Protein–nucleotide contacts in motor proteins detected by DNP-enhanced solid-state NMR

DNP (dynamic nuclear polarization)-enhanced solid-state NMR is employed to directly detect protein–DNA and protein–ATP interactions and identify the residue type establishing the intermolecular contacts. While conventional solid-state NMR can detect protein–DNA interactions in large oligomeric protein assemblies in favorable cases, it typically suffers from low signal-to-noise ratios. We show here, for the oligomeric DnaB helicase from Helicobacter pylori complexed with ADP and single-stranded DNA, that this limitation can be overcome by using DNP-enhanced spectroscopy. Interactions are established by DNP-enhanced ³¹P–¹³C polarization-transfer experiments followed by the recording of a 2D ¹³C–¹³C correlation experiment. The NMR spectra were obtained in less than 2 days and allowed the identification of residues of the motor protein involved in nucleotide binding.     

Differential Scanning Calorimetry of Unfreezable Water in Water–Protein–Polyol Systems

The amount of unfreezable water in lysozyme and bovine serum albumin in aqueous solutions of xylitol, sorbitol, glucose and sucrose was estimated by a differential scanning calorimeter according to new analytical methods. The antemelting point of aqueous polyol solutions seemed to shift to a higher temperature upon addition of protein, but the incipient melting point was not affected by the coexisting protein. The amount of unfreezable water in both proteins, as well as the heat of fusion of ice, decreased with increasing polyol concentration, regardless of the kind of polyols added. On the basis of these results, the solvation structure of the protein in these three-component systems and the mechanism of the polyol-induced stabilization of protein were discussed assuming protein–polyol interactions.     

Solid-State NMR Spectroscopy of the HIV gp41 Membrane Fusion Protein Supports Intermolecular Antiparallel β Sheet Fusion Peptide Structure in the Final Six-Helix Bundle State

The HIV gp41 protein catalyzes fusion between viral and target cell membranes. Although the ~ 20-residue N-terminal fusion peptide (FP) region is critical for fusion, the structure of this region is not well characterized in large gp41 constructs that model the gp41 state at different times during fusion. This paper describes solid-state NMR (SSNMR) studies of FP structure in a membrane-associated construct (FP-Hairpin), which likely models the final fusion state thought to be thermostable trimers with six-helix bundle structure in the region C-terminal of the FP. The SSNMR data show that there are populations of FP-Hairpin with either α helical or β sheet FP conformation. For the β sheet population, measurements of intermolecular ¹³C-¹³C proximities in the FP are consistent with a significant fraction of intermolecular antiparallel β sheet FP structure with adjacent strand crossing near L7 and F8. There appears to be negligible in-register parallel structure. These findings support assembly of membrane-associated gp41 trimers through interleaving of N-terminal FPs from different trimers. Similar SSNMR data are obtained for FP-Hairpin and a construct containing the 70 N-terminal residues of gp41 (N70), which is a model for part of the putative pre-hairpin intermediate state of gp41. FP assembly may therefore occur at an early fusion stage. On a more fundamental level, similar SSNMR data are obtained for FP-Hairpin and a construct containing the 34 N-terminal gp41 residues (FP34) and support the hypothesis that the FP is an autonomous folding domain.     

Protein–Protein and Protein–Membrane Associations in the Lignin Pathway

Supramolecular organization of enzymes is proposed to orchestrate metabolic complexity and help channel intermediates in different pathways. Phenylpropanoid metabolism has to direct up to 30% of the carbon fixed by plants to the biosynthesis of lignin precursors. Effective coupling of the enzymes in the pathway thus seems to be required. Subcellular localization, mobility, protein–protein, and protein–membrane interactions of four consecutive enzymes around the main branch point leading to lignin precursors was investigated in leaf tissues of Nicotiana benthamiana and cells of Arabidopsis thaliana. CYP73A5 and CYP98A3, the two Arabidopsis cytochrome P450s (P450s) catalyzing para- and meta-hydroxylations of the phenolic ring of monolignols were found to colocalize in the endoplasmic reticulum (ER) and to form homo- and heteromers. They moved along with the fast remodeling plant ER, but their lateral diffusion on the ER surface was restricted, likely due to association with other ER proteins. The connecting soluble enzyme hydroxycinnamoyltransferase (HCT), was found partially associated with the ER. Both HCT and the 4-coumaroyl-CoA ligase relocalized closer to the membrane upon P450 expression. Fluorescence lifetime imaging microscopy supports P450 colocalization and interaction with the soluble proteins, enhanced by the expression of the partner proteins. Protein relocalization was further enhanced in tissues undergoing wound repair. CYP98A3 was the most effective in driving protein association.     

Protein–RNA specificity by high-throughput principal component analysis of NMR spectra

Defining the RNA target selectivity of the proteins regulating mRNA metabolism is a key issue in RNA biology. Here we present a novel use of principal component analysis (PCA) to extract the RNA sequence preference of RNA binding proteins. We show that PCA can be used to compare the changes in the nuclear magnetic resonance (NMR) spectrum of a protein upon binding a set of quasi-degenerate RNAs and define the nucleobase specificity. We couple this application of PCA to an automated NMR spectra recording and processing protocol and obtain an unbiased and high-throughput NMR method for the analysis of nucleobase preference in protein–RNA interactions. We test the method on the RNA binding domains of three important regulators of RNA metabolism.     

Computational Assessment of Protein–protein Binding Affinity by Reversely Engineering the Energetics in Protein Complexes

The cellular functions of proteins are maintained by forming diverse complexes. The stability of these complexes is quantified by the measurement of binding affinity, and mutations that alter the binding affinity can cause various diseases such as cancer and diabetes. As a result, accurate estimation of the binding stability and the effects of mutations on changes of binding affinity is a crucial step to understanding the biological functions of proteins and their dysfunctional consequences. It has been hypothesized that the stability of a protein complex is dependent not only on the residues at its binding interface by pairwise interactions but also on all other remaining residues that do not appear at the binding interface. Here, we computationally reconstruct the binding affinity by decomposing it into the contributions of interfacial residues and other non-interfacial residues in a protein complex. We further assume that the contributions of both interfacial and non-interfacial residues to the binding affinity depend on their local structural environments such as solvent-accessible surfaces and secondary structural types. The weights of all corresponding parameters are optimized by Monte-Carlo simulations. After cross-validation against a large-scale dataset, we show that the model not only shows a strong correlation between the absolute values of the experimental and calculated binding affinities, but can also be an effective approach to predict the relative changes of binding affinity from mutations. Moreover, we have found that the optimized weights of many parameters can capture the first-principle chemical and physical features of molecular recognition, therefore reversely engineering the energetics of protein complexes. These results suggest that our method can serve as a useful addition to current computational approaches for predicting binding affinity and understanding the molecular mechanism of protein–protein interactions.     

Interaction of Pyrylium Dye with Self-Complementary DNA Oligomer as Studied by H NMR Spectroscopy‡

Abstract

Interaction of 2-methyl-4,6-bis-(4-N,N-dimethylaminophenyl) pyrylium salt (P2) with [d(CGACGTCG)]₂ was investigated by H NMR spectroscopy. The aromatic signals of P2 and the oligomer were shifted to the upfield by forming the complex, and intermolecular NOEs were also observed between P2 and the terminal CpG base steps but not between P2 and the central CpG. These results indicate that P2 binds to the weakly stacking CpG steps in an intercalation manner.

     

The Structure of the Cytochrome P450cam–Putidaredoxin Complex Determined by Paramagnetic NMR Spectroscopy and Crystallography

Cytochrome P450cam catalyzes the hydroxylation of camphor in a complex process involving two electron transfers (ETs) from the iron-sulfur protein putidaredoxin. The enzymatic control of the successive steps of catalysis is critical for a highly efficient reaction. The injection of the successive electrons is part of the control system. To understand the molecular interactions between putidaredoxin and cytochrome P450cam, we determined the structure of the complex both in solution and in the crystal state. Paramagnetic NMR spectroscopy using lanthanide tags yielded 446 structural restraints that were used to determine the solution structure. An ensemble of 10 structures with an RMSD of 1.3 Å was obtained. The crystal structure of the complex was solved, showing a position of putidaredoxin that is identical with the one in the solution structure. The NMR data further demonstrate the presence of a minor state or set of states of the complex in solution, which is attributed to the presence of an encounter complex. The structure of the major state shows a small binding interface and a metal-to-metal distance of 16 Å, with two pathways that provide strong electronic coupling of the redox centers. The interpretation of these results is discussed in the context of ET. The structure indicates that the ET rate can be much faster than the reported value, suggesting that the process may be gated.     

Resistance of Soybean Pectin–Protein Conjugate Pre-Adsorbed to the Air–Water Interface to Displacement by the Competitive Adsorption of Surfactant

Food Biophysics - The naturally occurring soybean pectin–protein conjugate pre-adsorbed to the air–water interface was shown to be displaced competitively from the interface when a...     

Solid state NMR sequential resonance assignments and conformational analysis of the 2×10.4 kDa dimeric form of the Bacillus subtilis protein Crh

Solid state NMR sample preparation and resonance assignments of the U-[¹³C,¹⁵N] 2×10.4 kDa dimeric form of the regulatory protein Crh in microcrystalline, PEG precipitated form are presented. Intra– and interresidue correlations using dipolar polarization transfer methods led to nearly complete sequential assignments of the protein, and to 88% of all ¹⁵N, ¹³C chemical shifts. For several residues, the resonance assignments differ significantly from those reported for the monomeric form analyzed by solution state NMR. Dihedral angles obtained from a TALOS-based statistical analysis suggest that the microcrystalline arrangement of Crh must be similar to the domain-swapped dimeric structure of a single crystal form recently solved using X-ray crystallography. For a limited number of protein residues, a remarkable doubling of the observed NMR resonances is observed indicative of local static or dynamic conformational disorder. Our study reports resonance assignments for the largest protein investigated by solid state NMR so far and describes the conformational dimeric variant of Crh with previously unknown chemical shifts.     

The Structure of 5-Cyclohexyl-2′-Deoxyuridine as Studied by X-Ray Crystallography and NMR Spectroscopy

Abstract

5-Cyclohexyl-2′-deoxyuridine (I) is an example of a 5-substituted pyrimidine 2′-deoxynucleoside which exhibits no antiviral activity and which is not a substrate for either cellular or viral (herpes) kinases. Despite the fact that a cursory inspection of NMR spectra of the compound, taken in DMSO-d ₆ solution, suggested that the compound had a normal conformation, we here show that in the crystal and in aqueous solution (analysed by 2D NMR techniques), the conformation of this nucleoside has a syn-glycosidic and C4′-exo (₄E) sugar pucker conformation.     

Molecular Features of the Zn2+ Binding Site in the Prion Protein Probed by 113Cd NMR

The cellular prion protein (PrP^C) is a zinc-binding protein that contributes to the regulation of Zn²⁺ and other divalent species of the central nervous system. Zn²⁺ coordinates to the flexible, N-terminal repeat region of PrP^C and drives a tertiary contact between this repeat region and a well-defined cleft of the C-terminal domain. The tertiary structure promoted by Zn²⁺ is thought to regulate inherent PrP^C toxicity. Despite the emerging consensus regarding the interaction between Zn²⁺ and PrP^C, there is little direct spectroscopic confirmation of the metal ion’s coordination details. Here, we address this conceptual gap by using Cd²⁺ as a surrogate for Zn²⁺. NMR finds that Cd²⁺ binds exclusively to the His imidazole side chains of the repeat segment, with a dissociation constant of ～1.2 mM, and promotes an N-terminal-C-terminal cis interaction very similar to that observed with Zn²⁺. Analysis of ¹¹³Cd NMR spectra of PrP^C, along with relevant control proteins and peptides, suggests that coordination of Cd²⁺ in the full-length protein is consistent with a three- or four-His geometry. Examination of the mutation E199K in mouse PrP^C (E200K in humans), responsible for inherited Creutzfeldt-Jakob disease, finds that the mutation lowers metal ion affinity and weakens the cis interaction. These findings not only provide deeper insight into PrP^C metal ion coordination but they also suggest new perspectives on the role of familial mutations in prion disease.     

Protein–protein interactions of huntingtin in the hippocampus

Molecular Biology - Huntingtin (HTT) occurs in the neuronal cytoplasm and can interact with structural elements of synapses. Huntington’s disease (HD) results from pathological expansion of a...