X-ray structure analysis of a metalloprotein with enhanced active-site resolution using in situ x-ray absorption near edge structure spectroscopy

X-ray absorption spectroscopy is exquisitely sensitive to the coordination geometry of an absorbing atom and therefore allows bond distances and angles of the surrounding atomic cluster to be measured with atomic resolution. By contrast, the accuracy and resolution of metalloprotein active sites obtainable from x-ray crystallography are often insufficient to analyze the electronic properties of the metals that are essential for their biological functions. Here, we demonstrate that the combination of both methods on the same metalloprotein single crystal yields a structural model of the protein with exceptional active-site resolution. To this end, we have collected an x-ray diffraction data set to 1.4-A resolution and Fe K-edge polarized x-ray absorption near edge structure (XANES) spectra on the same cyanomet sperm whale myoglobin crystal. The XANES spectra were quantitatively analyzed by using a method based on the multiple scattering approach, which yielded Fe-heme structural parameters with +/-(0.02-0.07)-A accuracy on the atomic distances and +/-7 degrees on the Fe-CN angle. These XANES-derived parameters were subsequently used as restraints in the crystal structure refinement. By combining XANES and x-ray diffraction, we have obtained an cyanomet sperm whale myoglobin structural model with a higher precision of the bond lengths and angles at the active site than would have been possible with crystallographic analysis alone.     

Accurate protein structure modeling using sparse NMR data and homologous structure information

While information from homologous structures plays a central role in X-ray structure determination by molecular replacement, such information is rarely used in NMR structure determination because it can be incorrect, both locally and globally, when evolutionary relationships are inferred incorrectly or there has been considerable evolutionary structural divergence. Here we describe a method that allows robust modeling of protein structures of up to 225 residues by combining , ¹³C, and ¹⁵N backbone and ¹³Cβ chemical shift data, distance restraints derived from homologous structures, and a physically realistic all-atom energy function. Accurate models are distinguished from inaccurate models generated using incorrect sequence alignments by requiring that (i) the all-atom energies of models generated using the restraints are lower than models generated in unrestrained calculations and (ii) the low-energy structures converge to within 2.0 Å backbone rmsd over 75% of the protein. Benchmark calculations on known structures and blind targets show that the method can accurately model protein structures, even with very remote homology information, to a backbone rmsd of 1.2–1.9 Å relative to the conventional determined NMR ensembles and of 0.9–1.6 Å relative to X-ray structures for well-defined regions of the protein structures. This approach facilitates the accurate modeling of protein structures using backbone chemical shift data without need for side-chain resonance assignments and extensive analysis of NOESY cross-peak assignments.     

High-resolution molecular structure of a peptide in an amyloid fibril determined by magic angle spinning NMR spectroscopy

Amyloid fibrils are self-assembled filamentous structures associated with protein deposition conditions including Alzheimer's disease and the transmissible spongiform encephalopathies. Despite the immense medical importance of amyloid fibrils, no atomic-resolution structures are available for these materials, because the intact fibrils are insoluble and do not form diffraction-quality 3D crystals. Here we report the high-resolution structure of a peptide fragment of the amyloidogenic protein transthyretin, TTR(105-115), in its fibrillar form, determined by magic angle spinning NMR spectroscopy. The structure resolves not only the backbone fold but also the precise conformation of the side chains. Nearly complete (13)C and (15)N resonance assignments for TTR(105-115) formed the basis for the extraction of a set of distance and dihedral angle restraints. A total of 76 self-consistent experimental measurements, including 41 restraints on 19 backbone dihedral angles and 35 (13)C-(15)N distances between 3 and 6 A were obtained from 2D and 3D NMR spectra recorded on three fibril samples uniformly (13)C, (15)N-labeled in consecutive stretches of four amino acids and used to calculate an ensemble of peptide structures. Our results indicate that TTR(105-115) adopts an extended beta-strand conformation in the amyloid fibrils such that both the main- and side-chain torsion angles are close to their optimal values. Moreover, the structure of this peptide in the fibrillar form has a degree of long-range order that is generally associated only with crystalline materials. These findings provide an explanation of the unusual stability and characteristic properties of this form of polypeptide assembly.     

De novo determination of peptide structure with solid-state magic-angle spinning NMR spectroscopy

The three-dimensional structure of the chemotactic peptide N-formyl-l-Met-l-Leu-l-Phe-OH was determined by using solid-state NMR (SSNMR). The set of SSNMR data consisted of 16 (13)C-(15)N distances and 18 torsion angle constraints (on 10 angles), recorded from uniformly (13)C,(15)N- and (15)N-labeled samples. The peptide's structure was calculated by means of simulated annealing and a newly developed protocol that ensures that all of conformational space, consistent with the structural constraints, is searched completely. The result is a high-quality structure of a molecule that has thus far not been amenable to single-crystal diffraction studies. The extensions of the SSNMR techniques and computational methods to larger systems appear promising.     

Consistent blind protein structure generation from NMR chemical shift data

Protein NMR chemical shifts are highly sensitive to local structure. A robust protocol is described that exploits this relation for de novo protein structure generation, using as input experimental parameters the (13)C(alpha), (13)C(beta), (13)C', (15)N, (1)H(alpha) and (1)H(N) NMR chemical shifts. These shifts are generally available at the early stage of the traditional NMR structure determination process, before the collection and analysis of structural restraints. The chemical shift based structure determination protocol uses an empirically optimized procedure to select protein fragments from the Protein Data Bank, in conjunction with the standard ROSETTA Monte Carlo assembly and relaxation methods. Evaluation of 16 proteins, varying in size from 56 to 129 residues, yielded full-atom models that have 0.7-1.8 A root mean square deviations for the backbone atoms relative to the experimentally determined x-ray or NMR structures. The strategy also has been successfully applied in a blind manner to nine protein targets with molecular masses up to 15.4 kDa, whose conventional NMR structure determination was conducted in parallel by the Northeast Structural Genomics Consortium. This protocol potentially provides a new direction for high-throughput NMR structure determination.     

The metal reductase activity of some multiheme cytochromes c: NMR structural characterization of the reduction of chromium(VI) to chromium(III) by cytochrome c(7)

The redox reaction between CrO(4)(2-) and the fully reduced three-heme cytochrome c(7) from Desulfuromonas acetoxidans to give chromium(III) and the fully oxidized protein has been followed by NMR spectroscopy. The hyperfine coupling between the oxidized protein protons and chromium(III), which remains bound to the protein, gives rise to line-broadening effects on the NMR resonances that can be transformed into proton-metal distance restraints. Structure calculations based on these unconventional constraints allowed us to demonstrate that chromium(III) binds at a unique site and to locate it on the protein surface. The metal ion is located 7.9 +/- 0.4 A from the iron of heme IV, 16.3 +/- 0.7 A from the iron of heme III, and 22.5 +/- 0.5 A from the iron of heme I. Shift changes caused by the presence of unreactive MoO(4)(2-), a CrO(4)(2-) analogue, indicate the involvement of the same protein area in the anion binding. The titration of the oxidation of cytochrome c(7) shows a detailed mechanism of action. The presence of a specific binding site supports the hypothesis of the biological role of this cytochrome as a metal reductase.     

DNA-nanotube-induced alignment of membrane proteins for NMR structure determination

Membrane proteins are encoded by 20-35% of genes but represent <1% of known protein structures to date. Thus, improved methods for membrane-protein structure determination are of critical importance. Residual dipolar couplings (RDCs), commonly measured for biological macromolecules weakly aligned by liquid-crystalline media, are important global angular restraints for NMR structure determination. For alpha-helical membrane proteins >15 kDa in size, Nuclear-Overhauser effect-derived distance restraints are difficult to obtain, and RDCs could serve as the main reliable source of NMR structural information. In many of these cases, RDCs would enable full structure determination that otherwise would be impossible. However, none of the existing liquid-crystalline media used to align water-soluble proteins are compatible with the detergents required to solubilize membrane proteins. We report the design and construction of a detergent-resistant liquid crystal of 0.8-microm-long DNA-nanotubes that can be used to induce weak alignment of membrane proteins. The nanotubes are heterodimers of 0.4-microm-long six-helix bundles each self-assembled from a 7.3-kb scaffold strand and >170 short oligonucleotide staple strands. We show that the DNA-nanotube liquid crystal enables the accurate measurement of backbone N(H) and C(alpha)H(alpha) RDCs for the detergent-reconstituted zeta-zeta transmembrane domain of the T cell receptor. The measured RDCs validate the high-resolution structure of this transmembrane dimer. We anticipate that this medium will extend the advantages of weak alignment to NMR structure determination of a broad range of detergent-solubilized membrane proteins.     

The des(1-6)antennapedia homeodomain: comparison of the NMR solution structure and the DNA-binding affinity with the intact Antennapedia homeodomain.

The nuclear magnetic resonance (NMR) solution structure of an N-terminally truncated mutant Antennapedia homeodomain, des(1-6)Antp(C39S), has been determined from 935 nuclear Overhauser effect upper distance constraints and 148 dihedral angle constraints by using the programs DIANA and OPAL. Twenty conformers representing the solution structure of des(1-6)Antp(C39S) have an average root-mean-square distance relative to the mean coordinates of 0.56 A for the backbone atoms of residues 8-59. Comparison with the intact Antp(C39S) homeodomain shows that the two proteins have identical molecular architectures. The removal of the N-terminal residues 1-6, which are flexibly disordered in the intact homeodomain, causes only strictly localized structure variations and does not noticeably affect the adjoining helix I from residues 10-21. The DNA-binding constant of des(1-6)Antp(C39S) is approximately 10-fold reduced relative to the intact Antp(C39S) homeodomain, which can now be attributed to the absence of the previously reported contacts of the N-terminal polypeptide segment of the intact Antp(C39S) homeodomain with the minor groove of the DNA duplex.     

Accurate and rapid docking of protein-protein complexes on the basis of intermolecular nuclear overhauser enhancement data and dipolar couplings by rigid body minimization

A simple and rapid method is presented for solving the three-dimensional structures of protein-protein complexes in solution on the basis of experimental NMR restraints that provide the requisite translational (i.e., intermolecular nuclear Overhauser enhancement, NOE, data) and orientational (i.e., backbone (1)H-(15)N dipolar couplings and intermolecular NOEs) information. Providing high-resolution structures of the proteins in the unbound state are available and no significant backbone conformational changes occur upon complexation (which can readily be assessed by analysis of dipolar couplings measured on the complex), accurate and rapid docking of the two proteins can be achieved. The method, which is demonstrated for the 40-kDa complex of enzyme I and the histidine phosphocarrier protein, involves the application of rigid body minimization using a target function comprising only three terms, namely experimental NOE-derived intermolecular interproton distance and dipolar coupling restraints, and a simple intermolecular van der Waals repulsion potential. This approach promises to dramatically reduce the amount of time and effort required to solve the structures of protein-protein complexes by NMR, and to extend the capabilities of NMR to larger protein-protein complexes, possibly up to molecular masses of 100 kDa or more.     

CLOUDS,a protocol for deriving a molecular proton density via NMR

We demonstrate the feasibility of computing realistic spatial proton distributions for proteins in solution from experimental NMR nuclear Overhauser effect data only and with minimal assignments. The method, CLOUDS, relies on precise and abundant interproton distance restraints calculated via a relaxation matrix analysis of sets of experimental nuclear Overhauser effect spectroscopy crosspeaks. The MIDGE protocol was adapted for this purpose. A gas of unassigned, unconnected H atoms is condensed into a structured proton distribution (cloud) via a molecular dynamics simulated-annealing scheme in which the internuclear distances and van der Waals repulsive terms are the only active restraints. Proton densities are generated by combining a large number of such clouds, each computed from a different trajectory. After filtering by reference to the cloud closest to the mean, a minimal dispersion proton density (foc) is identified. The latter affords a quasi-continuous hydrogen-only probability distribution that conveys immediate information on the protein surface topology (grooves, protrusions, potential binding site cavities, etc.), directly related to the molecular structure. Feasibility of the method was tested on NMR data measured on two globular protein domains of low regular secondary structure content, the col 2 domain of human matrix metalloproteinase-2 and the kringle 2 domain of human plasminogen, of 60 and 83 amino acid residues, respectively.     

Three-dimensional structure of proteins determined by molecular dynamics with interproton distance restraints: application to crambin.

Model calculations are performed to evaluate the utility of molecular dynamics with NMR interproton distance restraints for determining the three-dimensional structure of proteins. The system used for testing the method is the 1.5-A resolution crystal structure of crambin (a protein of 46 residues) from which a set of 240 approximate interproton distances of less than 4 A are derived. The convergence properties of the method are examined by using different dynamics protocols and starting from two initial structures; one is a completely extended beta-strand, and the other has residues 7-19 and 23-30 in the form of alpha-helices (as in the crystal structure) with the remaining residues in the form of extended beta-strands. In both cases global and local convergence to the correct final structure is achieved with rms atomic differences between the restrained dynamics structures and the crystal structure of 1.5-2.1 A for the backbone atoms and 2.1-2.8 A for all atoms; the averaged structure has backbone and all atom rms deviations of 1.3 and 1.9 A, respectively. Further, it is shown that a restrained dynamics structure with significantly larger deviations (i.e., 5.7 A for the backbone atoms) can be characterized as incorrect, independent of a knowledge of the crystal structure.     

Determination of DNA structures by NMR and distance geometry techniques: a computer simulation.

Computer simulations have been performed to determine how accurately and precisely structures of DNA oligomers can be generated from distance data obtained from two-dimensional NMR experiments. A hexamer fragment d(CGAATT) of the Dickerson dodecamer [Drew, H.R., Wing, R.M., Takano, T., Broka, C., Tanaha, S., Itakura, K. & Dickerson, R.E. (1981) Proc. Natl. Acad. Sci. USA 78, 2179-2183] was used as the model structure in these simulations. Protons were added to the coordinates of the original x-ray structure, which was then subjected to a regularization procedure to minimize deviations from standard bond lengths and bond angles. The proton-proton distances normally observed in NMR experiments were measured from this regularized target structure and used as input for a distance geometry algorithm. Distance geometry structures were generated from two distance sets, one with essentially exact distances (+/- 0.005 A) and one set with a precision (+/- 0.2 A) that simulates an optimal NMR experiment. The results of these calculations were used to judge how accurately and precisely the following helical parameters could be reproduced from this simulated NMR distance data: helical twist, helical rise, dislocation, roll, tilt, glycosidic angle, delta torsion angle, and pseudorotation angle. These data provide a basis from which to judge the quality of DNA structures produced from real NMR experiments.     

Molecular spectroscopy and dynamics of intrinsically fluorescent proteins: coral red (dsRed) and yellow (Citrine)

Gene expression of intrinsically fluorescent proteins in biological systems offers new noninvasive windows into cellular function, but optimization of these probes relies on understanding their molecular spectroscopy, dynamics, and structure. Here, the photophysics of red fluorescent protein (dsRed) from discosoma (coral), providing desired longer emission/absorption wavelengths, and an improved yellow fluorescent protein mutant (Citrine) (S65G/V68L/Q69 M/S72A/T203Y) for significant comparison, are characterized by using fluorescence correlation spectroscopy and time-correlated single-photon counting. dsRed fluorescence decays as a single exponential with a 3.65 +/- 0.07-ns time constant, indicating a single emitting state/species independent of pH 4.4-9.0, in contrast with Citrine. However, laser excitation drives reversible fluorescence flicker at 10(3)-10(4) Hz between dark and bright states with a constant partition fraction f(1) = 0.42 +/- 0.06 and quantum yield of approximately 3 x 10(-3). Unlike Citrine (pKa approximately 5.7), pH-dependent proton binding is negligible (pH 3. 9-11) in dsRed. Time-resolved anisotropy of dsRed reveals rapid depolarization (211 +/- 6 ps) plus slow rotational motion (53 +/- 8 ns), in contrast with a single rotational time (16 +/- 2 ns) for Citrine. The molecular dimensions, calculated from rotational and translational diffusion, indicate that dsRed is hydrodynamically 3.8 +/- 0.4 times larger than predicted for a monomer, which suggests an oligomer (possibly a tetramer) configuration even at approximately 10(-9) M. The fast depolarization is attributed to intraoligomer energy transfer between mobile nonparallel chromophores with the initial anisotropy implying a 24 +/- 3 degrees depolarization angle. Large two-photon excitation cross sections ( approximately 100 GM at 990 nm for dsRed and approximately 50 GM at 970 nm for Citrine), advantageous for two-photon-fluorescence imaging in cells, are measured.     

Structure of the coat protein in fd filamentous bacteriophage particles determined by solid-state NMR spectroscopy

The atomic resolution structure of fd coat protein determined by solid-state NMR spectroscopy of magnetically aligned filamentous bacteriophage particles differs from that previously determined by x-ray fiber diffraction. Most notably, the 50-residue protein is not a single curved helix, but rather is a nearly ideal straight helix between residues 7 and 38, where there is a distinct kink, and then a straight helix with a different orientation between residues 39 and 49. Residues 1-5 have been shown to be mobile and unstructured, and proline 6 terminates the helix. The structure of the coat protein in virus particles, in combination with the structure of the membrane-bound form of the same protein in bilayers, also recently determined by solid-state NMR spectroscopy, provides insight into the viral assembly process. In addition to their roles in molecular biology and biotechnology, the filamentous bacteriophages continue to serve as model systems for the development of experimental methods for determining the structures of proteins in biological supramolecular assemblies. New NMR results include the complete sequential assignment of the two-dimensional polarization inversion spin-exchange at the magic angle spectrum of a uniformly 15N-labeled 50-residue protein in a 1.6 x 107 Da particle in solution, and the calculation of the three-dimensional structure of the protein from orientational restraints with an accuracy equivalent to an rms deviation of approximately 1A.     

Determination of solution structures of proteins up to 40 kDa using CS-Rosetta with sparse NMR data from deuterated samples

We have developed an approach for determining NMR structures of proteins over 20 kDa that utilizes sparse distance restraints obtained using transverse relaxation optimized spectroscopy experiments on perdeuterated samples to guide RASREC Rosetta NMR structure calculations. The method was tested on 11 proteins ranging from 15 to 40 kDa, seven of which were previously unsolved. The RASREC Rosetta models were in good agreement with models obtained using traditional NMR methods with larger restraint sets. In five cases X-ray structures were determined or were available, allowing comparison of the accuracy of the Rosetta models and conventional NMR models. In all five cases, the Rosetta models were more similar to the X-ray structures over both the backbone and side-chain conformations than the “best effort” structures determined by conventional methods. The incorporation of sparse distance restraints into RASREC Rosetta allows routine determination of high-quality solution NMR structures for proteins up to 40 kDa, and should be broadly useful in structural biology.     

Antithrombotic effects of combining activated protein C and urokinase in nonhuman primates.

BACKGROUND. We have determined in vivo the relative antithrombotic efficacy and hemostatic safety of combining low-dose activated protein C (APC) and urokinase (urinary plasminogen activator, u-PA), two natural proteins that regulate thrombogenesis. METHODS AND RESULTS. To model acute thrombotic responses of native blood under conditions of arterial flow, thrombogenic segments of Dacron vascular graft (VG) were incorporated into chronic exteriorized femoral arteriovenous (AV) access shunts in baboons. Thrombus formation on VG was determined by measuring 1) the deposition of autologous 111In platelets using real-time scintillation camera imaging, 2) the accumulation of 125I fibrin, 3) segment patency by Doppler flow analysis, and 4) blood tests for thrombosis, including plasma concentrations of platelet factor 4, beta-thromboglobulin, fibrinopeptide A (FPA), and D-dimer. Treatments consisting of low-dose and intermediate-dose APC (0.07 or 0.25 mg/kg.hr), u-PA (25,000 or 50,000 IU/kg.hr), or the combination were administered for 1 hour by continuous intravenous infusion. In untreated controls, platelets and fibrin accumulated rapidly, reaching plateau values at 1 hour of 15.1 +/- 3.8 x 10(9) platelets and 7.8 +/- 2.2 mg fibrin. Although the low-dose APC or u-PA alone did not decrease either platelet or fibrin deposition significantly, this combination moderately reduced both platelet and fibrin accumulation (7.3 +/- 2.6 x 10(9) platelets, p less than 0.05; 3.9 +/- 0.6 mg fibrin, p less than 0.05). Furthermore, intermediate-dose APC or u-PA reduced thrombus formation by half when administered alone (p less than 0.001 for both platelet and fibrin deposition), and the combination markedly interrupted the accumulation of platelets (3.0 +/- 1.0 x 10(9) platelets, p less than 0.001) and fibrin (1.3 +/- 0.6 mg fibrin, p less than 0.001). During active treatments, all VG segments remained patent. Hemostatic plug forming capability, as measured by template bleeding times, remained normal during all experiments (p greater than 0.05). The T50 clearance time for APC activity was not affected by the concurrent administration of u-PA. u-PA alone increased the plasma levels of D-dimer, FPA, and, interestingly, APC, implying that during pharmacological activation of the fibrinolytic system, thrombin activity was released, and the protein C pathway was activated. CONCLUSIONS. A combination of intermediate-dose APC and u-PA produce substantial and efficient antithrombotic effects without impairing hemostatic function.     

Structure and backbone dynamics of a microcrystalline metalloprotein by solid-state NMR

We introduce a new approach to improve structural and dynamical determination of large metalloproteins using solid-state nuclear magnetic resonance (NMR) with (1)H detection under ultrafast magic angle spinning (MAS). The approach is based on the rapid and sensitive acquisition of an extensive set of (15)N and (13)C nuclear relaxation rates. The system on which we demonstrate these methods is the enzyme Cu, Zn superoxide dismutase (SOD), which coordinates a Cu ion available either in Cu(+) (diamagnetic) or Cu(2+) (paramagnetic) form. Paramagnetic relaxation enhancements are obtained from the difference in rates measured in the two forms and are employed as structural constraints for the determination of the protein structure. When added to (1)H-(1)H distance restraints, they are shown to yield a twofold improvement of the precision of the structure. Site-specific order parameters and timescales of motion are obtained by a gaussian axial fluctuation (GAF) analysis of the relaxation rates of the diamagnetic molecule, and interpreted in relation to backbone structure and metal binding. Timescales for motion are found to be in the range of the overall correlation time in solution, where internal motions characterized here would not be observable.     

Interindividual variability of multilead electrocardiographic recordings: influence of heart position.

The electrocardiogram (ECG) of normal, healthy subjects shows a large interindividual variability. Part of this variability is due to the heart position and orientation relative to the electrodes. In this report, the interindividual variability is quantified using the relative variability measure, computed as the averaged standard deviation in the ECGs, scaled by the average root mean square of the ECGs. The relative variability in the QRS complex is estimated as 0.52. The heart position and orientation relative to the lead positions is documented in 25 normal subjects. The long axis angle varies considerably among the subjects (27.1+/-8.8 degrees to the transversal plane and 38 degrees +/-5 degrees to the frontal plane). Moving the electrodes in the frontal plane to a position relative to a common reference point at the base of the heart (shift: 0.8+/-0.7 cm leftward and 2.4+/-2.3 cm downward) did not reduce the interindividual variability.     

NMR data collection and analysis protocol for high-throughput protein structure determination

A standardized protocol enabling rapid NMR data collection for high-quality protein structure determination is presented that allows one to capitalize on high spectrometer sensitivity: a set of five G-matrix Fourier transform NMR experiments for resonance assignment based on highly resolved 4D and 5D spectral information is acquired in conjunction with a single simultaneous 3D 15N,13C(aliphatic),13C(aromatic)-resolved [1H,1H]-NOESY spectrum providing 1H-1H upper distance limit constraints. The protocol was integrated with methodology for semiautomated data analysis and used to solve eight NMR protein structures of the Northeast Structural Genomics Consortium pipeline. The molecular masses of the hypothetical target proteins ranged from 9 to 20 kDa with an average of approximately 14 kDa. Between 1 and 9 days of instrument time were invested per structure, which is less than approximately 10-25% of the measurement time routinely required to date with conventional approaches. The protocol presented here effectively removes data collection as a bottleneck for high-throughput solution structure determination of proteins up to at least approximately 20 kDa, while concurrently providing spectra that are highly amenable to fast and robust analysis.