Protein-bound water molecule counting by resolution of (1)H spin-lattice relaxation mechanisms

Water proton spin-lattice relaxation is studied in dilute solutions of bovine serum albumin as a function of magnetic field strength, oxygen concentration, and solvent deuteration. In contrast to previous studies conducted at high protein concentrations, the observed relaxation dispersion is accurately Lorentzian with an effective correlation time of 41 +/- 3 ns when measured at low proton and low protein concentrations to minimize protein aggregation. Elimination of oxygen flattens the relaxation dispersion profile above the rotational inflection frequency, nearly eliminating the high field tail previously attributed to a distribution of exchange times for either whole water molecules or individual protons at the protein-water interface. The small high-field dispersion that remains is attributed to motion of the bound water molecules on the protein or to internal protein motions on a time scale of order one ns. Measurements as a function of isotope composition permit separation of intramolecular and intermolecular relaxation contributions. The magnitude of the intramolecular proton-proton relaxation rate constant is interpreted in terms of 25 +/- 4 water molecules that are bound rigidly to the protein for a time long compared with the rotational correlation time of 42 ns. This number of bound water molecules neglects the possibility of local motions of the water in the binding site; inclusion of these effects may increase the number of bound water molecules by 50%.     

31P NMR relaxation measurements of the phosphate backbone of a double stranded hexadeoxynucleotide in solution: determination of the chemical shift anisotropy

The five phosphates of the deoxynucleotide d(CpGpTpApCpG)₂ have been assigned by two-dimensional heteronuclear NMR spectroscopy. The chemical shift anisotropy and correlation time of each phosphate group has been determined from measurements of the spin-lattice, spin-spin relaxation rate constants and the ³¹P-{¹H} nuclear Overhauser enhancement (NOE) at three magnetic field strengths (4.7 T, 9.4 T, and 11.75 T) and two temperatures (288 K and 298 K). As expected, the relaxation data require two mechanisms to account for the observed rate constants, i.e. dipole-dipole and chemical shift anisotropy. At 9.4 T and 11.75 T, the latter mechanism dominates the relaxation, leading to insignificant NOE intensities. The correlation time, chemical shift anisotropy and effective P-H distance were obtained from least-squares fitting to the data. Comparison of the fitted value for the correlation time with that obtained from ¹H measurements shows that the molecule behaves essentially as rigid rotor on the nanosecond timescale. Large amplitude motions observed in long segments of DNA are due to bending motions that do not contribute significantly to relaxation in short oligonucleotides.Abbreviations CSA chemical shift anisotropy - NOE nuclear Overhauser enhancement Offprint requests to: A. N. Lane     

The residence time of the bound water in the hydrophobic minor groove of the carbocyclic-nucleoside analogs of Dickerson-Drew dodecamers

The residence time of the bound water molecules in the antisense oligodeoxyribonucleotides containing 7'-alpha-methyl (TMe) carbocyclic thymidines in duplex (I), d5'(1C2G3C4G5A6A7TMc8TMe9C10G11C12G)23', and 6'-alpha-hydroxy (TOH) carbocyclic thymidines in duplex (II), d5'(1C2G3C4G5AOH6AOH7TOH8 TOH9C10G11C12G)2(3), have been investigated using a combination of NOESY and ROESY experiments. Because of the presence of 7'-alpha-methyl groups of TMe in the centre of the minor groove in duplex (I), the residence time of the bound water molecule is shorter than 0.3 ns. The dramatic reduction of the residence time of the water molecule in the minor groove in duplex (I) compared with the natural counterpart has been attributed to the replacement of second shell of hydration and disruption of hydrogen-bonding with 04' in the minor groove by hydrophobic alpha-methyl groups, as originally observed in the X-ray study. This effect could not be attributed to the change of the width of the minor groove because a comparative NMR study of the duplex (I) and its natural counterpart showed that the widths of their minor grooves are more or less unchanged (r.m.s.d change in the core part is <0.63A). For duplex (II) with polar 6'-alpha-hydroxyl groups pointed to the minor groove, the correlation time is much longer than 0.36 ns as a result of the stabilising hydrogen-bonding interaction with N3 or 02 of the neighbouring nucleotides.     

Rotational motions in myoglobin assessed by carbon 13 relaxation measurements at two magnetic field strengths.

Proton-decoupled Fourier transform nuclear magnetic resonance spectroscopy of natural abundance 13C was used to obtain spectra of cyanoferrimyoglobin of sperm whale (Physeter catadon) at 14.1 and 23.5 kG. Comparison of the spin lattice relaxation times at these two field strengths allowed the unambiguous assignment of a rotational correlation time of 22 plus or minus 5 ns for the alpha carbon resonances. The spin lattice relaxation time value for a major band attributable to aromatic carbon atoms also corresponded to a single correlation time, attributable to over-all tumbling of the molecule. Certain narrower resonances reflect other modes of rotational motion in addition to the over-all tumbling. Observations of nuclear Overhauser enhancement and line widths accord with these conslusions.     

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.     

Nuclear magnetic resonance detection of bound water molecules in the active site of Lactobacillus casei dihydrofolate reductase in aqueous solution.

Proton nuclear magnetic resonance spectroscopy has been used to detect two water molecules bound to residues in the active site of the Lactobacillus casei dihydrofolate reductase (DHFR). Their presence was detected by measuring nuclear Overhauser effects between NH protons in protein residues and protons in the individual bound water molecules in two-dimensional nuclear Overhauser effect spectroscopy (NOESY), in nuclear Overhauser effect spectroscopy in the rotating frame (ROESY) and three-dimensional 1H-15N ROESY-heteronuclear multiple quantum coherence spectra recorded on samples containing appropriately 15N-labelled DHFR. For the DHFR-methotrexate-NADPH complex, two bound molecules were found, one close to the Trp5 amide NH proton and the other near to the Trp21 indole HE1 proton: these correspond to two of the water molecules (Wat201 and Wat253) detected in the crystal structure studies described by Bolin and co-workers. However, the nuclear magnetic resonance experiments did not detect any of the other bound water molecules observed in the X-ray studies. The nuclear magnetic resonance results indicate that the two bound water molecules that were detected have lifetimes in the solution state that are longer than approximately two nanoseconds. This is of considerable interest, since one of these water molecules (Wat253) has been implicated as the likely proton donor in the catalytic reduction of dihydrofolate to tetrahydrofolate.     

Dynamic characterization of the water binding loop in the P-type cardiotoxin: implication for the role of the bound water molecule.

Recent studies of cobra P-type cardiotoxins (CTXs) have shown that the water-binding loop (loop II) plays a crucial role in toxin binding to biological membranes and in their cytotoxicity. To understand the role of bound water in the loop, the structure and dynamics of the major P-type CTX from Taiwan cobra, CTX A3, were determined by a comprehensive NMR analysis involving (1)H NOESY/ROESY, (13)C[1)H]NOE/T(1) relaxation, and (17)O triple-quantum filtered NMR. A single water molecule was found to be tightly hydrogen bonded to the NH of Met26 with a correlation time (5-7 ns) approaching the isotropic tumbling time (3.8-4.5 ns) of the CTX A3 molecule. Surprisingly, despite the relatively long residence time (ca. 5 ns to 100 micros), the bound water molecule of CTX A3 is located within a dynamic (order parameter S(2) approximately 0.7) and solvent accessible loop. Comparison among several P-type CTXs suggests that proline residues in the consensus sequence of MxAxPxVPV should play an important role in the formation of the water binding loop. It is proposed that the exchange rate of the bound water may play a role in regulating the lipid binding mode of amphiphilic CTX molecules near membrane surfaces.     

Hydration of POPC bilayers studied by 1H-PFG-MAS-NOESY and neutron diffraction

The stability of lipid bilayers is ultimately linked to the hydrophobic effect and the properties of water of hydration. Magic angle spinning (MAS) nuclear Overhauser enhancement spectroscopy (NOESY) with application of pulsed magnetic field gradients (PFG) was used to study the interaction of water with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayers in the fluid phase. NOESY cross-relaxation between water and polar groups of lipids, but also with methylene resonances of hydrophobic hydrocarbon chains, has been observed previously. This observation led to speculations that substantial amounts of water may reside in the hydrophobic core of bilayers. Here, the results of a quantitative analysis of cross-relaxation in a lipid 1-palmitoyl-2-oleoyl-sn-glycero-3 phosphocholine (POPC)/water mixture are reported. Coherences were selected via application of pulsed magnetic field gradients. This technique shortens acquisition times of NOESY spectra to 20 min and reduces t ₁-spectral noise, enabling detection of weak crosspeaks, like those between water and lipids, with higher precision than with non-gradient NOESY methods. The analysis showed that water molecules interact almost exclusively with sites of the lipid–water interface, including choline, phosphate, glycerol, and carbonyl groups. The lifetime of lipid–water associations is rather short, on the order of 100 ps, at least one order of magnitude shorter than the lifetime of lipid–lipid associations. The distribution of water molecules over the lipid bilayer was measured at identical water content by neutron diffraction. Water molecules penetrate deep into the interfacial region of bilayers but water concentration in the hydrophobic core is below the detection limit of one water molecule per lipid, in excellent agreement with the cross-relaxation data. Dedicated to Prof. K. Arnold on the occasion of his 65th birthday.     

Time-resolved fluorescence studies on the ternary complex formed between bacterial elongation factor Tu, guanosine 5'-triphosphate, and phenylalanyl-tRNAPhe

Time-resolved fluorescence spectroscopy was used to investigate the solution dynamics of Escherichia coli tRNAPhe, Phe-tRNAPhe, and Phe-tRNAPhe associated with GTP and elongation factor Tu (EF-Tu) in a ternary complex. Two fluorescence probes were employed: fluorescein, covalently bound to Phe-tRNAPhe at the s4U8 base (Phe-tRNAPhe-Fl8), and ethidium bromide, noncovalently associated with the tRNA (EB.Phe-tRNAPhe). The lifetimes observed for ethidium bromide were 1.89 ns, free in solution, and 26.3 ns, bound to its tight binding site on tRNA. Fluorescein-labeled tRNA had a lifetime of 4.3 ns, with no significant difference among the values for aminoacylated, unacylated, and EF-Tu-bound Phe-tRNAPhe-Fl8. Differential phase and modulation data for each fluorophore-tRNA system were fit with local and global Debye rotational relaxation times. Local motion of the labeled fluorescein in Phe-tRNAPhe-Fl8, tRNAPhe-Fl8, and Phe-tRNAPhe-Fl8.EF-Tu.GTP was characterized by rotational relaxation times of 2.7 +/- 0.5, 2.4 +/- 0.4, and 2.4 +/- 0.1 ns, respectively. These values are equal, within experimental error, and suggest that the rotational mobility of the s4U8-conjugated dye is unaffected by either tRNAPhe aminoacylation or ternary complex formation. Global rotational relaxation times for Phe-tRNAPhe-Fl8, 97 ns, and EB.Phe-tRNAPhe, 140 ns, were equivalent to those determined for the unacylated species, denoting little change in the overall size or shape of the tRNA molecule upon aminoacylation. These values for (Phe-)tRNA were larger than expected for a hydrated sphere of equivalent volume, 83 ns, and therefore confirm the asymmetric nature of the tRNA structure in solution.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein reorientation and bound water molecules measured by 1H magnetic spin-lattice relaxation

The water-proton spin-lattice relaxation rate constant, 1/T(1), was measured as a function of magnetic field strength for several dilute protein solutions. By separating the intermolecular contributions from the intramolecular contributions to the water-proton spin-lattice relaxation, the number of water molecules that bind to the protein for a time long compared with the rotational correlation time may be measured. We find a good correlation between the number of long-lived water molecules and the predictions based on available free volume in the proteins studied. The rotational correlation times of these proteins are larger than predicted by the Stokes-Einstein-Debye (SED) model for a sphere reorienting in a viscous liquid. The discrepancy between experiment and theory is usually attributed to hydration effects increasing the effective radius of the particle. However, the average lifetime of water molecules at the protein interface is far too short to justify such a picture. We suggest that surface roughness may be responsible for the retardation of rotational mobility and find that the SED model provides a reasonable representation of experiment if the radius assumed for the reorienting particle is the arithmetic mean of the crystallographic packing radius and the radius deduced from the effective surface area of the protein.     

Dynamics of supercoiled and relaxed pTZ18U plasmids probed with a long-lifetime metal-ligand complex

[Ru(bpy)2(dppz)](2+) (bpy = 2,2'-bipyridine, dppz = dipyrido- [3,2-a:2',3'-c]phenazine) (RuBD), a long-lifetime metalligand complex, displays favorable photophysical properties. These include long lifetime, polarized emission, but no significant fluorescence from the complex that is not bound to DNA. To show the usefulness of this luminophore (RuBD) for probing the bending and torsional dynamics of nucleic acids, its intensity and anisotropy decays when intercalated into supercoiled and relaxed pTZ18U plasmids were examined using frequency-domain fluorometry with a blue light-emitting diode (LED) as the modulated light source. The mean lifetimes for the supercoiled plasmids (< tau > = 148 ns) were somewhat shorter than those for the relaxed plasmids (< tau > = 160 ns). This suggests that the relaxed plasmids were shielded more efficiently from water. The anisotropy decay data also showed somewhat shorter slow rotational correlation times for supercoiled plasmids (288 ns) than for the relaxed plasmids (355 ns). The presence of two rotational correlation times suggests that RuBD reveals both the bending and torsional motions of the plasmids. These results indicate that RuBD can be useful for studying both the bending and torsional dynamics of nucleic acids.     

Investigations of peptide hydration using NMR and molecular dynamics simulations: A study of effects of water on the conformation and dynamics of antamanide

Summary The influence of water binding on the conformational dynamics of the cyclic decapeptide antamanide dissolved in the model lipophilic environment chloroform is investigated by NMR relaxation measurements. The water-peptide complex has a lifetime of 35 s at 250 K, which is longer than typical lifetimes of water-peptide complexes reported in aqueous solution. In addition, there is a rapid intracomplex mobility that probably involves librational motions of the bound water or water molecules hopping between different binding sites. Water binding restricts the flexibility of antamanide. The experimental findings are compared with GROMOS molecular dynamics simulations of antamanide with up to eight bound water molecules. Within the simulation time of 600 ps, no water molecule leaves the complex. Additionally, the simulations show a reduced flexibility for the complex in comparison with uncomplexed antamanide. Thus, there is a qualitative agreement between the experimental NMR results and the computer simulations.     

Towards MRI contrast agents of improved efficacy. NMR relaxometric investigations of the binding interaction to HSA of a novel heptadentate macrocyclic triphosphonate Gd(III)-complex

 A novel heptacoordinating ligand consisting of a thirteen-membered tetraazamacrocycle containing the pyridine ring and bearing three methylenephosphonate groups (PCTP-[13]) has been synthesized. Its Gd(III) complex displays a remarkably high longitudinal water proton relaxivity (7.7 mM^–1 s^–1 at 25  °C, 20 MHz and pH 7.5) which has been accounted for in terms of contributions arising from (1) one water molecule bound to the metal ion, (2) hydrogen-bonded water molecules in the second coordination sphere, or (3) water molecules diffusing near the paramagnetic chelate. Variable-temperature ¹⁷O-NMR transverse relaxation data indicate that the residence lifetime of the metal-bound water molecule is very short (8.0 ns at 25  °C) with respect to the Gd(III) complexes currently considered as contrast agents for magnetic resonance imaging. Furthermore, GdPCTP-[13] interacts with human serum albumin (HSA), likely through electrostatic forces. By comparing water proton relaxivity data for the GdPCTP-[13]-HSA adduct, measured as a function of temperature and magnetic field strength, with those for the analogous adduct with GdDOTP (a twelve-membered tetraaza macrocyclic tetramethylenephosphonate complex lacking a metal-bound water molecule), it has been possible to propose a general picture accounting for the main determinants of the relaxation enhancement observed when a paramagnetic Gd(III) complex is bound to HSA. Basically, the relaxation enhancement in these systems arises from (1) water molecules in the hydration shell of the macromolecule and protein exchangeable protons which lie close to the interaction site of the paramagnetic complex and (2) the metal bound water molecule(s). As far as the latter contribution is concerned, the interaction with the protein causes an elongation of the residence lifetime of the metal-bound water molecule, which limits, to some extent, the potential relaxivity enhancement expected upon the binding of the paramagnetic complex to HSA. Received: 27 January 1997 / Accepted: 12 May 1997     

Quantitative measurement of water diffusion lifetimes at a protein/DNA interface by NMR

Hydration site lifetimes of slowly diffusing water molecules at the protein/DNA interface of the vnd/NK-2 homeodomain DNA complex were determined using novel three-dimensional NMR techniques. The lifetimes were calculated using the ratios of ROE and NOE cross-relaxation rates between the water and the protein backbone and side chain amides. This calculation of the lifetimes is based on a model of the spectral density function of the water-protein interaction consisting of three timescales of motion: fast vibrational/rotational motion, diffusion into/out of the hydration site, and overall macromolecular tumbling. The lifetimes measured ranged from approximately 400 ps to more than 5 ns, and nearly all the slowly diffusing water molecules detected lie at the protein/DNA interface. A quantitative analysis of relayed water cross-relaxation indicated that even at very short mixing times, 5 ms for ROESY and 12 ms for NOESY, relay of magnetization can make a small but detectable contribution to the measured rates. The temperature dependences of the NOE rates were measured to help discriminate direct dipolar cross-relaxation from chemical exchange. Comparison with several X-ray structures of homeodomain/DNA complexes reveals a strong correspondence between water molecules in conserved locations and the slowly diffusing water molecules detected by NMR. A homology model based on the X-ray structures was created to visualize the conserved water molecules detected at the vnd/NK-2 homeodomain DNA interface. Two chains of water molecules are seen at the right and left sides of the major groove, adjacent to the third helix of the homeodomain. Two water-mediated hydrogen bond bridges spanning the protein/DNA interface are present in the model, one between the backbone of Phe8 and a DNA phosphate, and one between the side chain of Asn51 and a DNA phosphate. The hydrogen bond bridge between Asn51 and the DNA might be especially important since the DNA contact made by the invariant Asn51 residue, seen in all known homeodomain/DNA structures, is critical for binding affinity and specificity.     

Applications of tritium NMR to macromolecules: A study of two nucleic acid molecules

Summary We have tritium labeled two nucleic acid molecules, an 8 kDa DNA oligomer and a 20 kDa hammerhead RNA for tritium NMR investigations. The DNA sequence studied has been previously used in homonuclear studies of DNA-bound water molecules and tritium NMR was expected to facilitate these investigations by eliminating the need to suppress the water resonance in tritium-detected ³H-¹H NOESY experiments. We observed the anticipated through-space interactions found in B-form DNA in the NOESY experiments and an unexpected antiphase cross-peak at the water frequency. T₁ measurements on the tritiated DNA molecule indicated that relaxation rates were also accelerated for tritium and protons. Tritium NMR spectra of the hammerhead RNA molecule indicated conformational dynamics in the conserved region of the molecule in the absence of Mg²⁺ and spermine, two components necessary for cleavage. The dynamics were also investigated by ¹⁵N-correlated ¹H spectroscopy and persisted after the addition of Mg²⁺ and spermine.     

Fluorescence lifetime and polarization anisotropy studies of membrane surfaces with pyridoxal 5'-phosphate

The fluorescence lifetime and depolarization of the pyridoxal 5'-phosphate label demonstrated different environments at the structure-solvent interface for micelles, liposomes, proteins and membranes. A short lifetime and rotational correlation time for the micelles and liposomes proved that the label was strongly associated with the water solvent and rotated freely about the covalent bond. The proteins provided a more buried or hydrophobic site as shown by an increase in the lifetimes. Rotational correlation times of 4-6 ns for sarcolemma and erythrocyte membranes suggested restricted rotation for the pyridoxal 5'-phosphate label. Lower values of the rotational correlation time for rod outer segment and myelin sheath proved that the protein epsilon-amino groups are at the solvent interface which allows for more rotation.     

Dielectric properties of hydrated collagen

Dielectric measurements have been carried out on partially hydrated collagen in the frequency ranges 100 kHz–5 MHz, 100 MHz–1 GHz, and 8–23 GHz. In the low-frequency range, a dispersion was observed around 100 kHz which results from inhomogeneous conductivity of the samples. A dielectric relaxation was observed aroud 0.3 GHz using time-domain-spectroscopy techniques. This relaxation can be considered to originate from mobile side chains. Microwave measurements indicate that the water relaxation may extend into the 10-GHz region. An apparent discrepancy between the main water relaxation time and the average rotational correlation time of water as measured by nmr line widths was resolved by the assumption that a fraction of the water molecules is bound to the collagen with residence times on the order of 10^?6 sec, whereas the remainder of the water is only weakly bound and exhibits rotational rates on the order of 10^?10 sec.     

Structure of the anthramycin-d(ATGCAT)2 adduct from one- and two-dimensional proton NMR experiments in solution

One- and two-dimensional 400-MHz proton NMR experiments are used to examine the solution structure of the covalent adduct formed by the interaction of anthramycin methyl ether with the self-complementary deoxyoligonucleotide d(ATGCAT)2. The concentration dependence of chemical shifts and nuclear Overhauser enhancement (NOE) experiments are utilized to assign the adenine H2 protons within the minor groove for both free d(ATGCAT)2 and the adduct. These studies demonstrate that one of the four adenine H2 protons is in close proximity to the bound anthramycin and this results in its upfield shift of 0.3 ppm compared to the adenine H2 protons of the free duplex. Effects of the covalent attachment of anthramycin to the d(ATGCAT)2 duplex result in an increased shielding of selected deoxyribose protons located within the minor groove of the adduct, as demonstrated by two-dimensional autocorrelated (COSY) NMR techniques. Interactions between the protons of the covalently attached anthramycin and the d(ATGCAT)2 duplex are determined by utilizing two-dimensional NOE (NOESY) techniques. Analysis of these data reveals NOE cross-peaks between the anthramycin methyl, H6, and H7 protons with specific deoxyoligonucleotide protons within the minor groove, thus allowing the orientation of the drug within the minor groove to be determined. Nonselective inversion recovery (T1) relaxation experiments are used to probe the structural and dynamic properties of the anthramycin-d(ATGCAT)2 adduct. These data suggest that the binding of anthramycin alters the correlation time of the d(ATGCAT)2 duplex and stabilizes both of the internal A X T base pairs with respect to solvent exchange.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure, dynamics and hydration of the nogalamycin-d(ATGCAT)2Complex determined by NMR and molecular dynamics simulations in solution.

The structure of the 1:1 nogalamycin:d(ATGCAT)2 complex has been determined in solution from high-resolution NMR data and restrained molecular dynamics (rMD) simulations using an explicit solvation model. The antibiotic intercalates at the 5'-TpG step with the nogalose lying along the minor groove towards the centre of the duplex. Many drug-DNA nuclear Overhauser enhancements (NOEs) in the minor groove are indicative of hydrophobic interactions over the TGCA sequence. Steric occlusion prevents a second nogalamycin molecule from binding at the symmetry-related 5'-CpA site, leading to the conclusion that the observed binding orientation in this complex is the preferred orientation free of the complication of end-effects (drug molecules occupy terminal intercalation sites in all X-ray structures) or steric interactions between drug molecules (other NMR structures have two drug molecules bound in close proximity), as previously suggested. Fluctuations in key structural parameters such as rise, helical twist, slide, shift, buckle and sugar pucker have been examined from an analysis of the final 500 ps of a 1 ns rMD simulation, and reveal that many sequence-dependent structural features previously identified by comparison of different X-ray structures lie within the range of dynamic fluctuations observed in the MD simulations. Water density calculations on MD simulation data reveal a time-averaged pattern of hydration in both the major and minor groove, in good agreement with the extensive hydration observed in two related X-ray structures in which nogalamycin is bound at terminal 5'-TpG sites. However, the pattern of hydration determined from the sign and magnitude of NOE and ROE cross-peaks to water identified in 2D NOESY and ROESY experiments identifies only a few "bound" water molecules with long residence times. These solvate the charged bicycloaminoglucose sugar ring, suggesting an important role for water molecules in mediating drug-DNA electrostatic interactions within the major groove. The high density of water molecules found in the minor groove in X-ray structures and MD simulations is found to be associated with only weakly bound solvent in solution.     

Quantitative internuclear distancesvia two-dimensional nuclear magnetic resonance spectra: A test case and a DNA octamer duplex

Two-dimensional proton nuclear magnetic resonance nuclear Overhauser effect experiments have been performed at a series of mixing times on proflavine and on a DNA octamer duplex [d-(GGAATTCC)]₂ in solution. Using the complete matrix approach recently explored theoretically (Keepers and James, 1984), proton-proton internuclear distances were determined quantitatively for proflavine from the two-dimensional nuclear Overhauser effect results. Since proflavine is a rigid molecule with X-ray crystal structure determined, interproton distances obtained from the two-dimensional nuclear Overhauser effect experiments in solution can be compared with those for the crystalline compound agreement is better than 10 %. Experimental two-dimensional nuclear Overhauser effect spectral data for [d-(GGAATTCC)]₂ were analyzed by comparison with theoretical two-dimensional nuclear Overhauser effect spectra at each mixing time calculated using the complete 70 × 70 relaxation matrix. The theoretical spectra were calculated using two structures: a standard B-form DNA structure and an energy-minimized structure based on similarity of the octamer's six internal residues with those of [d-(CGCGAATTCGCG)]₂, for which the crystal structure has been determined. Neither the standard B-DNA nor the energy-minimized structure yield theoretical two-dimensional nuclear Overhauser effect spectra which accurately reproduce all experimental peak intensities. But many aspects of the experimental spectra can be represented by both the B-DNA and the energy-minimized structure. In general, the energy-minimized structure yields theoretical two-dimensional nuclear Overhauser effect spectra which mimic many, if not all, features of the experimental, spectra including structural characteristics at the purine-pyrimidine junction.