Determination of Protein Backbone ¹³CO Chemical Shift Anisotropy Tensors in Solution

Determination of Protein Backbone 13CO Chemical Shift Anisotropy Tensors in Solutio

Temperature Dependence of Anisotropic Protein Backbone Dynamics

The measurement of 15N NMR spin relaxation, which reports the 15N−1H vector reorientational dynamics, is a widely used experimental method to assess the motion of the protein backbone. Here, we investigate whether the 15N−1H vector motions are representative of the overall backbone motions, by analyzing the temperature dependence of the 15N−1H and 13CO−13Cα reorientational dynamics for the small proteins binase and ubiquitin. The latter dynamics were measured using NMR cross-correlated relaxation experiments. The data show that, on average, the 15N−1H order parameters decrease only by 2.5% between 5 and 30 °C. In contrast, the 13CO−13Cα order parameters decrease by 10% over the same temperature trajectory. This strongly indicates that there are polypeptide-backbone motions activated at room temperature that are not sensed by the 15N−1H vector. Our findings are at variance with the common crank-shaft model for protein backbone dynamics, which predicts the opposite behavior. This study suggests that investigation of the 15N relaxation alone would lead to underestimation of the dynamics of the protein backbone and the entropy contained therein

Use of ¹³C−¹³C NOE for the Assignment of NMR Lines of Larger Labeled Proteins at Larger Magnetic Fields

Use of 13C−13C NOE for the Assignment of NMR Lines of Larger Labeled Proteins at Larger Magnetic Field

Characterization and Calculation of a Cytochrome <i>c</i>−Cytochrome <i>b</i>₅ Complex Using NMR Data^†

To identify the binding site for bovine cytochrome b5 (cyt b5) on horse cytochrome c (cyt c), cross-saturation transfer NMR experiments were performed with 2H- and 15N-enriched cyt c and unlabeled cyt b5. In addition, chemical shift changes of the cyt c backbone amide and side chain methyl resonances were monitored as a function of cyt b5 concentration. The chemical shift changes indicate that the complex is in fast exchange, and are consistent with a 1:1 stoichiometry. A Ka of (4 ± 3) × 105 M-1 was obtained with a lower limit of 855 s-1 for the dissociation rate of the complex. Mapping of the chemical shift variations and intensity changes upon cross-saturation NMR experiments in the complex reveals a single, contiguous interaction interface on cyt c. Using NMR data as constraints, a protein docking program was used to calculate two low-energy model complex clusters. Independent calculations of the effect of the cyt b5 heme ring current-induced magnetic dipole on cyt c were used to discriminate between the different models. The interaction surface of horse cyt c in the current experimentally constrained model of the cyt c−cyt b5 complex is similar but not identical to the interface predicted in yeast cyt c by Brownian dynamics and docking calculations. The occurrence of different amino acids at the protein−protein interface and the dissimilar assumptions employed in the calculations can largely account for the nonidentical interfaces

Experimental and Theoretical Evaluation of Multisite Cadmium(II) Exchange in Designed Three-Stranded Coiled-Coil Peptides

An important factor that defines the toxicity of elements such as cadmium­(II), mercury­(II), and lead­(II) with biological macromolecules is metal ion exchange dynamics. Intriguingly, little is known about the fundamental rates and mechanisms of metal ion exchange into proteins, especially helical bundles. Herein, we investigate the exchange kinetics of Cd­(II) using <i>de novo</i> designed three-stranded coiled-coil peptides that contain metal complexing cysteine thiolates as a model for the incorporation of this ion into trimeric, parallel coiled coils. Peptides were designed containing both a single Cd­(II) binding site, <b>Grand</b>L12AL16C [<b>Grand</b> = AcG-(LKALEEK)₅-GNH₂], <b>Grand</b>L26AL30C, and <b>Grand</b>L26AE28QL30C, as well as <b>Grand</b>L12AL16CL26AL30C with two Cd­(II) binding sites. The binding of Cd­(II) to any of these sites is of high affinity (<i>K</i>_A > 3 × 10⁷ M^–1). Using ¹¹³Cd NMR spectroscopy, Cd­(II) binding to these designed peptides was monitored. While the Cd­(II) binding is in extreme slow exchange regime without showing any chemical shift changes, incremental line broadening for the bound ¹¹³Cd­(II) signal is observed when excess ¹¹³Cd­(II) is titrated into the peptides. Most dramatically, for one site, L26AL30C, all ¹¹³Cd­(II) NMR signals disappear once a 1.7:1 ratio of Cd­(II)/(peptide)₃ is reached. The observed processes are not compatible with a simple “free-bound” two-site exchange kinetics at any time regime. The experimental results can, however, be simulated in detail with a multisite binding model, which features additional Cd­(II) binding site(s) which, once occupied, perturb the primary binding site. This model is expanded into differential equations for five-site NMR chemical exchange. The numerical integration of these equations exhibits progressive loss of the primary site NMR signal without a chemical shift change and with limited line broadening, in good agreement with the observed experimental data. The mathematical model is interpreted in molecular terms as representing binding of excess Cd­(II) to surface Glu residues located at the helical interfaces. In the absence of Cd­(II), the Glu residues stabilize the three-helical structure though salt bridge interactions with surface Lys residues. We hypothesize that Cd­(II) interferes with these surface ion pairs, destabilizing the helical structure, and perturbing the primary Cd­(II) binding site. This hypothesis is supported by the observation that the Cd­(II)-excess line broadening is attenuated in <b>Grand</b>L26AE28QL30C, where a surface Glu(28), close to the metal binding site, was changed to Gln. The external binding site may function as an entry pathway for Cd­(II) to find its internal binding site following a molecular rearrangement which may serve as a basis for our understanding of metal complexation, transport, and exchange in complex native systems containing α-helical bundles

Apoprotein Structure and Metal Binding Characterization of a <i>de Novo</i> Designed Peptide, α₃D<b>IV</b>, that Sequesters Toxic Heavy Metals

<i>De novo</i> protein design is a biologically relevant approach that provides a novel process in elucidating protein folding and modeling the metal centers of metalloproteins in a completely unrelated or simplified fold. An integral step in <i>de novo</i> protein design is the establishment of a well-folded scaffold with one conformation, which is a fundamental characteristic of many native proteins. Here, we report the NMR solution structure of apo α₃DIV at pH 7.0, a <i>de novo</i> designed three-helix bundle peptide containing a triscysteine motif (Cys18, Cys28, and Cys67) that binds toxic heavy metals. The structure comprises 1067 NOE restraints derived from multinuclear multidimensional NOESY, as well as 138 dihedral angles (ψ, φ, and χ₁). The backbone and heavy atoms of the 20 lowest energy structures have a root mean square deviation from the mean structure of 0.79 (0.16) Å and 1.31 (0.15) Å, respectively. When compared to the parent structure α₃D, the substitution of Leu residues to Cys enhanced the α-helical content of α₃D<b>IV</b> while maintaining the same overall topology and fold. In addition, solution studies on the metalated species illustrated metal-induced stability. An increase in the melting temperatures was observed for Hg­(II), Pb­(II), or Cd­(II) bound α₃D<b>IV</b> by 18–24 °C compared to its apo counterpart. Further, the extended X-ray absorption fine structure analysis on Hg­(II)-α₃D<b>IV</b> produced an average Hg­(II)–S bond length at 2.36 Å, indicating a trigonal T-shaped coordination environment. Overall, the structure of apo α₃D<b>IV</b> reveals an asymmetric distorted triscysteine metal binding site, which offers a model for native metalloregulatory proteins with thiol-rich ligands that function in regulating toxic heavy metals, such as ArsR, CadC, MerR, and PbrR

Experimental Characterization of Models for Backbone Picosecond Dynamics in Proteins. Quantification of NMR Auto- and Cross-correlation Relaxation Mechanisms Involving Different Nuclei of the Peptide Plane

NMR relaxation parameters were measured for the peptide-plane carbonyl and nitrogen nuclei for the protein Escherichia coli flavodoxin. A poor correlation between the general order parameters of the C‘−Cα vector (Zeng, L.; Fischer, M. W. F.; Zuiderweg, E. R. P. J. Biomol. NMR 1996, 7, 157−162) and the N−NH vector was observed. We interpret this lack of correlation in this nearly spherical protein as evidence of local or semilocal anisotropic motion. A new experiment is introduced from which the cross-correlation between the carbonyl chemical shift anisotropy relaxation and carbonyl-Cα dipole−dipole relaxation is obtained. We show theoretically that the three relaxation measurements, reporting on the dynamics of the C‘−Cα vector, N−NH vector, and CSA tensor components behave differently under anisotropic motion. The cross-correlation order parameter formalism for dipolar cross-correlation spectral densities, as introduced by Daragan and Mayo (Daragan, V. A.; Mayo, K. H. J. Magn. Reson. B 1995, 107, 274−278), has been extended to include cross-correlations between nonaxial chemical shift anisotropy and dipole−dipole relaxation. By analyzing our experimental data with the theoretical models for anisotropic local motion, dynamic models were obtained for the peptide planes of 32 residues of E. coli flavodoxin

Changes in Calmodulin Main-Chain Dynamics upon Ligand Binding Revealed by Cross-Correlated NMR Relaxation Measurements

The fast dynamics of protein backbones are often investigated by nuclear magnetic relaxation experiments that report on the degree of spatial restriction of the amide bond vector. By comparing calmodulin in the peptide-bound and peptide-free states with these classical methods, we observe little difference in the dynamics of the polypeptide main chain (average order parameter decrease of 0.01 unit upon binding). However, when using NMR methods that monitor the mobility of the CO−Cα bond vector, we reveal a significant reduction of dynamics of the protein main chain (average order parameter decrease of 0.048 units). Previous investigations have suggested that the side-chain dynamics is reduced by an average of 0.07 order parameter units upon ligand binding (Lee, A. L.; Kinnear, S. A.; Wand, A. J. Nat. Struct. Biol. 2000, 7, 72−77). The current findings suggest that the change of the CO−Cα bond vector dynamics is intermediate between the changes in NH and side-chain dynamics and report a previously undetected loss of main-chain entropy. Weak site-to-site correlations between the different motional indicators are also observed

Secondary Structure and Fold Homology of the ArsC Protein from the <i>Escherichia coli</i> Arsenic Resistance Plasmid R773^†

Resistance to several toxic anions in Escherichia coli is conferred by the ars operon carried on plasmid R773. The gene products of this operon catalyze extrusion of antimonials and arsenicals from cells. In this paper, we report the determination of the overall fold for ArsC, a 16 kDa protein of the ars operon involved in the reduction of arsenate to arsenite, using multidimensional, multinuclear NMR. The protein is found to contain large regions of extensive mobility, particularly in the active site. A model fold, computed on the basis of a preliminary set of NOEs, was found to be structurally homologous to E. coli glutaredoxin, thiol transferases, and glutathione S-transferase. Some kinship to the structure of low molecular weight tyrosine phosphatases, based on rough topological similarity but more so on the basis of a common anion-binding-loop motif H−CXnR, was also detected. Although functional, secondary, and tertiary structural homology is observed with these molecules, no significant homology in primary structure was detected. The mobilities of the active site of ArsC and of other enzymes are discussed

Inhibitors of Difficult Protein–Protein Interactions Identified by High-Throughput Screening of Multiprotein Complexes

Protein–protein interactions (PPIs) are important in all aspects of cellular function, and there is interest in finding inhibitors of these contacts. However, PPIs with weak affinities and/or large interfaces have traditionally been more resistant to the discovery of inhibitors, partly because it is more challenging to develop high-throughput screening (HTS) methods that permit direct measurements of these physical interactions. Here, we explored whether the functional consequences of a weak PPI might be used as a surrogate for binding. As a model, we used the bacterial ATPase DnaK and its partners DnaJ and GrpE. Both DnaJ and GrpE bind DnaK and catalytically accelerate its ATP cycling, so we used stimulated nucleotide turnover to indirectly report on these PPIs. In pilot screens, we identified compounds that block activation of DnaK by either DnaJ or GrpE. Interestingly, at least one of these molecules blocked binding of DnaK to DnaJ, while another compound disrupted allostery between DnaK and GrpE without altering the physical interaction. These findings suggest that the activity of a reconstituted multiprotein complex might be used in some cases to identify allosteric inhibitors of challenging PPIs