Accuracy of bound peptide structures determined by exchange transferred nuclear Overhauser data: A simulation study

The exchange-transferred NOE method to determine the three-dimensional structure of peptides bound to proteins, or other macromolecular systems, is becoming increasingly important in drug design efforts and for large or multicomponent assemblies, such as membrane receptors, where structural analysis of the full system is intractable. The exchange-transferred nuclear Overhauser effect spectroscopy (etNOESY) method allows the determination of the bound-state conformation of the peptide from the intra-molecular NOE interactions between ligand protons. Because only ligand–ligand NOEs are generally observable, the etNOESY method is restricted to fewer NOEs per residue than direct protein structure determination. In addition, the averaging of relaxation rates between free and bound states affects the measured cross-peak intensities, and possibly the accuracy of distance estimates. Accordingly, the study reported here was conducted to examine the conditions required to define a reliable structure. The program CORONA was used to simulate etNOE data using a rate-matrix including magnetic relaxation and exchange rates for two peptide–protein complexes derived from the reference complex of cAMP-dependent protein kinase ligated with a 24-residue inhibitor peptide. The results indicate that reasonably accurate peptide structures can be determined with relatively few NOE interactions when the interactions occur between non-neighboring residues. The reliability of the structural result is suggested from the pattern of NOE interactions. A structure with an accuracy of approximately 1.3 Å rms difference for the main-chain atoms can be obtained when etNOE interactions between non-neighboring residues occur over the length of the peptide. The global precision is higher (approximately 0.9 Å rms difference) but is not correlated to global accuracy. A local definition of precision along the backbone appears to be a good indicator of the local accuracy.     

Application of long-range order to predict unfolding rates of two-state proteins

Predicting the experimental unfolding rates of two-state proteins and models describing the unfolding rates of these proteins is quite limited because of the complexity present in the unfolding mechanism and the lack of experimental unfolding data compared with folding data. In this work, 25 two-state proteins characterized by Maxwell et al. (Protein Sci 2005;14:602–616) using a consensus set of experimental conditions were taken, and the parameter long-range order (LRO) derived from their three-dimensional structures were related with their experimental unfolding rates ln(k(u)). From the total data set of 30 proteins used by Maxwell et al. (Protein Sci 2005;14:602–616), five slow-unfolding proteins with very low unfolding rates were considered to be outliers and were not included in our data set. Except all beta structural class, LRO of both the all-alpha and mixed-class proteins showed a strong inverse correlation of r = -0.99 and -0.88, respectively, with experimental ln(k(u)). LRO shows a correlation of -0.62 with experimental ln(k(u)) for all-beta proteins. For predicting the unfolding rates, a simple statistical method has been used and linear regression equations were developed for individual structural classes of proteins using LRO, and the results obtained showed a better agreement with experimental results.     

On the conformation of luteinizing hormone-releasing hormone, nuclear Overhauser observations.

Observations of proton nuclear Overhauser effects in the molecule Luteinizing Hormone-Releasing Hormone indicate that a high population of a particular set of conformers exists in water solution. The results can be interpreted as two distinct conformers in which the pGlu¹ ring is in close proximity to aromatic residues further along the sequence of the linear structure. The observed nuclear Overhauser effects were in agreement with the enhancements calculated from models obtained by conformational energy calculations.     

Identification of localized redox states in plant-type two-iron ferredoxins using the nuclear Overhauser effect

The homonuclear Overhauser effect (NOE), in conjunction with nonselective spin-lattice relaxation measurements, has been employed to assign the contact-shifted resonances for the reduced form of two typical plant-type two-iron ferredoxins from the algae Spirulina platensis and Porphyra umbilicalis. These results demonstrate that the NOE should have broad general applicability for the assignments and electronic structural elucidation of diverse subclasses of paramagnetic iron-sulfur cluster proteins. NOE connectivities were detected only among sets of resonance exhibiting characteristically different deviations from Curie behavior, providing strong support for the applicability of the spin Hamiltonian formulation for the NMR properties of the antiferromagnetically coupled iron clusters [Dunham, W. R., Palmer, G., Sands, R. H., & Bearden, A. J. (1971) Biochim. Biophys. Acta 253, 373-384; Banci, L., Bertini, I., & Luchinat, C. (1989) Struct. Bonding (in press)]. The geminal beta-methylene protons for the two cysteines bound to the iron(II) center were clearly identified, as well as the C alpha H and one C beta H for each of the cysteines bound to the iron(III). The identification of the iron bound to cysteines 41 and 46 as the iron(II) in the reduced protein was effected on the basis of dipolar contacts between the bound cysteines, as predicted by crystal coordinates of S. platensis Fd [Tsukihara, T., Fukuyama, K., Nakamura, M., Katsube, Y., Tanaka, N., Kakudo, M., Wada, K., Hase, T., & Matsubara, H. (1981) J. Biochem. (Tokyo) 90, 1763-1773].(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective 31P(1H) nuclear Overhauser effect study on the polar headgroup conformation of phospholipids in micelles in organic solvents

The polar headgroup structure of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS) in inverted micelles in chloroform or benzene was investigated by the selective 31P(H) nuclear Overhauser effect (NOE). In the frequency dependence of the 31P(1H) NOE, PC micelles in CDCl3 showed two maxima. The larger maximum was located at the resonance of the glycerol-CH2OP protons and the smaller at the resonance of the N-methyl protons. In PC/PE mixed micelles in C6D6, both PC and PE showed three maxima which were located at the resonance of the CH2OP protons, the N-methyl protons and the amino protons in the frequency dependence of the 31P-NOE. The N-methyl protons of PC and the amino protons of PE were closely spaced to the phosphate groups of neighboring lipid molecules. The polar headgroups of PC and PE in the mixed micelles were concluded to lie in the plane perpendicular to the molecular axes. The frequency dependence of the 31P(H) NOE for PS micelles in C6D6 showed the maxima at the resonances of the amino protons and the CH2OP protons. The polar headgroups of PS molecules were not extended parallel to the molecular axes in the inverted micelles.     

Local and long-range structural effects caused by the removal of the N-terminal polypeptide fragment from immunoglobulin L chain lambda

The role of the N-terminal polypeptide fragment of the immunoglobulin l-chain in V domain packing stability, and the flexibility of the whole chain was approached by molecular dynamics simulation. The observations were supported by experimental analysis. The N-terminal polypeptide fragment appeared to be the low-stability packing element in the V domain. At moderately elevated temperature it may be replaced at its packing locus by Congo red and then removed by proteolysis. After removal of Congo red by adsorption to (diethylamino)ethyl (DEAE) cellulose, the stability of complete L chain and of L chain devoid of the N-terminal polypeptide fragment were compared. The results indicated that the N-terminal polypeptide fragment plays an essential role in the stability of the V domain. Its removal makes the domain accessible for ANS and Congo red dye binding without heating. The decreased domain stability was registered in particular as increased root mean square (RMS) fluctuation and higher susceptibility to proteolytic attack. The long-range effect was most clearly manifested at 340 K as independent V and C domain fluctuation in the l-chain devoid of the N-terminal polypeptide fragment. This is likely due to the lack of direct connections between the N- and C-termini of the V domain polypeptide. In a complete V domain the connection involves residues 8-12 and 106-110 in particular. Partial or complete disruption of this connection increases the freedom of V domain rotation, while its increased cohesion strengthens the coupling of the V and C domains, making the whole L chain less flexible.     

Solution conformation of the biantennary N-linked oligosaccharide of human serotransferrin using H NMR nuclear Overhauser effect measurements

The conformation in solution of the biantennary complex type oligosaccharide unit derived from human serotransferrin has been investigated using ¹H—¹H Nuclear Overhauser Effect (NOE) measurements at 300 MHz. From quantitation of the NOE, the (1–3) antenna is shown to exist in a preferred solution conformation with respect to the mannosyl-chitobiose core. The flexibility of the (1–6) arm, together with the absence of NOE data between this arm and the core, indicates that, in contrast to the (1–3) arm the (1–6) arm has no preferred conformation with respect to the core.     

Automated error-tolerant macromolecular structure determination from multidimensional nuclear Overhauser enhancement spectra and chemical shift assignments: improved robustness and performance of the PASD algorithm

We report substantial improvements to the previously introduced automated NOE assignment and structure determination protocol known as PASD (Kuszewski et al. (2004) J Am Chem Soc 26:6258-6273). The improved protocol includes extensive analysis of input spectral data to create a low-resolution contact map of residues expected to be close in space. This map is used to obtain reasonable initial guesses of NOE assignment likelihoods which are refined during subsequent structure calculations. Information in the contact map about which residues are predicted to not be close in space is applied via conservative repulsive distance restraints which are used in early phases of the structure calculations. In comparison with the previous protocol, the new protocol requires significantly less computation time. We show results of running the new PASD protocol on six proteins and demonstrate that useful assignment and structural information is extracted on proteins of more than 220 residues. We show that useful assignment information can be obtained even in the case in which a unique structure cannot be determined.     

Conformation of NAD+ bound to yeast and horse liver alcohol dehydrogenase in solution. The use of the proton-proton transferred nuclear Overhauser enhancement

Changes in reduced viscosity of nuclear lysates from rat liver cells have been studied, in conditions of very low shear stress by the use of an oscillating viscometer, as a function of incubation time in alkaline (pH 12.5) and neutral (pH 8.0) solutions. In non-denaturing conditions, nuclear DNA showed a stepwise, time-dependent increase of reduced viscosity, which suggests that it behaves as a single hydrodynamic unit that progressively changes its radius and viscoelastic properties because of a very slow unfolding, through discrete successive transitions, from a highly superpacked structure toward a linear relaxed B-form fiber. Experimental conditions shown to reduce chromatin-DNA superpacking without changing DNA length (e.g. G₁ cycling versus G₀ non-cycling liver cells, or young versus old rat liver cells) dramatically increased the initial value of reduced viscosity and its time-dependent increment. Conversely, in denaturing conditions, reduced viscosity increased in the initial phase (probably because DNA unfolding prevails on DNA unwinding), then exhibited a plateau level (when unfolding balances unwinding), and subsequently decreased progressively to the value of sheared DNA (when unwinding becomes more rapid due to the progressive breakage of phosphodiester bridges in alkali). Experimental conditions known to induce DNA single- or double-strand breaks (i.e. the use of liver cells from rats treated with dimethylnitrosamine or 2-acetylaminofluorene, or of liver cells exposed to X-rays) caused in both neutral and alkaline solution an increment in the initial reduced viscosity and in the slope of its time-dependent increase, which may be related to a reduction of chromatin-DNA superpacking. Moreover, it became evident in denaturing conditions that a decrease of the maximum viscosity and of the time taken to reach it both related to a reduced DNA length. These viscoelastic properties are constantly correlated with independent DNA structural measurements on the same nuclear lysates, to discriminate the effect due to mere aggregation and disaggregation.     

Polypeptide secondary structure determination by nuclear magnetic resonance observation of short proton-proton distances

The use of proton-proton nuclear Overhauser enhancement (NOE) distance information for identification of polypeptide secondary structures in non-crystalline proteins was investigated by stereochemical studies of standard secondary structures and by statistical analyses of the secondary structures in the crystal conformations of a group of globular proteins. Both regular helix and beta-sheet secondary structures were found to contain a dense network of short 1H-1H distances. The results obtained imply that the combined information on all these distances obtained from visual inspection of the two-dimensional NOE (NOESY) spectra is sufficient for determination of the helical and beta-sheet secondary structures in small globular proteins. Furthermore, cis peptide bonds can be identified from unique, short sequential proton-proton distances. Limitations of this empirical approach are that the exact start or end of a helix may be difficult to define when the adjoining residues form a tight turn, and that unambiguous identification of tight turns can usually be obtained only in the hairpins of antiparallel beta-structures. The short distances between protons in pentapeptide segments of the different secondary structures have been tabulated to provide a generally applicable guide for the analysis of NOESY spectra of proteins.     

一种百合甾体皂苷纯度及糖链结构核磁共振法分析

采用多种NMR分析技术,首次对百合甾体皂苷(25R,26R)-26-甲氧基螺甾烷-5-烯-3β-α-L-鼠李糖-(1→2)-[β-D-葡萄糖-(1→6)]-β-D-葡萄糖苷的1H和13C NMR信号进行了全归属,特别是应用选择性的1D TOCSY和1D NOESY核磁共振分析技术,对该化合物1中的氢谱信号严重重叠的糖链进行了详细的分析,提出了一套对甾体皂苷糖链信号进行全归属的核磁共振法.在确认其结构的基础上,建立了核磁共振法(1H NMR)测定该化合物1的纯度,给出了完整的实验条件,线性回归系数为0.9998,重复性实验RSD为0.58％,稳定性实验RSD为0.24％,操作简单、快速准确,且不需要其它对照品,是中药化学对照品纯度研究的一个有益补充.     

The active site and substrates binding mode of malonyl-CoA synthetase determined by transferred nuclear Overhauser effect spectroscopy, site-directed mutagenesis, and comparative modeling studies

The active sites and substrate bindings of Rhizobium trifolii molonyl-CoA synthetase (MCS) catalyzing the malonyl-CoA formation from malonate and CoA have been determined based on NMR spectroscopy, site-directed mutagenesis, and comparative modeling methods. The MCS-bound conformation of malonyl-CoA was determined from two-dimensional-transferred nuclear Overhauser effect spectroscopy data. MCS protein folds into two structural domains and consists of 16 alpha-helices, 24 beta-strands, and several long loops. The core active site was determined as a wide cleft close to the end of the small C-terminal domain. The catalytic substrate malonate is placed between ATP and His206 in the MCS enzyme, supporting His206 in its catalytic role as it generates reaction intermediate, malonyl-AMP. These findings are strongly supported by previous biochemical data, as well as by the site-directed mutagenesis data reported here. This structure reveals the biochemical role as well as the substrate specificity that conservative residues of adenylate-forming enzymes have.     

A nuclear Overhauser enhancement study on the imino proton resonances of a DNA pentadecamer comprising the specific target site of the cyclic AMP receptor protein in the ara BAD operon

A 500 MHz 1H-NMR study on a synthetic DNA pentadecamer comprising the specific target site of the cAMP receptor protein in the ara BAD operon is presented. Using pre-steady state NOE measurements, unambiguous assignments of all the imino proton resonances and associated adenine (H2) resonances are obtained. From the NOE data interbase pair interproton distances involving the imino and adenine (H2) protons are determined. It is shown that these distances are very similar to those expected for classical B DNA (RMS difference of 0.5 A), but are significantly different from those expected for classical A DNA (RMS difference of 1.1 A).     

Effects of internal motions on the development of the two-dimensional transferred nuclear Overhauser effect

Summary In this paper we address the influence of internal motions on the development of the transferred nuclear Overhauser effect in a ligand undergoing chemical exchange between a free and a bound state. We examine the effects of varying the effective correlation time as well as the motional order parameter for methyl group and phenyl ring rotations in the free and bound ligand conformations. The effect of decreasing the motional order for a proton pair on a methyl group or phenyl ring is to decrease the effective correlation time of the internuclear vector, and thus to decrease the cross-relaxation rate between the proton pair. This functions to dampen the effects of spin diffusion, especially in the bound ligand- where cross-relaxation rates are much faster than in the free ligand. The effect of changing the effective correlation time for methyl group motions has little effect on the build-up behaviour of the transferred nuclear Overhauser effect for small values of fraction bound, but a larger effect on how fast it decays. This effect is greater for internal motions in the free peptide than it is for internal motions in the bound peptide.Dedicated to the memory of Professor V.F. Bystrov     

Sequential resonance assignments as a basis for determination of spatial protein structures by high resolution proton nuclear magnetic resonance

A general scheme is proposed for the determination of spatial protein structures by proton nuclear magnetic resonance. The scheme relies on experimental observation by two-dimensional nuclear magnetic resonance techniques of complete throughbond and through-space proton-proton connectivity maps. These are used to obtain sequential resonance assignments for the individual residues in the amino acid sequence and to characterize the spatial polypeptide structure by a tight network of semi-quantitative, intramolecular distance constraints.     

Locating the stabilizing residues in (alpha/beta)8 barrel proteins based on hydrophobicity, long-range interactions, and sequence conservation

In nature, 1 out of every 10 proteins has an (alpha/beta)(8) (TIM)-barrel fold, and in most cases, pairwise comparisons show no sequence similarity between them. Hence, delineating the key residues that induce very different sequences to share a common fold is important for understanding the folding and stability of TIM-barrel domains. In this work, we propose a new consensus approach for locating these stabilizing residues based on long-range interactions, hydrophobicity, and conservation of amino acid residues. We have identified 957 stabilizing residues in 63 proteins from a nonredundant set of 71 TIM-barrel domains. Most of these residues are located in the 8-stranded beta-sheet, with nearly one half of them oriented toward the interior of the barrel and the other half oriented toward the surrounding alpha-helices. Several stabilizing residues are found in the N- and C-terminal loops, whereas very few appear in the alpha-helices that surround the internal beta-sheet. Further, these 957 residues are placed in 434 stabilizing segments of various sizes, and each domain contains 1-10 of these segments. We found that 8 segments per domain is the most abundant one, and two thirds of the proteins have 7-9 stabilizing segments. Finally, we verified the identified residues with experimental temperature factors and found that these residues are among the ones with less mobility in the considered proteins. We suggest that our new protocol serves as a powerful tool to identify the stabilizing residues in TIM-barrel domains, which can be used as potential candidates for studying protein folding and stability by means of protein engineering experiments.     

Direct assignment of the dihydrouridine-helix imino proton resonances in transfer ribonucleic acid nuclear magnetic resonance spectra by means of the nuclear Overhauser effect

The NMR resonances from the hydrogen-bonded ring NH protons in the dihydrouridine stem of Escherichia colt tRNA1Val have been assigned by experiments involving the nuclear Overhauser effect (NOE) between adjacent base pairs. Irradiation of the 8-14 tertiary resonance produced a NOE to base pair 13. Irradiation of the CG13 ring NH produced NOEs to base pairs 12 and 14. Similarly, base pair 12 was shown to be dipolar coupled to 11 and 13, and base pair 11 was found to be coupled to 10 and 12. These sequential connectivities led to the assignment of CG13 at -13.05 ppm, UA12 at -13.84 ppm, CG11 at -12.23 ppm, and GC10 at -12.60 ppm. The results are compared with previous, less direct assignments for these four base pairs and with the expected proton positions from the crystal structure coordinates for this helix.     

The task of determining the local structure of proteins by Overhauser effect nuclear spectroscopy addressed once more]

The testing of the earlier developed theoretical method for determining the backbone protein conformations (the local structure) on the basis of the two-dimensional nuclear Overhauser effect (NOE) spectroscopy has been fulfilled. The method approval has been carried out by the calculation (based upon spectral NOE parameters) of the local plastocyanin and bovine pancreatic trypsin inhibitor structures followed by the comparison of the received conformational parameters with the X-ray data. The comparison of the molecular conformations in solution and crystal has been implemented for different fragments of the polypeptide chain (beta-structures, alpha-helices, irregular segments) using the mathematical statistics methods. The verification of the "zero" hypothesis about the similarity of phi and psi variation rows which was carried out at the reliability level of 0.99 showed that in both cases there were no systematic deviations of dihedral angles of the compared conformations and that their dispersion differences were statistically indiscernible. It has been concluded that the approved method permits to determine the local structure of the conformationally rigid proteins (or their fragments) at the level close to that which provides the high resolution X-ray analysis.