Selective detection of rapid motions in spectrin by NMR

Human erythrocyte spectrin molecules exhibit relatively sharp (30-50 Hz) proton NMR signals in the aliphatic region. A standard solvent presaturation pulse sequence that also partially suppresses the broad envelope from protons with rigid structures in spectrin and selectively enhances the sharp resonances has been used to characterize the behavior of these resonances. The overall resonance pattern strongly resembles that of the denatured spectrin. The observed spectra are also quite similar to the line-broadened spectrum from a mixture of amino acids that corresponds to the composition of the spectrin molecule. These data indicate the existence of regions exhibiting rapid internal motions within the intact spectrin molecule, and suggest that the amino acid composition of the residues giving rise to the sharp resonances is quite similar to that of the full spectrin molecule.     

Dynamic structure of subtilisin-eglin c complex studied by normal mode analysis

Normal mode analysis of subtilisin-eglin c complex was performed to investigate the dynamics at the interface between the enzyme and the inhibitor. The internal motions of the complex calculated from the normal modes were divided into three parts: the internal motions changing the shape of each molecule, the external rigid-body motions changing their mutual dispositions, and the coupling between the internal and external motions. From the results of the analysis, the following characteristic features were found in the dynamics at the interface regions: 1) negative correlation between the internal and external motions within each molecule, and 2) positive correlation between the external motions of the two molecules. The former decreases the apparent amplitudes of motions at the interface. The latter minimizes the interference between individual motions of the two molecules. These dynamic characteristics allow the enzyme and the inhibitor to move as freely as possible. This finding suggests that the experimental evidence of the large entropy gain on binding should be attributed not only to strong hydrophobic interactions, but also to the dynamic structure of the complex, which is found to minimize an unavoidable loss of the conformational entropy on binding. Proteins 32:324–333, 1998. © 1998 Wiley-Liss, Inc.     

1H- and 2H-NMR study of bovine serum albumin solutions

Frozen, native and denatured bovine serum albumin solutions have been studied with a wide-band NMR pulse spectrometer. Both macromolecular and water protons spin-spin and spin-lattice relaxation times--t2m, t1m, t2w, t1w--have been measured between 170 and 360 K. In the native sample, the t2m process is the tumbling rate of the bovine serum albumin molecules. It gives to the spin-lattice relaxation an omega 0(-2) frequency dependence at room temperature in the studied frequency range, 6-90 MHz. An additional process contributes to t1m-1; it arises from internal backbone or segmental motions and provides a lower frequency behaviour. On denaturation, bovine serum albumin molecules lose their tumbling motion and form a rigid network, while internal backbone motions seem unaffected. Calorimetric Cp measurement confirms the occurrence of a phase transition upon denaturation. 1H and 2H spin-lattice relaxation times of water protons depend mainly on bound water mobility. 1H and 2H t2w depend also on the tertiary structure of bovine serum albumin and on its mobility, because of a fast exchange process between water and some protein protons (or deutons), while a cross-relaxation process between protein and water protons contributes to 1H t1w. Denaturation has no influence on bound water motional properties and bound water population.     

Measurements of water proton NMR spin-lattice relaxation time in the rotating frame (T1p) for studying motions in solutions of giant macro-molecules and supramolecular particles (T2 virus)

Spin-spin relaxation time (T2), spin-lattice relaxation time (T1), and spin-lattice relaxation time in the rotating frame (T1p) of water protons in solutions of bacteriophage T2 were studied by pulsed nuclear magnetic resonance. The frequency dependence of the measurements exhibits a dispersion implying existence of a fraction of water molecules in solution with a correlation time distribution centered at approximately 10(-5) sec which is strongly influenced by the reorientational motions of virus particles. Experiments were carried out with two forms of bacteriophage T2 existing at pH 5.4 and 7.8 respectively. The different structures of the virus at these two pH values are reflected in the NMR relaxation behavior of water protons.     

Extending the NMR spatial resolution limit for RNA by motional couplings

Experimental resolution of distinct dynamical processes in molecules can prove impossible when they are correlated to one another. In nuclear magnetic resonance (NMR) spectroscopy, couplings between internal and overall motions lead to intractable complexity, depriving insights into functionally important motions. Here we demonstrate that motional couplings can be used to anchor NMR frames of reference onto different parts of an RNA molecule, thus extending the spatial resolution limit for dynamical characterization.     

Spectral densities of nitrogen nuclei in Escherichia coli ribonuclease HI obtained by 15N NMR relaxation and molecular dynamics

Summary Spectral densities of the ¹⁵N amide in Escherichia coli ribonuclease HI, obtained from NMR relaxation experiments, were compared with those calculated using a molecular dynamics (MD) simulation. All calculations and comparisons assumed that the auto-correlation function describing the internal motions of the molecule was independent of the auto-correlation function associated with overall rotational diffusion. Comparisons were limited to those residues for which the auto-correlation function of internal motions rapidly relaxed and reached a steady state within 205 ps. The results show the importance of frequency components as well as amplitudes of internal motions in order to obtain a meaningful comparison of MD simulations with NMR data.     

Conserved nucleotides in an RNA essential for hepatitis B virus replication show distinct mobility patterns

The number of regulatory RNAs with identified non-canonical structures is increasing, and structural transitions often play a role in their biological function. This stimulates interest in internal motions of RNA, which can underlie structural transitions. Heteronuclear NMR relaxation measurements, which are commonly used to study internal motion, only report on local motions of few sites within the molecule. Here we have studied a 27-nt segment of the human hepatitis B virus (HBV) pregenomic RNA, which is essential for viral replication. We combined heteronuclear relaxation with the new off-resonance ROESY technique, which reports on internal motions of H,H contacts. Using off-resonance ROESY, we could for the first time detect motion of through-space H,H contacts, such as in intra-residue base-ribose contacts or inter-nucleotide contacts, both essential for NMR structure determination. Motions in non-canonical structure elements were found primarily on the sub-nanosecond timescale. Different patterns of mobility were observed among several mobile nucleotides. The most mobile nucleotides are highly conserved among different HBV strains, suggesting that their mobility patterns may be necessary for the RNA’s biological function.     

NMR studies of dynamics in RNA and DNA by 13C relaxation

RNA and DNA molecules experience motions on a wide range of time scales, ranging from rapid localized motions to much slower collective motions of entire helical domains. The many functions of RNA in biology very often require this molecule to change its conformation in response to biological signals in the form of small molecules, proteins or other nucleic acids, whereas local motions in DNA may facilitate protein recognition and allow enzymes acting on DNA to access functional groups on the bases that would otherwise be buried in Watson-Crick base pairs. Although these statements make a compelling case to study the sequence dependent dynamics in nucleic acids, there are few residue-specific studies of nucleic acid dynamics. Fortunately, NMR studies of dynamics of nucleic acids and nucleic acids-protein complexes are gaining increased attention. The aim of this review is to provide an update of the recent progress in studies of nucleic acid dynamics by NMR based on the application of solution relaxation techniques.     

Proton magnetic resonance studies of lipid bilayer membranes Experimental determination of inter- and intramolecular nuclear relaxation rates in sonicated phosphatidylcholine bilayer vesicles

We have determined the relative magnitudes of the intra- and intermolecular contributions to the nuclear magnetic relaxation rates of the methylene protons of the hydrocarbon chains in phosphatidylcholine bilayer vesicles over a range of temperatures and at two NMR frequencies (100 and 220 MHz). These measurements have been made by the isotopic dilution method using deuterated phosphatidylcholines containing fully deuterated hydrocarbon chains. The results showed that both the methylene linewidths and the spin-lattice relaxation rates are dominated by intramolecular dipolar interactions. Both the intra- and intermolecular contributions to the spin-lattice relaxation rate were found to decrease with increasing temperature and to exhibit a frequency dependence, the rates being higher at the lower NMR frequency in both cases. These observations indicate that both intra- and intermolecular dipolar interactions are modulated by anisotropic motions. In the case of the intermolecular dipolar fields, it is proposed that they are modulated both by the rapid rotational isomerization of the hydrocarbon chains as well as by lateral diffusion of the lipid molecules. That the hydrocarbon chain motion must be fairly effective in effecting efficient spin-lattice relaxation is evident from the negligible intramolecular interchain contribution to the relaxation found in the present work.     

An NMR study of d(CTACTGCTTTAG).d(CTAAAGCAGTAG) showing hydration water molecules in the minor groove of a TpA step

The hydration properties of the non-palindromic duplex d(CTACTGCTTTAG). d(CTAAAGCAGTAG) were investigated by NMR spectroscopy. The oligonucleotide possesses a heterogeneous B-DNA structure. The H2(n)-H1'(m+1) distances reflect a minor groove narrowing within the TTT/AAA segment (approximately 3.9A) and a sudden widening at the T10:A15 base-pair (approximately 5.3A), the standard B-DNA distance being approximately 5A. The facing T10pA11 and T14pA15 steps at the end of the TTTA/AAAT segment have completely different behaviors. Only A15 ending the AAA run displays NMR features comparable to those shown by adenines of TpA steps occupying the central position of TnAn (n> or =2) segments. These involve particular chemical shifts and line broadening of the H2 and H8 protons. Positive NOESY cross-peaks were measured between the water protons and the H2 protons of A15, A16 and A17 reflecting the occurrence of hydration water molecules with residence times longer than 500 picoseconds along the minor groove of the TTT/AAA segment. In contrast no water molecules with long residence times were observed neither for A3, A20 and A23 nor for A11 ending the 5'TTTA run. We confirm thus that the binding of water molecules with long residence time to adenine residues correlates with the minor groove narrowing. In contrast, the widening of the minor groove at the A11:T14 base-pair ending the TTTA/TAAA segment, likely associated to a high negative propeller twist value at this base-pair, prevents the binding of a water molecule with long residence time to A11 but not to A15 of the preceding T10:A15 base-pair. Thus, in our non-palindromic oligonucleotide the water molecules bind differently to A11 and A15 although both adenines are part of a TpA step. The slower motions occurring at A15 compared to A11 are also well explained by the present results.     

Internal motions in B- and Z-form poly(dG-dC).poly(dG-dC): 1H NMR relaxation studies

Proton NMR relaxation measurements are used to compare the molecular dynamics of 60 base pair duplexes of B- and Z-form poly(dG-dC).poly(dG-dC). The relaxation rates of the exchangeable guanine imino protons (Gim) in H2O and in 90% D2O show that below 20 degrees C spin-lattice relaxation is exclusively from proton-proton magnetic dipolar interactions while proton-nitrogen interactions contribute about 30% to the spin-spin relaxation. The observation that the spin-lattice relaxation is nonexponential and that the initial spin-lattice relaxation rate of the Gim, G-H8 and C-H6 protons depends on the selectivity of the exciting pulse shows that spin-diffusion dominates the spin-lattice relaxation. The relaxation rates of the Gim, C-H5, and C-H6 in B- and Z-form poly(dG-dC).poly(dG-dC) cannot be explained by assuming the DNA behaves as a rigid rod. The data can be fit by assuming large-amplitude out of plane motions (+/- 30-40 degrees, tau = 1-100 ns) and fast, large-amplitude local torsional motions (+/- 25-90 degrees, tau = 0.1-1.5 ns) in addition to collective torsional motions. The results for the B and Z forms show that the rapid internal motions are similar and large in both conformations although backbone motions are slightly slower, or of lower amplitude, in Z DNA. At high temperatures (greater than 60 degrees C), imino proton exchange with solvent dominates the spin-lattice relaxation of B-form poly(dG-dC).poly(dG-dC), but in the Z form no exchange contribution (less than 2 s-1) is observed at temperatures as high as 85 degrees C. Conformational fluctuations that expose the imino protons to the solvent are strikingly different in the B and Z forms. The results obtained here are compared with those previously reported for poly(dA-dT).poly(dA-dT).     

Interaction between the subunits of human erythrocyte spectrin using a fluorescence probe

Fluorescence labeling of spectrin subunits was performed with N-(1-anilinonaphthyl-4)maleimide (ANM) to study the interaction between alpha and beta subunits. The fluorescence anisotropy of both ANM alpha and ANM beta increased linearly with the addition of nonfluorescent beta or alpha subunit, and saturated at a protein ratio about 1, indicating that 1 mol alpha subunit binds to 1 mol beta subunit with high affinity in vitro. Furthermore, this binding seemed to be reversible, because the anisotropy value decreased when an excess fo nonfluorescent alpha was added to the ANM alpha/beta mixture. The anisotropy of ANM alpha attained a maximum level within l min after addition of the same quantity of nonfluorescent beta at 12 degrees C, and the anisotropy of this mixture decreased rapidly when an excess of nonfluorescent alpha was added. These findings suggested that both the binding process of beta to ANM alpha and the dissociation step of ANM alpha from the ANM alpha-beta complex were quite rapid. The results obtained here imply that dynamic interaction between alpha and beta subunits of spectrin should be taken into account in understanding the role of the spectrin molecule in the cytoskeletal mesh.     

Sequence-specific interaction of Hoechst 33258 with the minor groove of an adenine-tract DNA duplex studied in solution by 1H NMR spectroscopy.

The interaction of Hoechst 33258 with the minor groove of the adenine-tract DNA duplex d(CTTTTGCAAAAG)2 has been studied in both D2O and H2O solutions by 1D and 2D 1H NMR spectroscopy. Thirty-one nuclear Overhauser effects between drug and nucleotide protons within the minor groove of the duplex, together with ring-current induced perturbations to the chemical shifts of basepair and deoxyribose protons, define the position and orientation of the bound dye molecules. Two drug molecules bind cooperatively and in symmetry related orientations at the centre of the 5'-TTTT and 5'-AAAA sequences with the binding interactions spanning only the four A-T basepairs. The positively charged N-methylpiperazine moieties point towards the centre of the duplex while the phenol groups are disposed towards the 3'-ends of the sequence. Resonance averaging is apparent for both the D2/D6 and D3/D5 phenol protons and D2"'/D6"' and D3"'/D5"' of the N-methylpiperazine ring and is consistent with these groups being involved in rapid rotation or ring-flipping motions in the bound state. Interstrand NOEs between adenine H2s and deoxyribose H1' are consistent with a high degree of propeller twisting of the A-T basepairs at the binding site of the aromatic benzimidazole and phenol rings of Hoechst. The data imply that the minor groove is particularly narrow with many contacts between the complementary curved surfaces of the drug and DNA indicating that strong van der Waals interactions, involving the floor and the walls of the minor groove, stabilize the complex. In our model the NH groups of the benzimidazole rings are positioned to make a pair of bifurcated hydrogen bonds with the adenine N3 and thymine O2 on the floor of the minor groove.     

A carbon-13 nuclear magnetic resonance spectroscopic study of inter-proton pair order parameters: a new approach to study order and dynamics in phospholipid membrane systems.

We report a simple new nuclear magnetic resonance (NMR) spectroscopic method to investigate order and dynamics in phospholipids in which inter-proton pair order parameters are derived by using high resolution 13C cross-polarization/magic angle spinning (CP/MAS) NMR combined with 1H dipolar echo preparation. The resulting two-dimensional NMR spectra permit determination of the motionally averaged interpair second moment for protons attached to each resolved 13C site, from which the corresponding interpair order parameters can be deducted. A spin-lock mixing pulse before cross-polarization enables the detection of spin diffusion amongst the different regions of the lipid molecules. The method was applied to a variety of model membrane systems, including 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/sterol and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/sterol model membranes. The results agree well with previous studies using specifically deuterium labeled or predeuterated phospholipid molecules. It was also found that efficient spin diffusion takes place within the phospholipid acyl chains, and between the glycerol backbone and choline headgroup of these molecules. The experiment was also applied to biosynthetically 13C-labeled ergosterol incorporated into phosphatidylcholine bilayers. These results indicate highly restricted motions of both the sterol nucleus and the aliphatic side chain, and efficient spin exchange between these structurally dissimilar regions of the sterol molecule. Finally, studies were carried out in the lamellar liquid crystalline (L alpha) and inverted hexagonal (HII) phases of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). These results indicated that phosphatidylethanolamine lamellar phases are more ordered than the equivalent phases of phosphatidylcholines. In the HII (inverted hexagonal) phase, despite the increased translational freedom, there is highly constrained packing of the lipid molecules, particularly in the acyl chain region.     

Molecular dynamics study of a hyperthermophilic and a mesophilic rubredoxin

In recent years, increased interest in the origin of protein thermal stability has gained attention both for its possible role in understanding the forces governing the folding of a protein and for the design of new highly stable engineered biocatalysts. To study the origin of thermostability, we have performed molecular dynamics simulations of two rubredoxins, from the mesophile Clostridium pasteurianum and from the hyperthermophile Pyrococcus furiosus. The simulations were carried out at two temperatures, 300 and 373 K, for each molecule. The length of the simulations was within the range of 6-7.2 ns. The rubredoxin from the hyperthermophilic organism was more flexible than its mesophilic counterpart at both temperatures; however, the overall flexibility of both molecules at their optimal growth temperature was the same, despite 59% sequence homology. The conformational space sampled by both molecules was larger at 300 K than at 373 K. The essential dynamics analysis showed that the principal overall motions of the two molecules are significantly different. On the contrary, each molecule showed similar directions of motion at both temperatures.     

Clustering of the neural cell adhesion molecule (NCAM) at the neuronal cell surface induces caspase-8- and -3-dependent changes of the spectrin meshwork required for NCAM-mediated neurite outgrowth

Changes in neuronal morphology underlying neuronal differentiation depend on rapid and sustained cytoskeleton rearrangements in the growing neurites. Whereas cell adhesion molecules are well established as regulators of neuronal differentiation, less is known about the signaling mechanisms by which they influence the cytoskeleton. Here we show that the neural cell adhesion molecule (NCAM) associates with the active form of caspase-8 and that clustering of NCAM at the neuronal cell surface leads to activation of caspase-8 and -3 followed by the cleavage of the sub-membranous brain spectrin meshwork, but not of the actin or tubulin cytoskeleton. Inhibitors of caspase-8 and -3 specifically block the NCAM-dependent spectrin cleavage and abolish NCAM-dependent neurite outgrowth. NCAM-dependent rearrangements of the membrane associated spectrin meshwork via caspase-8 dependent caspase-3 activation are thus indispensable for NCAM-mediated neurite outgrowth.     

Rotational dynamics of erythrocyte spectrin

The rotational diffusion of erythrocyte spectrin has been measured using time-resolved phosphorescence anisotropy. The anisotropy of the spectrin dimer decays to zero with a time constant of 3 microseconds at 21 degrees C. The results are compared with the correlation times predicted for the anisotropy decay of an equivalent sphere and rigid rod. The data indicate that the ribbon-like spectrin molecule possesses considerable torsional and segmental flexibility. These motions are restricted, but not abolished, when spectrin is reconstituted into cross-linked cytoskeletal protein networks, or bound to spectrin-actin depleted erythrocyte membrane vesicles.     

Backbone dynamics of the oligomerization domain of p53 determined from 15N NMR relaxation measurements.

The backbone dynamics of the tetrameric p53 oligomerization domain (residues 319-360) have been investigated by two-dimensional inverse detected heteronuclear 1H-15N NMR spectroscopy at 500 and 600 MHz. 15N T1, T2, and heteronuclear NOEs were measured for 39 of 40 non-proline backbone NH vectors at both field strengths. The overall correlation time for the tetramer, calculated from the T1/T2 ratios, was found to be 14.8 ns at 35 degrees C. The correlation times and amplitudes of the internal motions were extracted from the relaxation data using the model-free formalism (Lipari G, Szabo A, 1982, J Am Chem Soc 104:4546-4559). The internal dynamics of the structural core of the p53 oligomerization domain are uniform and fairly rigid, with residues 327-354 exhibiting an average generalized order parameter (S2) of 0.88 +/- 0.08. The N- and C-termini exhibit substantial mobility and are unstructured in the solution structure of p53. Residues located at the N- and C-termini, in the beta-sheet, in the turn between the alpha-helix and beta-sheet, and at the C-terminal end of the alpha-helix display two distinct internal motions that are faster than the overall correlation time. Fast internal motions (< or = 20 ps) are within the extreme narrowing limit and are of uniform amplitude. The slower motions (0.6-2.2 ns) are outside the extreme narrowing limit and vary in amplitude.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure, calmodulin-binding, and calcium-binding properties of recombinant alpha spectrin polypeptides

We examined the structure and the distribution of binding activities within bacterially produced fragments of Drosophila alpha spectrin. By electron microscopy, purified spectrin fragments resembled the corresponding regions of native spectrin. The contour lengths of recombinant spectrin molecules were proportional to the length of their coding sequences, which is consistent with current models of spectrin structure in which individual segments of the polypeptide contribute independently to the structure of the native molecule. We localized two sites at which calcium may regulate spectrin function. First, a site responsible for calmodulin binding to Drosophila alpha spectrin was identified near the junction of repetitive segments 14 and 15. Second, a domain of Drosophila alpha spectrin that includes two EF hand calcium-binding sequences bound 45Ca in blot overlay assays. EF hand sequences from a homologous domain of Drosophila alpha actinin did not bind calcium under the same conditions.     

Effect of pressure on the tertiary structure and dynamics of folded basic pancreatic trypsin inhibitor

The on-line high-pressure cell NMR technique was used to study pressure-induced changes in the tertiary structure and dynamics of a globular protein, basic pancreatic trypsin inhibitor (BPTI). Practically all the proton signals of BPTI were observed with (1)H two-dimensional NMR spectroscopy at 750 MHz at variable pressure between 1 and 2000 bar. Chemical shifts, nuclear Overhauser effect (NOE), and line shapes were used to analyze conformational and dynamic changes of the protein as functions of pressure. Linear, reversible, but nonuniform pressure-induced chemical shift changes of practically all the C(alpha) protons and side chain protons showed that the entire secondary and tertiary structures are altered by pressure within the folded ensemble of BPTI. The high field shift tendency of most side chain proton signals and the increase in NOE intensities of some specific side chain protons indicated a site-specific compaction of the tertiary structure. Pressure dependence of ring flip rates was deduced from resonance line shapes of the slices of the two-dimensional NMR spectrum for ring proton signals of Tyr-35 and Phe-45. The rates of the flip-flop motions were considerably reduced at high pressure, from which activation volumes were determined to be 85 +/- 20 A(3) (or 51.2 ml/mol) and 46 +/- 9 A(3) (or 27.7 ml/mol) for Tyr-35 and Phe-45, respectively, at 57 degrees C. The present experiments confirm that pressure affects the entire secondary and tertiary structures of a globular protein with specific compaction of a core, leading to quite significant changes in slow internal dynamics of a globular protein.