Two-dimensional 1H and 31P NMR spectra and restrained molecular dynamics structure of an extrahelical adenosine tridecamer oligodeoxyribonucleotide duplex

Assignment of the 1H and 31P NMR spectra of an extrahelical adenosine tridecamer oligodeoxyribonucleotide duplex, d(CGCAGAATTCGCG)2, has been made by two-dimensional 1H-1H and heteronuclear 31P-1H correlated spectroscopy. The downfield 31P resonance previously noted by Patel et al. (1982) has been assigned by both 17O labeling of the phosphate as well as a pure absorption phase constant-time heteronuclear 31P-1H correlated spectrum and has been associated with the phosphate on the 3' side of the extrahelical adenosine. JH3'-P coupling constants for each of the phosphates of the tridecamer were obtained from the 1H-31P J-resolved selective proton-flip 2D spectrum. By use of a modified Karplus relationship the C4-C3'-O3-P torsional angles (epsilon) were obtained. There exists a good linear correlation between 31P chemical shifts and the epsilon torsional angle. The 31P chemical shifts and epsilon torsional angles follow the general observation that the more internal the phosphate is located within the oligonucleotide sequence, the more upfield the 31P resonance occurs. Because the extrahelical adenosine significantly distorts the deoxyribose phosphate backbone conformation even several bases distant from the extrahelical adenosine, 31P chemical shifts show complex site- and sequence-specific variations. Modeling and NOESY distance-restrained energy minimization and restrained molecular dynamics suggest that the extrahelical adenosine stacks into the duplex. However, a minor conformation is also observed in the 1H NMR, which could be associated with a structure in which the extrahelical adenosine loops out into solution.     

Two-dimensional 1H and 31P NMR spectra and restrained molecular dynamics structure of a covalent CPI-CDPI2-oligodeoxyribonucleotide decamer complex

CPI-CDPI2 is a synthetic analogue of CC-1065, which is a naturally occurring antitumor antibiotic. Assignment of the 1H NMR spectra of a CPI-CDPI2-oligodeoxyribonucleotide decamer, d-(CGCTTAAGCG)2, complex has been made by two-dimensional 1H/1H spectroscopy. The solution structure of the complex was calculated by an iterative hybrid relaxation matrix method combined with NOESY distance restrained molecular dynamics. Refinement proceeded in two steps in which the decamer was initially refined alone and then CPI-CDPI2 was added to the structure to allow initial estimates of drug-DNA contacts. A hybrid matrix/MD refinement was used to better take into account problems associated with spin diffusion. Thus the distances from the 2D NOESY spectra were calculated from the relaxation rate matrix which were evaluated from a hybrid NOESY volume matrix comprising elements from the experimental spectrum and those calculated from an initial structure. The hybrid matrix derived distances were then used in a restrained molecular dynamics procedure to obtain a new structure that better approximates the NOESY spectra. The resulting partially refined structure was then used to calculate an improved theoretical NOESY volume matrix which is once again merged with the experimental matrix until refinement is complete. The efficacy of CC-1065 has been attributed to its minor groove binding and alkylation to the N3 position of adenosine. CPI-CDPI2 appears to bind to the decamer in a similar manner. The effect of CPI-CDPI2 on the decamer's 1H and 31P spectrum was consistent with a minor groove binding motif with the drug alkylating at A17 with the CDPI rings oriented toward the 5'-end of the alkylated strand. In addition, the NMR data support one major adduct but also indicate the presence of a minor adduct. The latter could represent a drug alkylation of the DNA at a secondary site (or alternative orientation of the rings).     

Two-dimensional 1H and 31P NMR spectra of a decamer oligodeoxyribonucleotide duplex and a quinoxaline ((MeCys3, MeCys7]TANDEM) drug duplex complex

Assignment of the 1H and 31P NMR spectra of a decamer oligodeoxyribonucleotide duplex, d(CCCGATCGGG), and its quinoxaline ((MeCys3, MeCys7]TANDEM) drug duplex complex has been made by two-dimensional 1H-1H and heteronuclear 31P-1H correlated spectroscopy. The 31P chemical shifts of this 10 base pair oligonucleotide follow the general observation that the more internal the phosphate is located within the oligonucleotide sequence, the more upfield the 31P resonance occurs. While the 31P chemical shifts show sequence-specific variations, they also do not generally follow the Calladine "rules" previously demonstrated. 31P NMR also provides a convenient monitor of the phosphate ester backbone conformational changes upon binding of the drug to the duplex. Although the quinoxaline drug, [MeCys3, MeCys7]TANDEM, is generally expected to bind to duplex DNA by bis-intercalation, only small 31P chemical shift changes are observed upon binding the drug to duplex d(CCCGATCGGG). Additionally, only small perturbations in the 1H NMR and UV spectra are observed upon binding the drug to the decamer, although association of the drug stabilizes the duplex form relative to the other states. These results are consistent with a non-intercalative mode of association of the drug. Modeling and molecular mechanics energy minimization demonstrate that a novel structure in which the two quinoxaline rings of the drug binds in the minor groove of the duplex is possible.     

Solution structure of human calcitonin gene-related peptide by 1H NMR and distance geometry with restrained molecular dynamics

The structure of human calcitonin gene-related peptide 1 (hCGRP-1) has been determined by 1H NMR in a mixed-solvent system of 50% trifluoroethanol/50% H2O at pH 3.7 and 27 degrees C. Complete resonance assignment was achieved by using two-dimensional methods. Distance restraints for structure calculations were obtained by semiquantitative analysis of intra- and interresidue nuclear Overhauser effects; in addition, stereospecific or X1 rotamer assignments were obtained for certain side chains. Structures were generated from the distance restraints by distance geometry, followed by refinement using molecular dynamics, and were compared with experimental NH-C alpha H coupling constants and amide hydrogen exchange data. The structure of hCGRP-1 in this solvent comprises an amino-terminal disulfide-bonded loop (residues 2-7) leading into a well-defined alpha-helix between residues 8 and 18; thereafter, the structure is predominantly disordered, although there are indications of a preference for a turn-type conformation between residues 19 and 21. Comparison of spectra for the homologous hCGRP-2 with those of hCGRP-1 indicates that the conformations of these two forms are essentially identical.     

31P NMR spectra of oligodeoxyribonucleotide duplex lac operator-repressor headpiece complexes: importance of phosphate ester backbone flexibility in protein-DNA recognition.

The 31P NMR spectra of various 14-base-pair lac operators bound to both wild-type and mutant lac repressor headpiece proteins were analyzed to provide information on the backbone conformation in the complexes. The 31P NMR spectrum of a wild-type symmetrical operator, d(TGTGAGCGCTCACA)2, bound to the N-terminal 56-residue headpiece fragment of a Y7I mutant repressor was nearly identical to the spectrum of the same operator bound to the wild-type repressor headpiece. In contrast, the 31P NMR spectrum of the mutant operator, d(TATAGAGCGCTCATA)2, wild-type headpiece complex was significantly perturbed relative to the wild-type repressor-operator complex. The 31P chemical shifts of the phosphates of a second mutant operator, d(TGTGTGCGCACACA)2, showed small but specific changes upon complexation with either the wild-type or mutant headpiece. The 31P chemical shifts of the phosphates of a third mutant operator, d(TCTGAGCGCTCAGA)2, showed no perturbations upon addition of the wild-type headpiece. The 31P NMR results provide further evidence for predominant recognition of the 5'-strand of the 5'-TGTGA/3'-ACACT binding site in a 2:1 protein to headpiece complex. It is proposed that specific, strong-binding operator-protein complexes retain the inherent phosphate ester conformational flexibility of the operator itself, whereas the phosphate esters are conformationally restricted in the weak-binding operator-protein complexes. This retention of backbone torsional freedom in strong complexes is entropically favorable and provides a new (and speculative) mechanism for protein discrimination of different operator binding sites. It demonstrates the potential importance of phosphate geometry and flexibility on protein recognition and binding.     

Solution structure of regioselectively addressable functionalized templates: An NMR and restrained molecular dynamics investigation

Three cyclic peptides that are Regioselectively Addressable Functionalized Templates (RAFT) for use in protein de novo design have been investigated using a combination of nmr, restrained molecular dynamics, and CD spectroscopy. These peptides contain up to four selectively addressable sites (orthogonally protected lysine side chains) or have selectively addressable faces. The results show a common stable conformation for templates of this kind based on two type II β-turns and an associated β-sheet structure. A preferential orientation for the side chains is also demonstrated. The significance of these findings is discussed in the context of applications of RAFT that rely on their conformational rigidity and ability to present functionalities in a defined spatial arrangement. © 1996 John Wiley & Sons, Inc.     

Cholesterol dynamics in membranes of raft composition: a molecular point of view from 2H and 31P solid-state NMR

Lipidic membrane systems that have been reported to be composed of sphingomyelin (SM)-cholesterol (Chol) microdomains or "rafts" by Dietrich et al. [palmitoyloleoyl-phosphatidylcholine(POPC)/SM/Chol, 1/1/1; Dietrich, C., Bagatolli, L. A., Volovyk, Z. N., Thompson, N. L., Levi, M., Jacobson, K., and Gratton, E. (2001) Biophys. J. 80, 1417-1428] and by Schroeder et al. [SCRL: Liver-PC/Liver-phosphatidylethanolamine/SM/Cerebrosides/Chol, 1/1/1/1/2; Schroeder, R., London, E., and Brown, D. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 12130-12134] were investigated under the form of fully hydrated liposomes by the noninvasive solid-state (31)P and (2)H NMR method. Liposomes of binary lipid composition POPC/Chol and SM/Chol were also studied as boundary/control systems. All systems are found to be in the liquid-ordered phase (Lo) at physiological temperatures. Use of deuterium-labeled cholesterol afforded finding both the position of the sterol motional axis and its molecular order parameter. The axis of anisotropic rotation of cholesterol is such that the molecule is, on average, quasiperpendicular to the membrane plane, in all of the four systems investigated. Cholesterol order parameters greater than 0.8 are observed, indicating that the sterol is in a very motionally restricted environment in the temperature range 0-60 degrees C. The binary mixtures present "boundary" situations with the lowest values for POPC/Chol and the highest for SM/Chol. The SCRL raft mixture has the same ordering as the SM/Chol, i.e., the highest order parameter values over the temperature range. It demonstrates that in the SCRL mixture cholesterol dynamics is as in the binary system SM/Chol, therefore, suggesting that it might be depleted from the rest of the membrane to form complexes as if it were alone with SM. On the other hand, the mixture POPC/SM/Chol exhibits an intermediate ordering situation between those of SM/Chol and POPC/Chol. This strongly suggests that cholesterol could be in fast exchange, at the NMR time scale (milli- to microseconds), between two or more membrane regions of different dynamics and questions the statement of "rigid domains" made of SM and cholesterol in the model "raft" system POPC/SM/Chol.     

Structural study of DNA duplex containing an N-(2-deoxy-beta-D-erythro-pentofuranosyl) formamide frameshift by NMR and restrained molecular dynamics

The presence of an N-(2-deoxy-beta-D-erythro-pentofuranosyl) formamide (F) residue, a ring fragmentation product of thymine, in a frameshift context in the sequence 5'-d-(AGGACCACG)*d(CGTGGFTCCT) has been studied by 1H and 31P nuclear magnetic resonance (NMR) and molecular dynamics. Two-dimensional NMR studies show that the formamide residue, whether the cis or trans isomer, is rotated out of the helix and that the bases on either side of the formamide residue in the sequence, G14 and T16, are stacked over each other in a way similar to normal B-DNA. The cis and trans isomers were observed in the ratio 3:2 in solution. Information extracted from 31P NMR data reveal a modification of the phosphodiester backbone conformation at the extrahelical site, which is also observed during the molecular dynamics simulations.     

Solution structure of the orphan nuclear receptor rev-erb beta response element by 1H, 31P NMR and molecular simulation*

Rev-erb beta is an orphan receptor that binds as a homodimer or as a monomer to DNA. The solution structure of the non-palindromic 15 bp DNA duplex d(TAGAATGTAGGTCAG), the response element of Rev-erb beta for monomeric binding, was determined by 1H and 31P NMR, energy minimization with NMR-derived restraints for distances and NOE back-calculation methods. The refined final structures have the typical overall features of B-type DNA. However, titration of this 15 bp duplex with ReDBD, the DNA binding domain of Rev-erb beta, showed large shifts of imino protons and 31P signals, suggesting major conformational changes.     

31P NMR studies of the solution structure and dynamics of nucleosomes and DNA.

31P NMR studies of 140 base pair DNA fragments in nucleosomes and free in solution show no detectable change in the internucleotide 31P chemical shift or linewidth when DNA is packaged into nucleosomes. Measurements of 31P spin-lattice relaxation times T1 and 31P-[H] nuclear Overhauser enhancements revealed internal motion with a correlation time of about 4 x 10(-10) sec in double helical DNA, both free in solution and bound to nucleosomal core proteins. This result implies greater dynamic mobility in double helical DNA than has previously been supposed.     

Determination of the conformation of d(GGAAATTTCC)2 in solution by use of 1H NMR and restrained molecular dynamics

The conformation of the putative bent DNA d(GGAAATTTCC)2 in solution was studied by use of 1H NMR and restrained molecular dynamics. Most of the resonances were assigned sequentially. A total of 182 interproton distance restraints were determined from two-dimensional nuclear Overhauser effect spectra with short mixing times. Torsion angle restraints for each sugar moiety were determined by qualitative analysis of a two-dimensional correlated spectrum. Restrained molecular dynamics was carried out with the interproton distances and torsion angles incorporated into the total energy function of the system in the form of effective potential terms. As initial conformations for restrained molecular dynamics, classical A-DNA and B-DNA were adopted. The root mean square deviation (rmsd) between these two conformations is 5.5 A. The conformations obtained by use of restrained molecular dynamics are very similar to each other, the rmsd being 0.8 A. On the other hand, the conformations obtained by use of molecular dynamics without experimental restraints or restrained energy minimization depended heavily on the initial conformations, and convergence to a similar conformation was not attained. The conformation obtained by use of restrained molecular dynamics exhibits a few remarkable features. The second G residue takes on the BII conformation [Fratini, A. V., Kopka, M. L., Drew, H. R., & Dickerson, R. E. (1982) J. Biol. Chem. 257, 14686-14707] rather than the standard BI conformation. There is discontinuity of the sugar puckering between the eighth T and ninth C. The minor groove of the oligo(dA) tract is rather compressed. As a result, d(GGAAATTTCC)2 is bent.     

Conformation of an octapeptide fragment (2-9) of kaliocin-1 in DMSO-d6 by 1H NMR and restrained molecular dynamics

Kaliocin-1, a 31-residue synthetic peptide (FFSASCVPGADKGQFPNLCRLCA GTGENKCA), which has shown the antimicrobial activity forms the 152-182 fragment of human lactoferrin (HLf). As the octapeptide FSASCVPG forms the 2-9 fragment of kaliocin-1, in the present study, its conformation in dimethyl sulfoxide-d6 (DMSO-d6) has been determined using two-dimensional (2D) nuclear magnetic resonance (NMR) spectroscopy as well as restrained molecular dynamics. Sequence specific assignments of all the 1H resonances have been carried out using 2D correlation experiments (2D DQF-COSY, TOCSY and ROESY). In dimethyl sulfoxide-d6 at 25 degrees C, the octapeptide adopts a predominantly extended backbone conformation. The calculated structure resembles closely with the reported structure of the corresponding fragment of HLf. The peptide also has sequence and structural similarity with the corresponding fragments of lactoferrins from other organisms.     

Combined 1H and 31P NMR studies of cerebral metabolism in vivo

NMR spectroscopic methods have recently been developed for measurement of several concentrated cerebral metabolites in vivo. At present, 31P spectra from the brain permit detection of ATP, PCr, Pi, and certain sugar and lipid phosphates. The resonant frequency of Pi also provides a measure of cerebral pHi, and under some conditions ADP concentration can be calculated from information available in the 31P spectrum. The 1H spectrum of brain provides measurements of lactate, creatine, and several amino acids and choline-containing compounds. Both kinds of spectra can be obtained from the same subject. Our group at Yale used combined 31P and 1H methods to demonstrate that loss and recovery of phosphate energy stores and concomitant changes in cerebral amino acids during hypoglycemic coma in rodents could be observed in vivo. We then used the same methods to show that cerebral pHi can be normal while lactate is elevated in status epilepticus. NMR spectroscopy performed in vivo provides an array of chemically specific measurements unavailable by any other non-invasive method. It is thought to be entirely free of deleterious biological effects; hence, its potential for use in humans is considerable.     

Probing a CMP-Kdn synthetase by 1H, 31P, and STD NMR spectroscopy

CMP-Kdn synthetase catalyses the reaction of sialic acids (Sia) and cytidine-5'-triphosphate (CTP) to the corresponding activated sugar nucleotide CMP-Sia and pyrophosphate PP(i). STD NMR experiments of a recombinant nucleotide cytidine-5'-monophosphate-3-deoxy-d-glycero-d-galacto-nonulosonic acid synthetase (CMP-Kdn synthetase) were performed to map the binding epitope of the substrate CTP and the product CMP-Neu5Ac. The STD NMR analysis clearly shows that the anomeric proton of the ribose moiety of both investigated compounds is in close proximity to the protein surface and is likely to play a key role in the binding process. The relative rates of the enzyme reaction, derived from (1)H NMR signal integrals, show that Kdn is activated at a rate 2.5 and 3.1 faster than Neu5Ac and Neu5Gc, respectively. Furthermore, proton-decoupled (31)P NMR spectroscopy was successfully used to follow the enzyme reaction and clearly confirmed the appearance of CMP-Sia and the inorganic pyrophosphate by-product.     

Solution structure of phage lambda half-operator DNA by use of NMR, restrained molecular dynamics, and NOE-based refinement

The structure of a DNA decamer comprising the left half of the OR3 operator from bacteriophage lambda is determined in solution by using nuclear magnetic resonance spectroscopy and restrained molecular mechanics calculations. Nuclear magnetic resonance assignments for nonexchangeable protons are obtained by two-dimensional correlated and nuclear Overhauser effect (NOE) spectroscopies. Exchangeable proton resonances are assigned by one-dimensional NOE experiments. Coupling constant measurements from one- and two-dimensional experiments are used to determine approximate dihedral angles within the deoxyribose ring. Distances between protons are estimated by extrapolating distances derived from the time-dependent NOE intensities to initial mixing times. The sets of dihedral angles and distances form a basis for structure determination by restrained molecular dynamics. Separate runs start from classical A and from B DNA and converge to essentially identical structures (atomic root mean square difference of 0.8 A). The structures are improved by NOE-based refinement in which observed NOE intensities are compared to those calculated by using a full matrix analysis procedure. Final NOE residual (R) factors were less than 0.19. The resultant structures are generally B type in character, but display local sequence-dependent variations in dihedral angles and in the spatial arrangement of adjacent base pairs. Although the entire structure exhibits a small bend, the central core of the half-operator, which comprises the sequence-specific recognition site for cro repressor, is straight.     

Determination of solution conformations of PrP106-126, a neurotoxic fragment of prion protein, by 1H NMR and restrained molecular dynamics.

Experimental two-dimensional 1H NMR data have been obtained for PrP106-128 under the following solvent conditions: deionized water/2, 2,2-trifluoroethanol 50 : 50 (v/v) and dimethylsulfoxide. These data were analyzed by restrained molecular mechanics calculations to determine how changes in solvation affect the conformation of the peptide. In deionized water at pH 3.5, the peptide adopted a helical conformation in the hydrophobic region spanning residues Met112-Leu125, with the most populated helical region corresponding to the Ala115-Ala119 segment ( approximately 10%). In trifluoroethanol/H2O, the alpha-helix increased in population especially in the Gly119-Val122 tract ( approximately 25%). The conformation of this region was found to be remarkably sensitive to pH, as the Ala120-Gly124 tract shifted to an extended conformation at pH 7. In dimethylsulfoxide, the hydrophobic cluster adopted a prevalently extended conformation. For all tested solvents the region spanning residues Asn108-Met112 was present in a 'turn-like' conformation and included His111, situated just before the starting point of the alpha-helix. Rather than by conformational changes, the effect of His111 is exerted by changes in its hydrophobicity, triggering aggregation. The amphiphilic properties and the pH-dependent ionizable side-chain of His111 may thus be important for the modulation of the conformational mobility and heterogeneity of PrP106-126.     

Solution structure of human insulin-like growth factor 1: a nuclear magnetic resonance and restrained molecular dynamics study

The solution structure of human insulin-like growth factor 1 has been investigated with a combination of nuclear magnetic resonance and restrained molecular dynamics methods. The results show that the solution structure is similar to that of insulin, but minor differences exist. The regions homologous to insulin are well-defined, while the remainder of the molecule exhibits greater disorder. The resultant structures have been used to visualize the sites for interaction with a number of physiologically important proteins.