Backbone Amide Protons Research Articles

Anion-Binding Properties of Short Linear Homopeptides.

A comprehensive thermodynamic and structural study of the complexation affinities of tetra (L1), penta (L2), and hexaphenylalanine (L3) linear peptides towards several inorganic anions in acetonitrile (MeCN) and N,N-dimethylformamide (DMF) was carried out. The influence of the chain length on the complexation thermodynamics and structural changes upon anion binding are particularly addressed here. The complexation processes were characterized by means of spectrofluorimetric, 1H NMR, microcalorimetric, and circular dichroism spectroscopy titrations. The results indicate that all three peptides formed complexes of 1:1 stoichiometry with chloride, bromide, hydrogen sulfate, dihydrogen phosphate (DHP), and nitrate anions in acetonitrile and DMF. In the case of hydrogen sulfate and DHP, anion complexes of higher stoichiometries were observed as well, namely those with 1:2 and 2:1 (peptide:anion) complexes. Anion-induced peptide backbone structural changes were studied by molecular dynamic simulations. The anions interacted with backbone amide protons and one of the N-terminal amine protons through hydrogen bonding. Due to the anion binding, the main chain of the studied peptides changed its conformation from elongated to quasi-cyclic in all 1:1 complexes. The accomplishment of such a conformation is especially important for cyclopeptide synthesis in the head-to-tail macrocyclization step, since it is most suitable for ring closure. In addition, the studied peptides can act as versatile ionophores, facilitating transmembrane anion transport.

Metal interactions of α-synuclein probed by NMR amide-proton exchange.

The aberrant aggregation of α-synuclein (αS), a disordered protein primarily expressed in neuronal cells, is strongly associated with the underlying mechanisms of Parkinson's disease. It is now established that αS has a weak affinity for metal ions and that these interactions alter its conformational properties by generally promoting self-assembly into amyloids. Here, we characterised the nature of the conformational changes associated with metal binding by αS using nuclear magnetic resonance (NMR) to measure the exchange of the backbone amide protons at a residue specific resolution. We complemented these experiments with 15N relaxation and chemical shift perturbations to obtain a comprehensive map of the interaction between αS and divalent (Ca2+, Cu2+, Mn2+, and Zn2+) and monovalent (Cu+) metal ions. The data identified specific effects that the individual cations exert on the conformational properties of αS. In particular, binding to calcium and zinc generated a reduction of the protection factors in the C-terminal region of the protein, whereas both Cu(II) and Cu(I) did not alter the amide proton exchange along the αS sequence. Changes in the R2/R1 ratios from 15N relaxation experiments were, however, detected as a result of the interaction between αS and Cu+ or Zn2+, indicating that binding to these metals induces conformational perturbations in distinctive regions of the protein. Collectively our data suggest that multiple mechanisms of enhanced αS aggregation are associated with the binding of the analysed metals.

Localising individual atoms of tryptophan side chains in the metallo-β-lactamase IMP-1 by pseudocontact shifts from paramagnetic lanthanoid tags at multiple sites.

The metallo--lactamase IMP-1 features a flexible loop near the active site that assumes different conformations in single crystal structures, which may assist in substrate binding and enzymatic activity. To probe the position of this loop, we labelled the tryptophan residues of IMP-1 with 7-C-indole and the protein with lanthanoid tags at three different sites. The magnetic susceptibility anisotropy () tensors were determined by measuring pseudocontact shifts (PCSs) of backbone amide protons. The tensors were subsequently used to identify the atomic coordinates of the tryptophan side chains in the protein. The PCSs were sufficient to determine the location of Trp28, which is in the active site loop targeted by our experiments, with high accuracy. Its average atomic coordinates showed barely significant changes in response to the inhibitor captopril. It was found that localisation spaces could be defined with better accuracy by including only the PCSs of a single paramagnetic lanthanoid ion for each tag and tagging site. The effect was attributed to the shallow angle with which PCS isosurfaces tend to intersect if generated by tags and tagging sites that are identical except for the paramagnetic lanthanoid ion.

Molecular Dynamics Insight into the Lipid II Recognition by Type A Lantibiotics: Nisin, Epidermin, and Gallidermin

Lanthionine-containing peptides (lantibiotics) have been considered as pharmaceutical candidates for decades, although their clinical application has been restricted. Most lantibiotics kill bacteria via targeting and segregating of the cell wall precursor—membrane-inserted lipid II molecule—in some cases accompanied by pores formation. Nisin-like lantibiotics specifically bind to pyrophosphate (PPi) moiety of lipid II with their structurally similar N-terminal thioether rings A and B. Although possessing higher pore-forming capability, nisin, in some cases, is 10-fold less efficient in vivo as compared to related epidermin and gallidermin peptides, differing just in a few amino acid residues within their target-binding regions. Here, using molecular dynamics simulations, we investigated atomistic details of intermolecular interactions between the truncated analogues of these peptides (residues 1–12) and lipid II mimic (dimethyl pyrophosphate, DMPPi). The peptides adopt similar conformation upon DMPPi binding with backbone amide protons orienting into a single center capturing PPi moiety via simultaneous formation of up to seven hydrogen bonds. Epidermin and gallidermin adopt the complex-forming conformation twice as frequent as nisin does, enhancing the binding by the lysine 4 side chain. Introduction of the similar residue to nisin in silico improves the binding, providing ideas for further design of prototypic antibiotics.

Advances in Hydrogen/Deuterium Exchange Mass Spectrometry and the Pursuit of Challenging Biological Systems.

Solution-phase hydrogen/deuterium exchange (HDX) coupled to mass spectrometry (MS) is a widespread tool for structural analysis across academia and the biopharmaceutical industry. By monitoring the exchangeability of backbone amide protons, HDX-MS can reveal information about higher-order structure and dynamics throughout a protein, can track protein folding pathways, map interaction sites, and assess conformational states of protein samples. The combination of the versatility of the hydrogen/deuterium exchange reaction with the sensitivity of mass spectrometry has enabled the study of extremely challenging protein systems, some of which cannot be suitably studied using other techniques. Improvements over the past three decades have continually increased throughput, robustness, and expanded the limits of what is feasible for HDX-MS investigations. To provide an overview for researchers seeking to utilize and derive the most from HDX-MS for protein structural analysis, we summarize the fundamental principles, basic methodology, strengths and weaknesses, and the established applications of HDX-MS while highlighting new developments and applications.

ESCASA: Analytical estimation of atomic coordinates from coarse-grained geometry for nuclear-magnetic-resonance-assisted protein structure modeling. I. Backbone and Hβ protons.

A method for the estimation of coordinates of atoms in proteins from coarse-grained geometry by simple analytical formulas (ESCASA), for use in nuclear-magnetic-resonance (NMR) data-assisted coarse-grained simulations of proteins is proposed. In this paper, the formulas for the backbone Hα and amide (HN ) protons, and the side-chain Hβ protons, given the Cα -trace, have been derived and parameterized, by using the interproton distances calculated from a set of 140 high-resolution non-homologous protein structures. The mean standard deviation over all types of proton pairs in the set was 0.44 Å after fitting. Validation against a set of 41 proteins with NMR-determined structures, which were not considered in parameterization, resulted in average standard deviation from average proton-proton distances of the NMR-determined structures of 0.25 Å, compared to 0.21 Å obtained with the PULCHRA all-atom-chain reconstruction algorithm and to the 0.12 Å standard deviation of the average-structure proton-proton distance of NMR-determined ensembles. The formulas provide analytical forces and can, therefore, be used in coarse-grained molecular dynamics.

Confronting the Invisible: Assignment of Protein 1HN Chemical Shifts in Cases of Extreme Broadening.

NMR studies of intrinsically disordered proteins (IDPs) at neutral pH values are hampered by the rapid exchange of backbone amide protons with solvent. Although exchange rates can be modulated by changes in pH, interactions between IDPs that lead to phase separation sometimes only occur at neutral pH values or higher, where backbone amide-based experiments fail. Here we describe a simple NMR experiment for measuring amide proton chemical shifts in cases where 1HN spectra cannot be obtained. The approach uses a weak 1H B1 field, searching for elusive 1HN resonance frequencies that become encoded in the intensities of cross-peaks in three-dimensional 1Hα-detect spectra. Applications to the CAPRIN1 protein in both dilute- and phase-separated states highlight the utility of the method, establishing that accurate 1HN chemical shifts can be obtained even in cases where solvent hydrogen exchange rates are on the order of 1500 s-1.

High-Resolution Diffusion Measurements of Proteins by NMR under Near-Physiological Conditions.

Measuring the translational diffusion of proteins under physiological conditions can be very informative, especially when multiple diffusing species can be distinguished. Diffusion NMR or diffusion-ordered spectroscopy (DOSY) is widely used to study molecular diffusion, where protons are used as probes, which can be further edited by the proton-attached heteronuclei to provide additional resolution. For example, the combination of the backbone amide protons (1HN) to measure diffusion with the well-resolved 1H/15N correlations has afforded high-resolution DOSY experiments. However, significant amide-water proton exchange at physiological temperature and pH can affect the accuracy of diffusion data or cause complete loss of DOSY signals. Although aliphatic protons do not exchange with water protons, and thus are potential probes to measure diffusion rates, 1H/13C correlations are often in spectral overlap or masked by the water signal, which hampers the use of these correlations. In this report, a method was developed that separates the nuclei used for diffusion (α protons, 1Hα) and those used for detection (1H/15N and 13C'/15N correlations). This approach enables high-resolution diffusion measurements of polypeptides in a mixture of biomolecules, thereby providing a powerful tool to investigate coexisting species under physiologically relevant conditions.

Determining methyl sidechain conformations in a CS-ROSETTA model using methyl 1H-13C residual dipolar couplings.

Modelling of protein structures based on backbone chemical shifts, using programs such as CS-ROSETTA, is becoming increasingly popular, especially for systems where few restraints are available or where homologous structures are already known. While the reliability of CS-ROSETTA calculations can be improved by incorporation of some additional backbone NMR data such as those afforded by residual dipolar couplings or minimal NOE data sets involving backbone amide protons, the sidechain conformations are largely modelled by statistical energy terms. Here, we present a simple method based on methyl residual dipolar couplings that can be used to determine the rotameric state of the threefold symmetry axis of methyl groups that occupy a single rotamer, determine rotameric distributions, and identify regions of high flexibility. The method is demonstrated for methyl side chains of a deletion variant of the human chaperone DNAJB6b.

Three-Dimensional Protein Structure Determination Using Pseudocontact Shifts of Backbone Amide Protons Generated by Double-Histidine Co2+-Binding Motifs at Multiple Sites.

Pseudocontact shifts (PCSs) generated by paramagnetic metal ions contribute highly informative long-range structure restraints that can be measured in solution and are ideally suited to guide structure prediction algorithms in determining global protein folds. We recently demonstrated that PCSs, which are relatively small but of high quality, can be generated by a double-histidine (dHis) motif in an α-helix, which provides a well-defined binding site for a single Co2+ ion. Here we show that PCSs of backbone amide protons generated by dHis-Co2+ motifs positioned in four different α-helices of a protein deliver excellent restraints to determine the three-dimensional (3D) structure of a protein in a way akin to the global positioning system (GPS). We demonstrate the approach with GPS-Rosetta calculations of the 3D structure of the C-terminal domain of the chaperone ERp29 (ERp29-C). Despite the relatively small size of the PCSs generated by the dHis-Co2+ motifs, the structure calculations converged readily. Generating PCSs by the dHis-Co2+ motif thus presents an excellent alternative to the use of lanthanide tags.

High-Sensitivity Detection of Nanometer 1H-19F Distances for Protein Structure Determination by 1H-Detected Fast MAS NMR.

Protein structure determination by solid-state NMR requires the measurement of many interatomic distances through dipole-dipole couplings. To obtain multiple long-range distance restraints rapidly and with high sensitivity, here we demonstrate a new 1H-detected fast magic-angle-spinning NMR technique that yields many long distances in a two-dimensional (2D)-resolved fashion. The distances are measured up to ∼15 Å, with an accuracy of better than 10%, between 1H and 19F, two nuclear spins that have the highest gyromagnetic ratios. Exogenous fluorines are sparsely introduced into the aromatic residues of the protein, which is perdeuterated and back-exchanged to give amide protons. This 1H-19F distance experiment, termed 2D heteronuclear single-quantum coherence rotational-echo double-resonance (HSQC-REDOR), is demonstrated on the singly fluorinated model protein, GB1. We extracted 33 distances between 5-19F-Trp43 and backbone amide protons, using 2D spectral series that were measured in less than 3 days. Combining these 1H-19F distance restraints with 13C-19F distances and chemical shifts, we calculated a GB1 structure with a backbone root-mean-square deviation of 1.73 Å from the high-resolution structure. This 1H-detected 1H-19F distance technique promises to provide a highly efficient tool for constraining the three-dimensional structures of proteins and protein-ligand complexes, with not only precise and fast measurements but also access to truly long-range distances.

Characterization of H/D exchange in type 1 pili by proton-detected solid-state NMR and molecular dynamics simulations

Uropathogenic Escherichia coli invades and colonizes hosts by attaching to cells using adhesive pili on the bacterial surface. Although many biophysical techniques have been used to study the structure and mechanical properties of pili, many important details are still unknown. Here we use proton-detected solid-state NMR experiments to investigate solvent accessibility and structural dynamics. Deuterium back-exchange at labile sites of the perdeuterated, fully proton back-exchanged pili was conducted to investigate hydrogen/deuterium (H/D) exchange patterns of backbone amide protons in pre-assembled pili. We found distinct H/D exchange patterns in lateral and axial intermolecular interfaces in pili. Amide protons protected from H/D exchange in pili are mainly located in the core region of the monomeric subunit and in the lateral intermolecular interface, whereas the axial intermolecular interface and the exterior region of pili are highly exposed to H/D exchange. Additionally, we performed molecular dynamics simulations of the type 1 pilus rod and estimated the probability of H/D exchange based on hydrogen bond dynamics. The comparison of the experimental observables and simulation data provides insights into stability and mechanical properties of pili.

Tracking Higher Order Protein Structure by Hydrogen-Deuterium Exchange Mass Spectrometry.

Structural biology has provided a fundamental understanding of protein structure and mechanistic insight into their function. However, high-resolution structures alone are insufficient for a complete understanding of protein behavior. Higher energy conformations, conformational changes, and subtle structural fluctuations that underlie the proper function of proteins are often difficult to probe using traditional structural approaches. Hydrogen/Deuterium Exchange with Mass Spectrometry (HDX-MS) provides a way to probe the accessibility of backbone amide protons under native conditions, which reports on local structural dynamics of solution protein structure that can be used to track complex structural rearrangements that occur in the course of a protein's function. In the last 20 years the advances in labeling techniques, sample preparation, instrumentation, and data analysis have enabled HDX to gain insights into very complex biological systems. Analysis of challenging targets such as membrane protein complexes is now feasible and the field is paving the way to the analysis of more and more complex systems.

Identification of highly potent competence stimulating peptide-based quorum sensing activators in Streptococcus mutans through the utilization of N-methyl and reverse alanine scanning

Quorum sensing (QS) controls the pathogenic behavior of Streptococcus mutans, a primary cause of dental caries. S. mutans uses the competence stimulating peptide (CSP) to control mutacin production, a bacteriocin utilized by S. mutans to outcompete different commensal bacteria in mixed biofilm environments. In this study, we performed an N-methyl scan of an 18-CSP-based scaffold lacking the first two amino acid residues that were shown to be dispensable, to gain important mechanistic insight as to the role of backbone amide protons in the interaction between CSP and the ComD receptor. We then utilized the reverse alanine approach to develop CSP-based analogs with enhanced activities. The two most potent analogs were found to induce bacteriocin production at sub-nanomolar concentration using an interspecies inhibition assay. Overall, our analysis revealed that the 18-CSP sequence is not optimized and can be improved by replacement of multiple positions with alanine. Our results further suggest that the hydrophobic residues in S. mutans 18-CSP are involved in both receptor binding and activation.

Nuclear magnetic resonance-based determination of dioxygen binding sites in protein cavities.

The location and ligand accessibility of internal cavities in cysteine-free wild-type T4 lysozyme was investigated using O2 gas-pressure NMR spectroscopy and molecular dynamics (MD) simulation. Upon increasing the concentration of dissolved O2 in solvent to 8.9 mM, O2 -induced paramagnetic relaxation enhancements (PREs) to the backbone amide and side chain methyl protons were observed, specifically around two cavities in the C-terminal domain. To determine the number of O2 binding sites and their atomic coordinates from the 1/r6 distance dependence of the PREs, we established an analytical procedure using Akaike's Information Criterion, in combination with a grid-search. Two O2 -accessible sites were identified in internal cavities: One site was consistent with the xenon-binding site in the protein in crystal, and the other site was established to be a novel ligand-binding site. MD simulations performed at 10 and 100 mM O2 revealed dioxygen ingress and egress as well as rotational and translational motions of O2 in the cavities. It is therefore suggested that conformational fluctuations within the ground-state ensemble transiently develop channels for O2 association with the internal protein cavities.

NMR spectra of PB2 627, the RNA-binding domain in influenza A virus RNA polymerase that contains the pathogenicity factor lysine 627, and improvement of the spectra by small osmolytes

The influenza A virus, which has an RNA genome, requires RNA-dependent RNA polymerase for transcription and replication. The polymerase is comprised of the subunits PA, PB1, and PB2. The C-terminal RNA-binding domain in PB2 contains lysine 627 (PB2 627), which is associated with pathogenicity and host range. However, the structure and molecular mechanism of PB2 627 in solution remain obscure. Here, we investigated PB2 627 in solution by nuclear magnetic resonance (NMR) and detected inhomogeneity in the intensities of backbone amide proton signals due to local fluctuations in structure. To characterize the effects of chemical chaperones on spectral data and improve the data quality, we tested 20 different additives, including L-arginine L-glutamate salt, (L-arginine)2SO4, glycerol, β-octylglucoside, 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate, Na2SO4, 1,5-diaminopentane, 1,4-diaminobutane, trehalose, sucrose, glycine, trimethylamine N-oxide, β-alanine, L-α-alanine, hydroxyectoine, betaine, L-proline, and non-detergent sulfobetaine 195, 201, and 256. We evaluated the quality of the resulting spectra by calculating the standard deviation and average of the ratio of signal intensities to noise level of amide peaks, as well as the ratio of the standard deviation to the average. NMR-profile analysis revealed diverse effects of additives on the dynamic properties of PB2 627. Based on such criteria, we found that small osmolytes such as glycine and L-α-alanine reduced structural fluctuations and improved the quality of spectral data, which is likely to facilitate a detailed NMR-based structural analysis. The methodology developed here may also be more generally useful for evaluating the effects of chemical chaperones on the structural integrity of proteins.

Protein Structure Determination by Assembling Super-Secondary Structure Motifs Using Pseudocontact Shifts

Computational and nuclear magnetic resonance hybrid approaches provide efficient tools for 3D structure determination of small proteins, but currently available algorithms struggle to perform with larger proteins. Here we demonstrate a new computational algorithm that assembles the 3D structure of a protein from its constituent super-secondary structural motifs (Smotifs) with the help of pseudocontact shift (PCS) restraints for backbone amide protons, where the PCSs are produced from different metal centers. The algorithm, DINGO-PCS (3D assembly of Individual Smotifs to Near-native Geometry as Orchestrated by PCSs), employs the PCSs to recognize, orient, and assemble the constituent Smotifs of the target protein without any other experimental data or computational force fields. Using a universal Smotif database, the DINGO-PCS algorithm exhaustively enumerates any given Smotif. We benchmarked the program against ten different protein targets ranging from 100 to 220 residues with different topologies. For nine of these targets, the method was able to identify near-native Smotifs.

Synthesis of N‐Methylated Pseudodesmin A Analogues: on the Structural Importance of N‐H Hydrogen Bonds

AbstractThe structural importance of backbone amide protons in the bacterial cyclic lipopeptide pseudodesmin A, a viscosin group member, is studied by selective introduction of N‐methylated amino acids. Site‐selective N‐methylation was conveniently achieved by introduction of N−Me‐L–Leu at positions 1 and 7 during solid phase peptide synthesis. The impact of this modification on conformation, antibiotic activity and supramolecular behaviour allowed to establish the significance of intra‐ and intermolecular hydrogen bonds.

DNA Interactions Probed by Hydrogen-Deuterium Exchange (HDX) Fourier Transform Ion Cyclotron Resonance Mass Spectrometry Confirm External Binding Sites on the Minichromosomal Maintenance (MCM) Helicase

The archaeal minichromosomal maintenance (MCM) helicase from Sulfolobus solfataricus (SsoMCM) is a model for understanding structural and mechanistic aspects of DNA unwinding. Although interactions of the encircled DNA strand within the central channel provide an accepted mode for translocation, interactions with the excluded strand on the exterior surface have mostly been ignored with regard to DNA unwinding. We have previously proposed an extension of the traditional steric exclusion model of unwinding to also include significant contributions with the excluded strand during unwinding, termed steric exclusion and wrapping (SEW). The SEW model hypothesizes that the displaced single strand tracks along paths on the exterior surface of hexameric helicases to protect single-stranded DNA (ssDNA) and stabilize the complex in a forward unwinding mode. Using hydrogen/deuterium exchange monitored by Fourier transform ion cyclotron resonance MS, we have probed the binding sites for ssDNA, using multiple substrates targeting both the encircled and excluded strand interactions. In each experiment, we have obtained >98.7% sequence coverage of SsoMCM from >650 peptides (5-30 residues in length) and are able to identify interacting residues on both the interior and exterior of SsoMCM. Based on identified contacts, positively charged residues within the external waist region were mutated and shown to generally lower DNA unwinding without negatively affecting the ATP hydrolysis. The combined data globally identify binding sites for ssDNA during SsoMCM unwinding as well as validating the importance of the SEW model for hexameric helicase unwinding.

Monitoring Backbone Hydrogen-Bond Formation in β-Barrel Membrane Protein Folding.

β-barrel membrane proteins are key components of the outer membrane of bacteria, mitochondria and chloroplasts. Their three-dimensional structure is defined by a network of backbone hydrogen bonds between adjacent β-strands. Here, we employ hydrogen-deuterium (H/D) exchange in combination with NMR spectroscopy and mass spectrometry to monitor backbone hydrogen bond formation during folding of the outer membrane protein X (OmpX) from E. coli in detergent micelles. Residue-specific kinetics of interstrand hydrogen-bond formation were found to be uniform in the entire β-barrel and synchronized to formation of the tertiary structure. OmpX folding thus propagates via a long-lived conformational ensemble state in which all backbone amide protons exchange with the solvent and engage in hydrogen bonds only transiently. Stable formation of the entire OmpX hydrogen bond network occurs downhill of the rate-limiting transition state and thus appears cooperative on the overall folding time scale.