Magnetic relaxation dispersion studies of biomolecular solutions.

Although the MRD method has a long record in biomolecular systems, it has undergone a renaissance in the past few years as methodological developments have provided access to new types of information. In particular, MRD studies of quadrupolar nuclei such as 17O and 23Na have yielded valuable insights about the interactions of proteins and oligonucleotides with their solvent environment. The biomolecular MRD literature is still dominated by hydration studies, but the method has also been used to study the interaction of organic cosolvents and inorganic counterions with biomolecules. The MRD method can potentially make important contributions to the understanding of the mechanisms whereby protein conformational stability is affected by nonaqueous solvent components, such as denaturants, stabilizers, and helix promoters. Residence times of water molecules and other low molecular weight species in association with biomolecules can be determined by MRD. Such residence times are of general interest for understanding the kinetics of biomolecule-ligand interactions and, when exchange is gated by the biomolecule, can be used to characterize large-scale conformational fluctuations on nanosecond-millisecond time scales. By monitoring the integrity and specific internal hydration sites as well as the global solvent exposure, the MRD method can also shed light on the structure and dynamics of biomolecules in fluctuating nonnative states. Because it does not rely on high resolution, the MRD method is also applicable to very large biomolecules and complexes and has even been used to investigate protein crystals, gels, and biological tissues. In fact, dynamic studies of solids and liquid crystals were among the earliest applications of the MRD method. In many of its diverse applications, the MRD method provides unique information, complementing that available from high-resolution NMR.

Sequence-specific binding of counterions to B-DNA.

Recent studies by x-ray crystallography, NMR, and molecular simulations have suggested that monovalent counterions can penetrate deeply into the minor groove of B form DNA. Such groove-bound ions potentially could play an important role in AT-tract bending and groove narrowing, thereby modulating DNA function in vivo. To address this issue, we report here (23)Na magnetic relaxation dispersion measurements on oligonucleotides, including difference experiments with the groove-binding drug netropsin. The exquisite sensitivity of this method to ions in long-lived and intimate association with DNA allows us to detect sequence-specific sodium ion binding in the minor groove AT tract of three B-DNA dodecamers. The sodium ion occupancy is only a few percent, however, and therefore is not likely to contribute importantly to the ensemble of B-DNA structures. We also report results of ion competition experiments, indicating that potassium, rubidium, and cesium ions bind to the minor groove with similarly weak affinity as sodium ions, whereas ammonium ion binding is somewhat stronger. The present findings are discussed in the light of previous NMR and diffraction studies of sequence-specific counterion binding to DNA.

Dissection of the structural and functional role of a conserved hydration site in RNase T1.

The reoccurrence of water molecules in crystal structures of RNase T1 was investigated. Five waters were found to be invariant in RNase T1 as well as in six other related fungal RNases. The structural, dynamical, and functional characteristics of one of these conserved hydration sites (WAT1) were analyzed by protein engineering, X-ray crystallography, and (17)O and 2H nuclear magnetic relaxation dispersion (NMRD). The position of WAT1 and its surrounding hydrogen bond network are unaffected by deletions of two neighboring side chains. In the mutant Thr93Gln, the Gln93N epsilon2 nitrogen replaces WAT1 and participates in a similar hydrogen bond network involving Cys6, Asn9, Asp76, and Thr91. The ability of WAT1 to form four hydrogen bonds may explain why evolution has preserved a water molecule, rather than a side-chain atom, at the center of this intricate hydrogen bond network. Comparison of the (17)O NMRD profiles from wild-type and Thr93Gln RNase T1 yield a mean residence time of 7 ns at 27 degrees C and an orientational order parameter of 0.45. The effects of mutations around WAT1 on the kinetic parameters of RNase T1 are small but significant and probably relate to the dynamics of the active site.

Hydration of denatured and molten globule proteins.

The hydration of nonnative states is central to protein folding and stability but has been probed mainly by indirect methods. Here we use water 17O relaxation dispersion to monitor directly the internal and external hydration of alpha-lactalbumin, lysozyme, ribonuclease A, apomyoglobin and carbonic anhydrase in native and nonnative states. The results show that nonnative proteins are more structured and less solvent exposed than commonly believed. Molten globule proteins preserve most of the native internal hydration sites and have native-like surface hydration. Proteins denatured by guanidinium chloride are not fully solvent exposed but contain strongly perturbed occluded water. These findings shed new light on hydrophobic stabilization of proteins.

Water molecules in DNA recognition I: hydration lifetimes of trp operator DNA in solution measured by NMR spectroscopy.

The present NMR study investigates the residence times of the hydration water molecules associated with uncomplexed trp operator DNA in solution by measuring intermolecular nuclear Overhauser effects (NOE) between water and DNA protons, and the nuclear magnetic relaxation dispersion (NMRD) of the water 2H and 17O resonances. Both methods indicate that the hydration water molecules exchange with bulk water on the sub-nanosecond time scale at 4 degreesC. No evidence was obtained for water molecules bound with longer residence times. In particular, the water molecules at the sites of interfacial hydration in the trp repressor/operator complex do not seem kinetically stabilized in the uncomplexed DNA. Analysis of the crystal structures of two different trp repressor/operator complexes shows very similar structural environments for the water molecules mediating specific contacts between the protein and the DNA, whereas much larger variations are observed for the location of corresponding water molecules detected in the crystal structure of an uncomplexed trp operator DNA duplex. Therefore, it appears unlikely that the hydration characteristics of the uncomplexed DNA target would be a major determinant of trp repressor/operator recognition.

Thermal denaturation of ribonuclease A characterized by water 17O and 2H magnetic relaxation dispersion.

Water oxygen-17 and deuteron nuclear magnetic relaxation dispersion (NMRD) measurements were used to characterize ribonuclease A (RNase A) in the course of thermal denaturation at pH 2 and 4. The structure and dynamics of the protein were probed by specific long-lived water molecules, by the short-lived surface hydration, and by labile side-chain hydrogens. The NMRD data show that native RNase A contains at least three water molecules with a mean residence time of 8 ns at 27 degreesC and an activation enthalpy of ca. 40 kJ mol-1. These water molecules are identified with some or all of six ordered water molecules partly buried in surface pockets in the crystal structure of RNase A. The loss of the 17O dispersion at higher temperatures demonstrates that, in the thermally denatured protein, these surface pockets are either not present or undergoing large structural fluctuations on a subnanosecond time scale. The relaxation dispersion step vanishes monotonically and essentially in concert with the CD denaturation curves, thus ruling out the existence of equilibrium intermediates with a substantial amount of non-native and long-lived hydration water. The NMRD data show that thermally denatured RNase A has a relatively compact but highly flexible structure. The global solvent exposure and the hydrodynamic volume of the denatured protein are much less than for maximally unfolded disulfide-intact RNase A. The NMRD data show that thermal denaturation is accompanied by a large reduction of the mean-square orientational order parameter of side-chain O-H bonds, implying that, in the denatured state, these side chains sample a wide distribution of conformational states on a subnanosecond time scale.

Water and monovalent ions in the minor groove of B-DNA oligonucleotides as seen by NMR.

During the past 8 years, two complementary nmr techniques-magnetic relaxation dispersion and nuclear Overhauser effect spectroscopy-have been applied extensively to the study of water and monovalent ions in the minor groove of B-DNA oligonucleotides in solution. In this review, the possibilities and limitations of the two methods are outlined, with emphasis on the interpretational steps whereby molecular-level information is extracted from the primary data. The results on sequence-dependent hydration and ion-DNA interactions obtained so far by these methods is summarized and critically assessed. The nmr results are also compared with structural data from x-ray crystallography.

Dimethyl sulfoxide binding to globular proteins: a nuclear magnetic relaxation dispersion study.

The 2H magnetic relaxation dispersion (NMRD) technique was used to characterize interactions of dimethyl sulfoxide (DMSO) with globular proteins. A difference NMRD experiment involving the N-acetylglucosamine trisaccharide inhibitor, demonstrated that the DMSO 2H NMRD profile in lysozyme solution is due to a single DMSO molecule bound in the active cleft, with a molecular order parameter of 0.47 +/- 0.05 and a residence time in the range 10 ns to 5 ms. With the aid of transverse 2H relaxation data, the upper bound of the residence time was further reduced to 100 microns. A 1H shift titration experiment was also performed, yielding a binding constant of 2.3 +/- 0.3 M-1 at 27 degrees C. In contrast to lysozyme, no DMSO dispersion was observed for bovine pancreatic trypsin inhibitor (BPTI), indicating that a stable DMSO-protein complex requires a cleft of appropriate geometry in addition to hydrogen-bond and hydrophobic interactions.

Kinetics of DNA hydration.

The hydration of the d(CGCGAATTCGCG) B-DNA duplex in solution was studied by nuclear magnetic relaxation dispersion (NMRD) of the water nuclei 1H, 2H, and 17O, and by nuclear Overhauser effects (NOEs) in high-resolution two-dimensional 1H NMR spectra. By comparing results from the free duplex with those from its complex with netropsin, water molecules in the "spine of hydration" in the AATT region of the minor groove could be distinguished from hydration water elsewhere in the duplex. The 2H and 17O relaxation dispersions yield a model-independent residence time of 0.9(+/-0.1) ns at 4 degrees C for five highly ordered water molecules in the spine. When corrected for frequency offset effects, the NOE data yield the same residence time as the NMRD data, giving credence to both methods. At 27 degrees C, the residence time is estimated to 0.2 ns, a factor of 40 shorter than the tumbling time of the duplex. The NMRD data show that all water molecules associated with the duplex, except the five molecules in the spine, have residence times significantly shorter than 1 ns at 4 degrees C. There is thus no long-lived hydration structure associated with the phosphate backbone. In contrast to 2H and 17O, the 1H relaxation dispersion is dominated by labile DNA protons and therefore provides little information about DNA hydration.

Using buried water molecules to explore the energy landscape of proteins.

Buried water molecules constitute a highly conserved, integral part of nearly all known protein structures. Such water molecules exchange with external solvent as a result of protein conformational fluctuations. We report here the results of water (17)O and (2)H magnetic relaxation dispersion measurements on wild-type and mutant bovine pancreatic trypsin inhibitor in aqueous solution at 4-80 degrees C. These data lead to the first determination of the exchange rate of a water molecule buried in a protein. The strong temperature dependence of this rate is ascribed to large-scale conformational fluctuations in an energy landscape with a statistical ruggedness of approximately 10 kJ mol(-1).

Protein hydration dynamics in aqueous solution.

Water oxygen-17 and deuteron spin relaxation rates, measured as a function of resonance frequency, have been used to study the dynamics of protein hydration in aqueous solutions of ribonuclease A, lysozyme, myoglobin, trypsin and serum albumin. The relaxation data conform to the picture of protein hydration dynamics, proposed on the basis of previous studies of smaller proteins, where the long-lived water molecules responsible for the relaxation dispersion are identified with a small number of integrat water molecules seen in the crystal structures. These integral water molecules, with residence times in the range 10(-9)-10(-3) s, are either buried in internal cavities, trapped in narrow clefts or coordinated to metal ions. For the water molecules in the traditional hydration layer at the protein surface, the relaxation data suggest an average residence time in the range 10-50 ps, consistent with high-resolution 1H spectroscopy and computer simulations. The relaxation data also reveal some more specific features of protein hydration, relating to hydration of cavities that appear empty by crystallography, entrapment of water between structural domains of large proteins and subnanosecond 180 degrees flips in buried water clusters.

A new view of water dynamics in immobilized proteins.

The inflection frequency of the deuteron magnetic relaxation dispersion from water in rotationally immobilized protein samples has recently been found to be essentially independent of temperature and protein structure. This remarkable invariance has been interpreted in terms of a universal residence time of 1 microseconds for protein-associated water molecules. We demonstrate here that this interpretation is an artifact of the conventional perturbation theory of spin relaxation, which is not valid for rotationally immobile proteins. Using a newly developed non-perturbative, stochastic theory of spin relaxation, we identify the apparent correlation time of 1 microseconds with the inverse of the nuclear quadrupole frequency, thus explaining its invariance. The observed dispersion profiles are consistent with a broad distribution of residence times, spanning the microseconds range. Furthermore, we argue that the deuteron dispersion is due to buried water molecules rather than to the traditional surface hydration previously invoked, and that the contribution from rapidly exchanging protein hydrogens cannot be neglected. The conclusions of the present work are also relevant to proton relaxation in immobilized protein samples and to magnetic resonance imaging of soft tissue.

Residence times of the buried water molecules in bovine pancreatic trypsin inhibitor and its G36S mutant.

The three-dimensional structure of the bovine pancreatic trypsin inhibitor (BPTI) contains 4 internal water molecules, denoted W111, W112, W113, and W122, the latter being replaced by a seryl side chain in the BPTI(G36S) analogue. To investigate the effect of the exchange between these explicit water sites and the bulk solvent, we have measured water 17O and 2H nuclear magnetic relaxation in solutions of BPTI and the G36S mutant over the Larmor frequency range 2.6-49 MHz. A comparison of the data from the two nuclei shows unequivocally that the isolated buried water molecule, W122, of BPTI contributes only to 2H, but not to 17O relaxation, while the other 3 waters contribute fully to the relaxation of both nuclei. This demonstrates that the residence time of W122 is in the range 10-200 microseconds, while the residence times of W111-W113 are in the range 15 ns-1 microseconds. The slower exchange of W122 indicates that the functionally active region of BPTI, near the Cys14-Cys38 disulfide bond, is less flexible than the central region of BPTI, where the other 3 buried waters are located.

Protein hydration dynamics in aqueous solution: a comparison of bovine pancreatic trypsin inhibitor and ubiquitin by oxygen-17 spin relaxation dispersion.

Water oxygen-17 spin relaxation was used to study hydration and dynamics of the globular proteins bovine pancreatic trypsin inhibitor (BPTI) and ubiquitin in aqueous solution. The frequency dispersion of the longitudinal and transverse relaxation rates was measured over the Larmor frequency range 2.6 to 49 MHz in the pD range 2 to 11 at 27 degrees C. While the protein-induced relaxation enhancement was similar for the two proteins at high frequencies, it was an order of magnitude smaller for ubiquitin than for BPTI at low frequencies. This difference was ascribed to the absence, in ubiquitin, of highly ordered internal water molecules, which are known to be present in BPTI and in most other globular proteins. These observations demonstrate that the water relaxation dispersion in protein solutions is essentially due to a few structural water molecules buried within the protein matrix, but exchanging rapidly with the external water. The relaxation data indicate that the internal water molecules of BPTI exchange with bulk water on the time-scale 10(-8) to 10(-6) second thus lowering the recently reported upper bound on the residence time of these internal water molecules by four orders of magnitude, and implying that local unfolding occurs on the submicrosecond time-scale. The water molecules residing at the surface of the two proteins were found to be highly mobile, with an average rotational correlation time of approximately 20 picoseconds. For both proteins, the oxygen-17 relaxation depended only very weakly on pD, showing that ionic residues do not perturb hydration water dynamics more than other surface residues. We believe that the present results resolve the long-standing controversy regarding the mechanism behind the spin relaxation dispersion of water nuclei in protein solutions, thus establishing oxygen-17 relaxation as a powerful tool for studies of structurally and functionally important water molecules in proteins and other biomolecules.

Hydrogen exchange and protein hydration: the deuteron spin relaxation dispersions of bovine pancreatic trypsin inhibitor and ubiquitin.

Water deuteron (2H) spin relaxation was used to study hydrogen exchange, hydration, and protein dynamics in aqueous solutions of the globular proteins bovine pancreatic trypsin inhibitor (BPTI) and ubiquitin. The frequency dispersion of the longitudinal 2H relaxation rate was measured in the pD range 2 to 11 at 27 degrees C. In contrast to the previously reported water 17O relaxation dispersion from the same samples, the 2H dispersion depends strongly on pD. This pD dependence is due to labile protein deuterons in acidic side-chains and surface peptide groups, which exchange rapidly with water deuterons. The pD dependence of the 2H relaxation in BPTI solutions could be quantitatively accounted for in terms of known pK values and hydrogen exchange rate constants. For ubiquitin, labile protein deuterons contribute importantly to the 2H relaxation dispersion even in the neutral pD range. The 2H relaxation data also provided information about the orientational order and internal motion of OD and ND bonds in side-chains and surface peptides. A comparison of the water contribution to the 2H dispersion with the 17O dispersion indicates that one of the four internal water molecules of BPTI, presumably the deeply buried water molecule W122, exchanges more slowly (10(-6) to 10(-4) second) than the other three (10(-8) to 10(-6) second).

[Proton magnetic resonance spectra of bound water in muscles]. / Spektry protonnogo magnitnogo razonansa sviazannoi vody v myshtse.

The state of water in a muscle tissue was investigated using 1H-NMR. Water spectra in the muscle were found after holding it out at variable humidity and after the muscle species had been pressed with different effort. The NMR-spectra demonstrated the existence of two different water fractions with poor exchange between them. One of them was associated with structured water in the sarcoplasm connected with the contractile proteins. The second fraction was related with the myoplasm. The absorption isotherms for two fractions of water were observed.

[Ultrasonic skull trepanation]. / Ul'trazvukovaia trepanatsiia cherepa.

Experimental ultrasonic cranial trepanation was performed on 60 albino rats by means of a semi-automatic device, Y3T-1. The trepanation is altraumatic and does not need any considerable physical effort. A histological examination showed that the zone of bone necrosis along the edge of the cut surface was one third to one half that developing with the use of electric and pneumatic surgical instruments. The Y3T-1 device was used in operations on the skull in 78 patients. Resection trepanation of the skull was carried out in 55 patients, osteoplastic in 23. With the use of the ultrasonic device the time of the operation is shorter, a good hemostatic effect is produced, and no considerable physical effort is required on the part of the surgeon. A special catch incorporated in the device ensures safety of the operation. In reimplantation of an osteoperiosteal flap or in primary-delayed cranioplasty the use of an ultrasonic chisel for punctate osteosynthesis creates good conditions for bone tissue regeneration.