Protein structure refinement based on paramagnetic NMR shifts: applications to wild-type and mutant forms of cytochrome c.

A new approach to NMR solution structure refinement is introduced that uses paramagnetic effects on nuclear chemical shifts as constraints in energy minimization or molecular dynamics calculations. Chemical shift differences between oxidized and reduced forms of horse cytochrome c for more than 300 protons were used as constraints to refine the structure of the wild-type protein in solution and to define the structural changes induced by a Leu 94 to Val mutation. A single round of constrained minimization, using the crystal structure as the starting point, converged to a low-energy structure with an RMS deviation between calculated and observed pseudo-contact shifts of 0.045 ppm, 7.5-fold lower than the starting structure. At the same time, the procedure provided stereospecific assignments for more than 45 pairs of methylene protons and methyl groups. Structural changes caused by the mutation were determined to a precision of better than 0.3 A. Structure determination based on dipolar paramagnetic (pseudocontact) shifts is applicable to molecules containing anisotropic paramagnetic centers with short electronic relaxation times, including numerous naturally occurring metalloproteins, as well as proteins or nucleic acids to which a paramagnetic metal ion or ligand may be attached. The long range of paramagnetic shift effects (up to 20 A from the iron in the case of cytochrome c) provides global structural constraints, which, in conjunction with conventional NMR distance and dihedral angle constraints, will enhance the precision of NMR solution structure determination.     

Structural and orientational information of the membrane embedded M13 coat protein by (13)C-MAS NMR spectroscopy

Oriented and unoriented M13 coat protein, incorporated into dimyristoyl phosphatidylcholine bilayers, has been studied by (13)C-magic angle spinning nuclear magnetic resonance (MAS NMR) spectroscopy. Rotational resonance experiments provided two distance constraints between Calpha and C&z.dbnd6;O positions of the labelled residues Val-29/Val-30 (0.4+/-0.5nm) and Val-29/Val-31 (0.45+/-0. 5nm) in its hydrophobic domain. The derived dihedral angles (Phi, Psi) for Val-30 revealed a local alpha-helical conformation. (13)C-CP-MAS experiments on uniformly aligned samples (MAOSS experiments) using the (13)C&z.dbnd6;O labelled site of Val-30 allowed the determination of the helix tilt (20 degrees +/-10 degrees ) in the membrane. It is shown that one uniform MAS high-resolution solid state NMR approach can be used to obtain structural and orientational data.     

Calibration of the angular dependence of the amide proton-C alpha proton coupling constants, 3JHN alpha, in a globular protein. Use of 3JHN alpha for identification of helical secondary structure

The vicinal amide proton-C alpha proton spin-spin coupling constants, JHN alpha, in the globular protein basic pancreatic trypsin inhibitor (BPTI) have been measured using phase-sensitive correlated spectroscopy at high digital resolution. In conjunction with the crystal structure of BPTI, these data were used to calibrate the correlation between 3JHN alpha and the dihedral angle phi. The resulting "BPTI curve" is 3JHN alpha = 6.4 cos2 theta - 1.4 cos theta + 1.9 (theta = [phi - 60 degrees]). It is further shown that measurement of the spin-spin couplings 3JHN alpha presents an independent, reliable method for identification of the location of helical structure in the amino acid sequence of proteins.     

Analysis of dihedral angle preferences for alanine and glycine residues in alpha and beta transmembrane regions

For the past 50?years, the Ramachandran map has been used effectively to study the protein structure and folding. However, though extensive analysis has been done on dihedral angle preferences of residues in globular proteins, related studies and reports of membrane proteins are limited. It is of interest to explore the conformational preferences of residues in transmembrane regions of membrane proteins which are involved in several important and diverse biological processes. Hence, in the present work, a systematic comparative computational analysis has been made on dihedral angle preferences of alanine and glycine in alpha and beta transmembrane regions (the two major classes of transmembrane proteins) with the aid of the Ramachandran map. Further, the conformational preferences of residues in transmembrane regions were compared with the non-transmembrane regions. We have extracted cation-pi interacting residues present in transmembrane regions and explored the dihedral angle preferences. From our observations, we reveal the higher percentage of occurrences of glycine in alpha and beta transmembrane regions than other hydrophobic residues. Further, we noted a clear shift in ψ-angle preferences of glycine residues from negative bins in alpha transmembrane regions to positive bins in beta transmembrane regions. Also, cation-pi interacting residues in beta transmembrane regions avoid preferring ψ-angles in the range of ?59° to ?30°. In this article, we insist that the studies on preferences of dihedral angles in transmembrane regions, thorough understanding of structure and folding of transmembrane proteins, can lead to modeling of novel transmembrane regions towards designing membrane proteins.     

The Nuclear Magnetic Resonance Solution Structure of the Mixed Disulfide between Escherichia coli Glutaredoxin(C14S) and Glutathione

The determination of the nuclear magnetic resonance (NMR) solution structure of the mixed disulfide between the mutant Escherichia coli glutaredoxin Grx(C14S) and glutathione (GSH), Grx(C14S)-SG, is described, the binding site for GSH on Grx(C14S) is located, and the non-bonding interactions between -SG and the protein are characterized. Based on nearly complete sequence-specific NMR assignments, 1010 nuclear Overhauser enhancement upper distance constraints and 116 dihedral angle constraints were obtained as the input for the structure calculations, for which the distance geometry program DIANA was used followed by energy minimization in a waterbath with the AMBER force field in the program OPAL. The -SG moiety was found to be localized on the surface of the protein in a cleft bounded by the amino acid residues Y13, T58, V59, Y72, T73 and D74. Hydrogen bonds have been identified between -SG and the residues V59 and T73 of Grx(C14S), and the formation of an additional hydrogen bond with Y72 and electrostatic interactions with the side-chains of D74 and K45 are also compatible with the NMR, conformational constraints. Comparison of the reduced and oxidized forms of Grx with Grx(C14S)-SG shows that the mixed disulfide more closely resembles the oxidized form of the protein. Functional implications of this observation are discussed. Comparisons are also made with the related proteins bacteriophage T4 glutaredoxin and glutathione S-transferase.     

Solution structure of alpha t alpha, a helical hairpin peptide of de novo design.

alpha t alpha is a 38-residue peptide designed to adopt a helical hairpin conformation in solution (Fezoui Y, Weaver DL Osterhout JJ, 1995, Protein Sci 4:286-295). A previous study of the carboxylate form of alpha t alpha by CD and two-dimensional NMR indicated that the peptide was highly helical and that the helices associated in approximately the intended orientation (Fezoui Y, Weaver DL, Osterhout JJ, 1994, Proc Natl Acad Sci USA 91:3675-3679). Here, the solution structure of alpha t alpha as determined by two-dimensional NMR is reported. A total of 266 experimentally derived distance restraints and 20 dihedral angle restraints derived from J-couplings were used. One-hundred initial structures were generated by distance geometry and refined by dynamical simulated annealing. Twenty-three of the lowest-energy structures consistent with the experimental restraints were analyzed. The results presented here show that alpha t alpha is comprised of two associating helices connected by a turn region.     

Dipole moment in TIM alpha/beta fold proteins

TIM proteins of alpha/beta barrel fold from alpha/beta class as given in SCOP database were taken for dipole moment analysis. In all, 32 structures were analyzed for their dipole moment contributions. Representative structures from 20 super families in the alpha/beta fold, with different enzyme functions and 12 protein domains of TIM family in TIM super family were considered. The active sites of these proteins are located on the C-terminal side of the beta-strands. The molecules of same alpha/beta fold, but differing in their functionality also showed a common electrostatic field pattern along the barrel axis and had the dipole moment along the barrel axis and towards C-terminal end of the beta-strands. However, it is observed from our calculations that the dipole moment direction is possibly a consequence of the structural fold, with distribution of charges playing a modulatory role, and does not contribute to the location of active site. We show here that apart from the commonly held view as proposed by Hol et al [Hol W G L, van Duijnen PT and Berendsen H J C (1978) Nature (London), 273, 443-446] of the role of the alpha helical dipole moment, the beta-sheets in the barrel can also have a considerable dipole moment contribution. Taken together with our dipole moment analysis on integral membrane proteins [Vasanthi G and Krishnaswamy S (2002) Indian J Biochem Biophys 39, 93-100], this suggests the need to examine the role of dipole moment in the case of especially beta sheets forming barrels.     

Membrane‐induced structure of novel human tachykinin hemokinin‐1 (hHK1)

PPT‐C encoded hemokinin‐1(hHK‐1) of Homo sapiens (TGKASQFFGLM) is a structurally distinct neuropeptide among the tachykinin family that participate in the NK‐1 receptor downstream signaling processes. Subsequently, signal transduction leads to execution of various effector functions which includes aging, immunological, and central nervous system (CNS) regulatory actions. However the conformational pattern of ligand receptor binding is unclear. The three‐dimensional structure of the hemokinin‐1 in aqueous and micellar environment has been studied by one and two‐dimensional proton nuclear magnetic resonance (2D 1H‐NMR spectroscopy) and distance geometry calculations. Data shows that hemokinin‐1 was unstructured in aqueous environment; anionic detergent SDS induces α‐helix formation. Proton NMR assignments have been carried out with the aid of correlation spectroscopy (gradient‐COSY and TOCSY) and nuclear Overhauser effect spectroscopy (NOESY and ROESY) experiments. The inter proton distances and dihedral angle constraints obtained from the NMR data have been used in torsion angle dynamics algorithm for NMR applications (CYANA) to generate a family of structures, which have been refined using restrained energy minimization and dynamics. Helical conformation is observed from residue K3‐M11. The conformational range of the peptide revealed by NMR studies has been analyzed in terms of characteristic secondary features. Observed conformational features have been compared to that of Substance P potent NK1 agonist. Thus the report provides a structural insight to study hHK‐1‐NK1 interaction that is essential for hHK1 based signaling events. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 702–710, 2015.     

Solution conformation of asparagine-linked oligosaccharides: alpha(1-6)-linked moiety

The solution conformation is presented for representatives of each of the major classes of asparaginyl oligosaccharides. In this report the conformation of the alpha(1-6)-linked moiety is described. The conformational properties of these glycopeptides were determined by high-resolution 1H nuclear magnetic resonance in conjunction with potential energy calculations. The NMR parameters that were used in this analysis were chemical shifts and nuclear Overhauser enhancements. Potential energy calculations were used to evaluate the preferred conformers available for the different linkages in glycopeptides and to draw conclusions and to draw conclusions about the behavior in solution of these molecules. For all classes, identical conformations were found for the 6-arm except for the torsional angle, omega, about the C5-C6 bond of the alpha 1-6 linkage. For high mannose and hybrid structures omega was found to be -60 degrees, for bisected biantennary complex structures omega was 180 degrees, and for complex biantennary structures averaging between -60 degrees and 180 degrees occurs.     

The electric dipole moment of DNA-binding HU protein calculated by the use of NMR database

Electric birefringence measurements indicated the presence of a large permanent dipole moment in HU protein–DNA complex. In order to substantiate this observation, numerical computation of the dipole moment of HU protein homodimer was carried out by using NMR protein databases. The dipole moments of globular proteins have hitherto been calculated with X-ray databases and NMR data have never been used before. The advantages of NMR databases are: (a) NMR data are obtained, unlike X-ray databases, using protein solutions. Accordingly, this method eliminates the bothersome question as to the possible alteration of the protein structure due to the transition from the crystalline state to the solution state. This question is particularly important for proteins such as HU protein which has considerable internal flexibility’s; (b) the three dimensional coordinates of hydrogen atoms in protein molecules can be determined with a sufficient resolution and this enables the N–H as well as C=O bond moments to be calculated. Since the NMR database of HU protein from Bacillus stearothermophilus consists of 25 models, the surface charge as well as the core dipole moments were computed for each of these structures. The results of these calculations show that the net permanent dipole moments of HU protein homodimer is approximately 500–530 D (1 D=3.33×10⁻³⁰ Cm) at pH 7.5 and 600–630 D at the isoelectric point (pH 10.5). These permanent dipole moments are unusually large for a small protein of the size of 19.5 kDa. Nevertheless, the result of numerical calculations is compatible with the electro-optical observation, confirming a very large dipole moment in this protein.     

Global folds of proteins with low densities of NOEs using residual dipolar couplings: application to the 370-residue maltodextrin-binding protein

The global fold of maltose-binding protein in complex with the substrate beta-cyclodextrin was determined by solution NMR methods. The two-domain protein is comprised of a single polypeptide chain of 370 residues, with a molecular mass of 42 kDa. Distance information in the form of H(N)-H(N), H(N)-CH(3) and CH(3)-CH(3) NOEs was recorded on (15)N, (2)H and (15)N, (13)C, (2)H-labeled proteins with methyl protonation in Val, Leu, and Ile (C(delta1) only) residues. Distances to methyl protons, critical for the structure determination, comprised 77 % of the long-range restraints. Initial structures were calculated on the basis of 1943 NOEs, 48 hydrogen bond and 555 dihedral angle restraints. A global pair-wise backbone rmsd of 5.5 A was obtained for these initial structures with rmsd values for the N and C domains of 2.4 and 3.8 A, respectively. Direct refinement against one-bond (1)H(N)-(15)N, (13)C(alpha)-(13)CO, (15)N-(13)CO, two-bond (1)H(N)-(13)CO and three-bond (1)H(N)-(13)C(alpha) dipolar couplings resulted in structures with large numbers of dipolar restraint violations. As an alternative to direct refinement against measured dipolar couplings we have developed an approach where discrete orientations are calculated for each peptide plane on the basis of the dipolar couplings described above. The orientation which best matches that in initial NMR structures calculated from NOE and dihedral angle restraints exclusively is used to refine further the structures using a new module written for CNS. Modeling studies from four different proteins with diverse structural motifs establishes the utility of the methodology. When applied to experimental data recorded on MBP the precision of the family of structures generated improves from 5.5 to 2.2 A, while the rmsd with respect to the X-ray structure (1dmb) is reduced from 5.1 to 3.3 A.     

Transmembrane signal transduction of the alpha(IIb)beta(3) integrin

Integrins are composed of noncovalently bound dimers of an alpha- and a beta-subunit. They play an important role in cell-matrix adhesion and signal transduction through the cell membrane. Signal transduction can be initiated by the binding of intracellular proteins to the integrin. Binding leads to a major conformational change. The change is passed on to the extracellular domain through the membrane. The affinity of the extracellular domain to certain ligands increases; thus at least two states exist, a low-affinity and a high-affinity state. The conformations and conformational changes of the transmembrane (TM) domain are the focus of our interest. We show by a global search of helix-helix interactions that the TM section of the family of integrins are capable of adopting a structure similar to the structure of the homodimeric TM protein Glycophorin A. For the alpha(IIb)beta(3) integrin, this structural motif represents the high-affinity state. A second conformation of the TM domain of alpha(IIb)beta(3) is identified as the low-affinity state by known mutational and nuclear magnetic resonance (NMR) studies. A transition between these two states was determined by molecular dynamics (MD) calculations. On the basis of these calculations, we propose a three-state mechanism.     

One- and two-dimensional NMR investigations of the heme pocket in free alpha(CO) chains from human hemoglobin

Two-dimensional nuclear magnetic resonance techniques were used to assign resonances corresponding to heme pocket residues of the isolated alpha(CO) subunits of the human adult hemoglobin (HbA). The assignment procedure was based on the partial identification of the amino acid spin system from the J-correlated (COSY) spectrum and on the nuclear Overhauser effect connectivities (from NOSEY spectra) with the heme substituents. We present here partial assignments corresponding to five amino acid residues: Leu86, Leu-91, Val-93, Leu-101 and Leu-136. Starting from the known crystallographic structure of the alpha subunit in the hemoglobin tetramer, we applied a dipolar model to compute the ring-current shift of the protons from fifteen amino acid residues in the heme pocket. Comparison of the predicted and observed chemical shifts suggests that there is a very close similarity between the heme pocket tertiary structure of the alpha(CO) subunits in crystals of HbA(CO) and of the free alpha(CO) chains. The one-dimensional NMR spectra were used to monitor the pH-induced structural changes, the effects of chemical modification and of ligand substitution. Upon increasing the pH from 5.6 to 9.0 the structure of the heme environment appears to be invariant with the exception of some residues in the CD corner. The structure is also largely conserved when p-chloromercuribenzoate is bound to Cys-104. In contrast, the substitution of CO by O2 as ligand induces many large changes in the heme cavity which can be partially characterized by NMR spectroscopy.     

Combined use of homo- and heteronuclear coupling constants as restraints in molecular dynamics simulations.

A penalty function for scalar coupling constants has been applied in molecular dynamics simulations as an experimental constraint. The function is based on the difference between the coupling constant calculated from the dihedral angle and the experimentally measured coupling constant. The method is illustrated on a model cyclic pentapeptide for which 3JHN-H alpha and 3JHN-C beta, both about the phi backbone dihedral angle, have been measured. The function is efficient in producing structures consistent with the scalar couplings, but removed from the conformation observed in solution. This arises from the lack of J restraints for the psi dihedral angle. Simulations with both nuclear Overhauser effect (NOE) and J-coupling restraints illustrates small but significant differences from simulations using only NOEs.     

Nearest-neighbor effects on backbone alpha and beta carbon chemical shifts in proteins

We present a method for analyzing the chemical shift database to yield information on nearest-neighbor effects on carbon-13 chemical shift values for alpha and beta carbons of amino acids in proteins. For each amino acid sequence XYZ, we define two correction factors, Delta(XY) s and Delta(YZ) s , representing the effects on (delta13 Calpha-delta13 Cbeta) for residue Y from the preceding residue (X) and the following residue (Z), where X, Y, and Z represent one of the 20 naturally occurring amino acids, Delta designates the change in value or the correction factor (in ppm), and s is an index standing for one of three "pseudo secondary structure states" derived from chemical shift dispersions, which we show represent residues in primarily alpha-helix, beta-strand, and non-alphabeta(coil). The correction factors were obtained from maximum likelihood fitting of (delta13 Calpha-delta13 Cbeta) values from the chemical shifts of 651 proteins to a mixture of three Gaussians. These correction factors were derived strictly from the analysis of assigned chemical shifts, without regard to the three-dimensional structures of these proteins. The corrections factors were found to differ according to the secondary structural environment of the central residue (deduced from the chemical shift distribution) as well as by different identities of the nearest neighboring residues in the sequence. The areas subsumed by the sequence-dependent chemical shift distributions report on the relative energies of the sequences in different pseudo secondary structural environments, and the positions of the peaks indicate the chemical shifts of lowest energy conformations. As such, these results have potential applications to the determination of dihedral angle restraints from chemical shifts for structure determination and to more accurate predictions of chemical shifts in proteins of known structure. From a database of chemical shifts associated well-defined three-dimensional structures, comparisons were made between DSSP designations derived from three-dimensional structure and pseudo secondary structure designations derived from nearest-neighbor corrected chemical shift analysis. The high level of agreement between the two approaches to classifying secondary structure provides a measure of confidence in this chemical shift-based approach to the analysis of protein structure.     

In-cell NMR for protein-protein interactions (STINT-NMR)

We describe an in-cell NMR-based method for mapping the structural interactions (STINT-NMR) that underlie protein-protein complex formation. This method entails sequentially expressing two (or more) proteins within a single bacterial cell in a time-controlled manner and monitoring their interactions using in-cell NMR spectroscopy. The resulting NMR data provide a complete titration of the interaction and define structural details of the interacting surfaces at atomic resolution. Unlike the case where interacting proteins are simultaneously overexpressed in the labeled medium, in STINT-NMR the spectral complexity is minimized because only the target protein is labeled with NMR-active nuclei, which leaves the interactor protein(s) cryptic. This method can be combined with genetic and molecular screens to provide a structural foundation for proteomic studies. The protocol takes 4 d from the initial transformation of the bacterial cells to the acquisition of the NMR spectra.     

Solution conformation of asparagine-linked oligosaccharides: alpha(1-2)-, alpha(1-3)-, beta(1-2)-, and beta(1-4)-linked units

The solution conformation is presented for representatives of each of the major classes of asparaginyl oligosaccharides. In this report the conformation of alpha(1-3)-, alpha(1-2)-, beta(1-2)-, and beta(1-4)-linked units is described. The conformational properties of these glycopeptides were determined by high-resolution 1H nuclear magnetic resonance in conjunction with potential energy calculations. The NMR parameters that were used in this analysis were chemical shifts and nuclear Overhauser enhancements. Potential energy calculations were used to evaluate the preferred conformers available for the different linkages in glycopeptides and to draw conclusions about the behavior in solution of these molecules. It was found that the linkage conformation of the Man alpha 1-3 residues was not affected by substitution either at the 2-position by alpha Man or beta GlcNAc or at the 4-position by beta GlcNAc or by the presence of a bisecting GlcNAc on the adjacent beta Man residue.     

Expression, purification, and characterization of Thermotoga maritima membrane proteins for structure determination

Structural studies of integral membrane proteins typically rely upon detergent micelles as faithful mimics of the native lipid bilayer. Therefore, membrane protein structure determination would be greatly facilitated by biophysical techniques that are capable of evaluating and assessing the fold and oligomeric state of these proteins solubilized in detergent micelles. In this study, an approach to the characterization of detergent-solubilized integral membrane proteins is presented. Eight Thermotoga maritima membrane proteins were screened for solubility in 11 detergents, and the resulting soluble protein-detergent complexes were characterized with small angle X-ray scattering (SAXS), nuclear magnetic resonance (NMR) spectroscopy, circular dichroism (CD) spectroscopy, and chemical cross-linking to evaluate the homogeneity, oligomeric state, radius of gyration, and overall fold. A new application of SAXS is presented, which does not require density matching, and NMR methods, typically used to evaluate soluble proteins, are successfully applied to detergent-solubilized membrane proteins. Although detergents with longer alkyl chains solubilized the most proteins, further characterization indicates that some of these protein-detergent complexes are not well suited for NMR structure determination due to conformational exchange and protein oligomerization. These results emphasize the need to screen several different detergents and to characterize the protein-detergent complex in order to pursue structural studies. Finally, the physical characterization of the protein-detergent complexes indicates optimal solution conditions for further structural studies for three of the eight overexpressed membrane proteins.     

The structure and dipole moment of globular proteins in solution and crystalline states: use of NMR and X-ray databases for the numerical calculation of dipole moment

The large dipole moment of globular proteins has been well known because of the detailed studies using dielectric relaxation and electro-optical methods. The search for the origin of these dipolemoments, however, must be based on the detailed knowledge on protein structure with atomic resolutions. At present, we have two sources of information on the structure of protein molecules: (1) x-ray databases obtained in crystalline state; (2) NMR databases obtained in solution state. While x-ray databases consist of only one model, NMR databases, because of the fluctuation of the protein folding in solution, consist of a number of models, thus enabling the computation of dipole moment repeated for all these models. The aim of this work, using these databases, is the detailed investigation on the interdependence between the structure and dipole moment of protein molecules. The dipole moment of protein molecules has roughly two components: one dipole moment is due to surface charges and the other, core dipole moment, is due to polar groups such as N--H and C==O bonds. The computation of surface charge dipole moment consists of two steps: (A) calculation of the pK shifts of charged groups for electrostatic interactions and (B) calculation of the dipole moment using the pK corrected for electrostatic shifts. The dipole moments of several proteins were computed using both NMR and x-ray databases. The dipole moments of these two sets of calculations are, with a few exceptions, in good agreement with one another and also with measured dipole moments.