOESY cross peaks observed between the resonances of the water and the adenine 2H protons of the central d-(AATT)2 segment in the duplex d-(GTGGAATTCCAC)2 indicate the presence of a 'spine of hydration' with water molecules exhibiting residence times on the DNA longer than 1 nanosecond. In contrast, no positive intermolecular NOESY cross peaks were detected in the d-(TTAA)2 segment of the duplex d-(GTGGTTAACCAC)2, indicating that no water molecules bound with similarly long residence times occur in the minor groove of this fragment. These results can be correlated with the larger width of the minor groove in d-(TTAA)2 segments as compared to that in d-(AATT)2 segments, as observed previously in single crystal structures of related oligonucleotide duplexes in B type conformation. The present experiments confirm earlier experimental results from single crystal studies and theoretical predictions that a 5'-dTA-3' step in the nucleotide sequence interrupts the spine of hydration in the minor groove.

Base Sequence↗

PubMed1994

Solution structure and dynamics of PEC-60, a protein of the Kazal type inhibitor family, determined by nuclear magnetic resonance spectroscopy.

The three-dimensional solution structure of porcine PEC-60, a 60 amino acid residue protein of the Kazal type family of proteinase inhibitors, was determined by nuclear magnetic resonance (NMR) spectroscopy. The structure determination is based on nearly complete 1H, 13C and 15N resonance assignments including stereospecific 1H resonance assignments for 40 pairs of methylene protons and isopropyl methyl groups. The stereospecific resonance assignments of the beta-protons were supported by heteronuclear long-range correlation experiments recorded at natural 13C and 15N isotopic abundances. A group of 20 conformers were calculated using the experimentally derived NMR constraints with the program DIANA, and energy-minimized in a 4 A water shell using the program OPAL. The average of the root-mean-square deviations relative to the mean structure of the 20 conformers selected to represent the solution structure of PEC-60 is 0.55 A for the backbone atoms of residues 6 to 10 and 24 to 60. Disordered conformations are observed for the amino-terminal pentapeptide and the polypeptide segment containing residues 11 to 23. The NMR structure confirms the structural similarity of PEC-60 to the Kazal type family of proteinase inhibitors which had been previously suggested on the basis of amino acid homology. The well-defined part of PEC-60 contains a short three-stranded anti-parallel beta-sheet involving the residues 27 to 29, 33 to 35 and 53 to 56 with a beta-bulge at residue 55, a type I turn comprising residues 29 to 32, and an alpha-helix involving the residues 37 to 48. T1(13C) relaxation measurements of the alpha-carbons and linewidth measurements of the amide proton signals indicate substantially increased mobility on the pico- to nanosecond time-scale for the amino-terminal pentapeptide as well as within the loop comprising residues 11 to 23. The structure of PEC-60 is compared to the X-ray crystal structures of homologous Kazal type proteinase inhibitors and the dynamic properties of PEC-60 are discussed with respect to the observed lack of any substantial trypsin inhibiting activity.

Amino Acid Sequence↗

PubMed1993

Hydration of proteins. A comparison of experimental residence times of water molecules solvating the bovine pancreatic trypsin inhibitor with theoretical model calculations.

A 1 ns trajectory from a molecular dynamics study of 1.4 ns total length was used for a detailed analysis of the residence times of water molecules located near 227 selected bovine pancreatic trypsin inhibitor (BPTI) atoms. The simulation was performed using the GROMOS force field, with apolar hydrogen atoms treated as united atoms, and the SPC/E water model. The system consisted of 568 BPTI atoms and 2371 water molecules. The theoretical results are in good agreement with experimental data available from nuclear magnetic resonance spectroscopy. The residence times of individual water molecules coming near a given BPTI atom, as obtained from the simulation, vary greatly and range between 10 and 500 ps. The effective residence time, calculated using a correlation function technique from the presence of all individual water molecules visiting the hydration shell of a given BPTI atom, never exceeds 200 ps. The average residence time near backbone and side-chain atoms is approximately 39 ps and 24 ps, respectively. The shortest residence times, on average, are found near charged atoms (19 ps), whereas near non-polar and polar side-chain atoms the residence times are 25 ps and 36 ps, respectively. There is no apparent correlation between the residence times of the hydration water molecules of solvent-accessible residues and their location in different regular or non-regular secondary structures.

Amino Acid Sequence↗

PubMed1993

Disulfide bond isomerization in BPTI and BPTI(G36S): an NMR study of correlated mobility in proteins.

Two conformational isomers were observed in the 1H nuclear magnetic resonance (NMR) spectra of the basic pancreatic trypsin inhibitor (BPTI) and of a mutant protein with Gly 36 replaced by Ser, BPTI(G36S). The less abundant isomer differs from the major conformation by different chirality of the Cys 14-Cys 38 disulfide bond. In BPTI, the population of the minor conformer increases from about 1.5% at 4 degrees C to 8% at 68 degrees C. In BPTI(G36S), the population of the minor conformation is about 15% of the total protein, so that a detailed structural study was technically feasible; a trend toward increasing population of the minor conformer at higher temperatures was observed also for this mutant protein. The activation parameters for the exchange between the two conformations were measured in the temperature range 4-68 degrees C, using uniformly 15N-enriched protein samples. Below room temperature the exchange rate of the disulfide flip follows an Arrhenius-type temperature dependence, with negative activation entropy in both proteins. At higher temperatures the exchange rates are governed by a different set of activation parameters, which are similar to those for the ring flips of Tyr 35 about the C beta-C gamma bond. Although the equilibrium enthalpy and entropy were found to be largely temperature independent, the activation entropy changes sign and is positive at higher temperatures. These results suggest that, above room temperature, the disulfide flips are coupled to the same protein structure fluctuations as the ring flips of Tyr 35.

Amino Acid Sequence↗

PubMed1993

Contributions from hydration of carboxylate groups to the spectrum of water-polypeptide proton-proton Overhauser effects in aqueous solution.

Nuclear Overhauser effects (NOE) were measured between water protons and protons of the glutamic acid side chain of the bicyclic decapeptide c-[Arg-Arg-Cys-Asn-Ala-Glu-Ala-Cys-Lys-Gly] in aqueous solution. Positive NOEs were observed between the gamma CH2 group of Glu and the water resonance, with similar NOE intensities at pH 2.0 and pH 6.3 in both the laboratory frame and the rotating frame of reference. These results indicate that the residence times of the hydration water molecules near the side-chain methylene protons are shorter than 500 ps for both the charged form and the uncharged form of Glu, and hence comparable to the water residence times near uncharged amino acid side chains. Furthermore, this study shows that the acidic proton in protonated carboxylic acid groups is not likely to interfere with the observation of polypeptide-hydration water NOEs, which is in contrast to the hydroxyl protons of the side chains of serine, threonine and tyrosine.

Algorithms↗

PubMed1992

NMR observation of individual molecules of hydration water bound to DNA duplexes: direct evidence for a spine of hydration water present in aqueous solution.

The residence times of individual hydration water molecules in the major and minor grooves of DNA were measured by nuclear magnetic resonance (NMR) spectroscopy in aqueous solutions of d-(CGCGAATTCGCG)2 and d-(AAAAATTTTT)2. The experimental observations were nuclear Overhauser effects (NOE) between water protons and the protons of the DNA. The positive sign of NOEs with the thymine methyl groups shows that the residence times of the hydration water molecules near these protons in the major groove of the DNA must be shorter than about 500 ps, which coincides with the behavior of surface hydration water in peptides and proteins. Negative NOEs were observed with the hydrogen atoms in position 2 of adenine in both duplexes studied. This indicates that a 'spine of hydration' in the minor groove, as observed by X-ray diffraction in DNA crystals, is present also in solution, with residence times significantly longer than 1 ns. Such residence times are reminiscent of 'interior' hydration water molecules in globular proteins, which are an integral part of the molecular architecture both in solution and in crystals.

Base Sequence↗

PubMed1992

NMR spectroscopy of hydroxyl protons in aqueous solutions of peptides and proteins.

Hydroxyl groups of serine and threonine, and to some extent also tyrosine are usually located on or near the surface of proteins. NMR observations of the hydroxyl protons is therefore of interest to support investigations of the protein surface in solution, and knowledge of the hydroxyl NMR lines is indispensable as a reference for studies of protein hydration in solution. In this paper, solvent suppression schemes recently developed for observation of hydration water resonances were used to observe hydroxyl protons of serine, threonine and tyrosine in aqueous solutions of small model peptides and the protein basic pancreatic trypsin inhibitor (BPTI). The chemical shifts of the hydroxyl protons of serine and threonine were found to be between 5.4 and 6.2 ppm, with random-coil shifts at 4 degrees C of 5.92 ppm and 5.88 ppm, respectively, and those of tyrosine between 9.6 and 10.1 ppm, with a random-coil shift of 9.78 ppm. Since these spectral regions are virtually free of other polypeptide 1H NMR signals, cross peaks with the hydroxyl protons are usually well separated even in homonuclear two-dimensional 1H NMR spectra. To illustrate the practical use of hydroxyl proton NMR in polypeptides, the conformations of the side-chain hydroxyl groups in BPTI were characterized by measurements of nuclear Overhauser effects and scalar coupling constants involving the hydroxyl protons. In addition, hydroxyl proton exchange rates were measured as a function of pH, where simple first-order rate processes were observed for both acid- and base-catalysed exchange of all but one of the hydroxyl-bearing residues in BPTI. For the conformations of the individual Ser, Thr and Tyr side chains characterized in the solution structure with the use of hydroxyl proton NMR, both exact coincidence and significant differences relative to the corresponding BPTI crystal structure data were observed.

Amino Acids↗

PubMed1992

Protein hydration in aqueous solution.

Proton nuclear magnetic resonance was used to study individual molecules of hydration water bound to the protein basic pancreatic trypsin inhibitor (BPTI) and to the nonapeptide oxytocin in aqueous solution. The experimental observations are nuclear Overhauser effects (NOE) between protons of individual amino acid residues of the protein and those of hydration water. These NOEs were recorded by two-dimensional (2D) and three dimensional (3D) NOE spectroscopy (NOESY) in the laboratory frame, and by the corresponding experiments in the rotating frame (ROESY). The studies show that there are two qualitatively different types of hydration sites. Four water molecules in the interior of the BPTI molecule are in identical locations in the crystal structure and in solution. Their NOEs with the protein protons are characterized by large negative cross-relaxation rates sigma NOE, which indicates that the residence times of the water molecules in these hydration sites are longer than ca. 10 ns. Additional experiments with extrinsic shift reagents established an upper limit of 20 ms at 4 degrees C for these residence times. Surface hydration of both the globular protein BPTI and the flexibly disordered polypeptide oxytocin is by water molecules with residence times in the subnanosecond range, as evidenced by small positive sigma NOE values observed for their NOEs with nearby polypeptide protons. Short residence times prevail for all surface hydration sites, independent of whether or not they are occupied by well ordered, X-ray observable water in the protein single crystals.

Aprotinin↗

PubMed1991

Protein hydration in aqueous solution.

High-resolution proton nuclear magnetic resonance studies of protein hydration in aqueous solution show that there are two qualitatively different types of hydration sites. A well-defined, small number of water molecules in the interior of the protein are in identical locations in the crystal structure and in solution, and their residence times are in the range from about 10(-2) to 10(-8) second. Hydration of the protein surface in solution is by water molecules with residence times in the subnanosecond range, even when they are located in hydration sites that contain well-ordered water in the x-ray structures of protein single crystals.

Animals↗

PubMed1991

Protein hydration studied with homonuclear 3D 1H NMR experiments.

Homonuclear 3D 1H NOESY-TOCSY and 3D 1H ROESY-TOCSY experiments were used to resolve and assign nuclear Overhauser effect (NOE) cross peaks between the water signal and individual polypeptide proton resonances in H2O solutions of the basic pancreatic trypsin inhibitor. Combined with a novel, robust water-suppression technique, positive and negative intermolecular NOEs were detected at 4 degrees C. The observation of positive NOEs between water protons and protein protons enables more precise estimates of the very short residence times of the water molecules in the hydration sites on the protein surface.

Amino Acid Sequence↗