Backbone Chemical Shifts Research Articles

PH-Dependent Protonation of Surface Carboxylate Groups in PsbO Enables Local Buffering and Triggers Structural Changes.

Photosystem II (PSII) catalyzes the splitting of water, releasing protons and dioxygen. Its highly conserved subunit PsbO extends from the oxygen‐evolving center (OEC) into the thylakoid lumen and stabilizes the catalytic Mn4CaO5 cluster. The high degree of conservation of accessible negatively charged surface residues in PsbO suggests additional functions, as local pH buffer or by affecting the flow of protons. For this discussion, we provide an experimental basis, through the determination of pK a values of water‐accessible aspartate and glutamate side‐chain carboxylate groups by means of NMR. Their distribution is strikingly uneven, with high pK a values around 4.9 clustered on the luminal PsbO side and values below 3.5 on the side facing PSII. pH‐dependent changes in backbone chemical shifts in the area of the lumen‐exposed loops are observed, indicating conformational changes. In conclusion, we present a site‐specific analysis of carboxylate group proton affinities in PsbO, providing a basis for further understanding of proton transport in photosynthesis.

Conditional Disorder in Small Heat-shock Proteins

Small heat-shock proteins (sHSPs) are molecular chaperones that respond to cellular stresses to combat protein aggregation. HSP27 is a critical human sHSP that forms large, dynamic oligomers whose quaternary structures and chaperone activities depend on environmental factors. Upon exposure to cellular stresses, such as heat shock or acidosis, HSP27 oligomers can dissociate into dimers and monomers, which leads to significantly enhanced chaperone activity. The structured core of the protein, the α-crystallin domain (ACD), forms dimers and can prevent the aggregation of substrate proteins to a similar degree as the full-length protein. When the ACD dimer dissociates into monomers, it partially unfolds and exhibits enhanced activity. Here, we used solution-state NMR spectroscopy to characterize the structure and dynamics of the HSP27 ACD monomer. Web show that the monomer is stabilized at low pH and that its backbone chemical shifts, 15N relaxation rates, and 1H–15N residual dipolar couplings suggest structural changes and rapid motions in the region responsible for dimerization. By analyzing the solvent accessible and buried surface areas of sHSP structures in the context of a database of dimers that are known to dissociate into disordered monomers, we predict that ACD dimers from sHSPs across all kingdoms of life may partially unfold upon dissociation. We propose a general model in which conditional disorder—the partial unfolding of ACDs upon monomerization—is a common mechanism for sHSP activity.

Structure and Topology of the SERCA Regulator DWORF in Lipid Bilayers by Oriented Sample Solid-State NMR

Calcium (Ca2+) dysregulation is a hallmark of heart failure and is characterized by impaired Ca2+ sequestration into the sarcoplasmic reticulum (SR) by the SR-Ca2+-ATPase (SERCA). Regulins, single-pass membrane proteins, bind and inhibit SERCA by allosterically modulating the affinity of its Ca2+ binding sites. DWarf Open Reading Frame (DWORF) is a 35 amino acid regulin that enhances SERCA2a activity in cardiomyocytes. As a first step to understanding DWORF regulation of SERCA, we used Oriented-Sample Solid-State NMR spectroscopy (OS-ssNMR) and computational methods to determine the structure and topology of DWORF in liquid crystalline lipid bilayers. We found that DWORF adopts a helical structure, with a pronounced kink at the N-terminus. Restrained molecular dynamics calculations using backbone dipolar couplings and chemical shift anisotropy show the dynamic topology of DWORF, with the N-terminal helix spanning tilt angles ranging from 50-60 degrees and C-terminal helix ranging from 30-40 degrees. The two helical domains are anchored to both membrane leaflets by polar residues. This topology differs from that of phospholamban, a main regulator of SERCA, and might explain the opposite effects on the ATPase's apparent affinity for Ca2+ ions of these two regulins.

1H, 13C, and 15N resonance assignment and secondary structure of the pheromone-binding protein2 from the agricultural pest Ostrinia furnacalis (OfurPBP2).

Ostrinia furnacalis, a lepidopteran moth, is an invasive pest found in Asia, Australia, Africa, and parts of the United States. The O. furnacalis pheromone-binding protein2 (OfurPBP2), present in the male moth antenna, plays a role in the detection of female-secreted pheromone in a process that leads to mating. To understand the structural mechanism of pheromone binding and release in this pest, we have initiated characterization of OfurPBP2 by solution NMR. Here, we report the backbone resonance assignments and the secondary structural elements of OfurPBP2 at pH 6.5 using uniformly 13C, 15N-labeled protein with various triple resonance NMR experiments. The assignments are 97% completed for backbone and 88% completed for side-chain resonances. The secondary structure of OfurPBP2, based on backbone chemical shifts, consists of eight α-helices, including a well-structured C-terminal helix.

Boosting the resolution of low-field [Formula: see text] relaxation experiments on intrinsically disordered proteins with triple-resonance NMR.

Improving our understanding of nanosecond motions in disordered proteins requires the enhanced sampling of the spectral density function obtained from relaxation at low magnetic fields. High-resolution relaxometry and two-field NMR measurements of relaxation have, so far, only been based on the recording of one- or two-dimensional spectra, which provide insufficient resolution for challenging disordered proteins. Here, we introduce a 3D-HNCO-based two-field NMR experiment for measurements of protein backbone [Formula: see text] amide longitudinal relaxation rates. The experiment provides accurate longitudinal relaxation rates at low field (0.33 T in our case) preserving the resolution and sensitivity typical for high-field NMR spectroscopy. Radiofrequency pulses applied on six different radiofrequency channels are used to manipulate the spin system at both fields. The experiment was demonstrated on the C-terminal domain of [Formula: see text] subunit of RNA polymerase from Bacillus subtilis, a protein with highly repetitive amino-acid sequence and very low dispersion of backbone chemical shifts.

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.

Backbone chemical shifts of the PICK1 PDZ domain with the GluA2 C-terminal

Backbone chemical shifts of the PICK1 PDZ domain with the GluA2 C-terminal

Backbone and side-chain chemical shift assignments of the ribosome-inactivating protein trichobakin (TBK) in solution

Backbone and side-chain chemical shift assignments of the ribosome-inactivating protein trichobakin (TBK) in solution

Backbone and side chain 1H, 15N and 13C chemical shift assignments of the molten globule state of L94G mutant of horse cytochrome-c.

Proteins fold via a number of intermediates that help them to attain their unique native 3D structure. These intermediates can be trapped under extreme conditions of pH, temperature and chemical denaturants. Similar states can also be achieved by other processes like chemical modification, site directed mutagenesis (or point mutation) and cleavage of covalent bonds of natural proteins under physiological conditions usually taken as dilute buffer (near neutral pH) and 25°C. Structural characterization of molten globules is hampered due to (i) their transient nature, (ii) very low population at equilibrium, and (iii) prone to aggregation at high concentration. Furthermore, the dynamic nature of these folding intermediates makes them unsuitable for X-ray diffraction. Hence, understanding their structures at the atomic level is often a challenge. However, characterization of these intermediates at the atomic level is possible by NMR, which could possibly unravel new details of the protein folding process. We have previously shown that the L94G mutant of horse cytochrome-c displays characteristics of the molten globule (MG) state at pH 6.0 and 25°C. As a first step towards characterizing this MG state at the atomic level by NMR, we report its complete backbone, side chain and heme chemical shift assignments.

1H, 13C, and 15N backbone and side chain chemical shift assignment of YdaS, a monomeric member of the HigA family.

The cryptic prophage CP-933P in Escherichia coli O157:H7 contains a parDE-like toxin-antitoxin module, the operator region of which is recognized by two flanking transcription regulators: PaaR2 (ParE associated Regulator), which forms part of the paaR2-paaA2-parE2 toxin-antitoxin operon and YdaS (COG4197), which is encoded in the opposite direction but shares the operator. Here we report the 1H, 15N and 13C backbone and side chain chemical shift assignments of YdaS from Escherichia coli O157:H7 in its free state. YdaS is a distinct relative to HigA antitoxins but behaves as a monomer in solution. The BMRB Accession Number is 27917.

Integrative Protein Modeling in RosettaNMR from Sparse Paramagnetic Restraints

Computational methods to predict protein structure from nuclear magnetic resonance (NMR) restraints that only require assignment of backbone signals, hold great potential to study larger proteins. Ideally, computational methods designed to work with sparse data need to add atomic detail that is missing in the experimental restraints. We introduce a comprehensive framework into the Rosetta suite that uses NMR restraints derived from paramagnetic labeling. Specifically, RosettaNMR incorporates pseudocontact shifts, residual dipolar couplings, and paramagnetic relaxation enhancements. It continues to use backbone chemical shifts and nuclear Overhauser effect distance restraints. We assess RosettaNMR for protein structure prediction by folding 28 monomeric proteins and 8 homo-oligomeric proteins. Furthermore, the general applicability of RosettaNMR is demonstrated on two protein-protein and three protein-ligand docking examples. Paramagnetic restraints generated more accurate models for 85% of the benchmark proteins and, when combined with chemical shifts, sampled high-accuracy models (≤2Å) in 50% of the cases.

Solution NMR backbone assignment reveals interaction-free tumbling of human lineage-specific Olduvai protein domains.

Olduvai protein domains, encoded primarily by NBPF genes, have been linked to both human brain evolution and cognitive diseases such as autism and schizophrenia. There are six primary domains that comprise the Olduvai family: three conserved domains (CON1-3) and three human lineage-specific domains (HLS1-3), which typically occur as a triplet (HLS1, HLS2 and HLS3). Herein, we present the solution NMR assignment of the backbone chemical shifts of the separate HLS1, 2 and 3 domains of NBPF15. Our data suggest that there is no change in the structure of the separate domains when compared to the full-length triplet (HLS1-HLS2-HLS3). We also demonstrate that there is no direct interaction between the three domains.

Backbone (1H and 15N) Chemical Shift Assignments and Relaxation Parameters for the low complexity domain of FUS in the condensed phase

Backbone (1H and 15N) Chemical Shift Assignments and Relaxation Parameters for the low complexity domain of FUS in the condensed phase

I-PINE web server: an integrative probabilistic NMR assignment system for proteins.

Various methods for understanding the structural and dynamic properties of proteins rely on the analysis of their NMR chemical shifts. These methods require the initial assignment of NMR signals to particular atoms in the sequence of the protein, a step that can be very time-consuming. The probabilistic interaction network of evidence (PINE) algorithm for automated assignment of backbone and side chain chemical shifts utilizes a Bayesian probabilistic network model that analyzes sequence data and peak lists from multiple NMR experiments. PINE, which is one of the most popular and reliable automated chemical shift assignment algorithms, has been available to the protein NMR community for longer than a decade. We announce here a new web server version of PINE, called Integrative PINE (I-PINE), which supports more types of NMR experiments than PINE (including three-dimensional nuclear Overhauser enhancement and four-dimensional J-coupling experiments) along with more comprehensive visualization of chemical shift based analysis of protein structure and dynamics. The I-PINE server is freely accessible at http://i-pine.nmrfam.wisc.edu . Help pages and tutorial including browser capability are available at: http://i-pine.nmrfam.wisc.edu/instruction.html . Sample data that can be used for testing the web server are available at: http://i-pine.nmrfam.wisc.edu/examples.html .

Structural insight into the unique dsDNA binding topology of the human ORC2 wing helix domain.

The origin recognition complex (ORC) is indispensable for the initiation of DNA replication during the cell cycle. The DNA-binding modes of the human ORC winged helix domain (WHD) remain enigmatic, as the dsDNA recognition sites of archaeal and Saccharomycescerevisiae ORC WHDs are distinct. Here, we solved the high-resolution crystal structure of the human ORC2 WHD, although its complex with dsDNA is difficult to crystallize due to its weak binding affinities. The near-complete NMR backbone assignments and chemical shift perturbations reveal a new dsDNA binding topology in addition to the conserved β-sheet hairpin region, in which residues show higher dynamics. The key interacting residues (R540, K548, and K549) were validated by mutagenesis studies. Our data suggest that the ORC2 WHD recognizes dsDNA sequences through its flexible β-sheet hairpin as an anchor point, while the rest of the protein adopts various orientations in different species. This weak but real interaction module identified by NMR is useful for the structural reconstruction of large biomolecular complexes using cryo-EM. The binding topology and dynamics of ORC2 WHDs were also underpinned by molecular dynamics simulations.

Probing initial transient oligomerization events facilitating Huntingtin fibril nucleation at atomic resolution by relaxation-based NMR

The N-terminal region of the huntingtin protein, encoded by exon-1, comprises an amphiphilic domain (httNT), a polyglutamine (Q n ) tract, and a proline-rich sequence. Polyglutamine expansion results in an aggregation-prone protein responsible for Huntington's disease. Here, we study the earliest events involved in oligomerization of a minimalistic construct, httNTQ7, which remains largely monomeric over a sufficiently long period of time to permit detailed quantitative NMR analysis of the kinetics and structure of sparsely populated [Formula: see text] oligomeric states, yet still eventually forms fibrils. Global fitting of concentration-dependent relaxation dispersion, transverse relaxation in the rotating frame, and exchange-induced chemical shift data reveals a bifurcated assembly mechanism in which the NMR observable monomeric species either self-associates to form a productive dimer (τex ∼ 30 μs, K diss ∼ 0.1 M) that goes on to form a tetramer ([Formula: see text] μs; K diss ∼ 22 μM), or exchanges with a "nonproductive" dimer that does not oligomerize further (τex ∼ 400 μs; K diss ∼ 0.3 M). The excited state backbone chemical shifts are indicative of a contiguous helix (residues 3-17) in the productive dimer/tetramer, with only partial helical character in the nonproductive dimer. A structural model of the productive dimer/tetramer was obtained by simulated annealing driven by intermolecular paramagnetic relaxation enhancement data. The tetramer comprises a D 2 symmetric dimer of dimers with largely hydrophobic packing between the helical subunits. The structural model, validated by EPR distance measurements, illuminates the role of the httNT domain in the earliest stages of prenucleation and oligomerization, before fibril formation.

GPU Accelerated Computation of Isotropic Chemical Shifts Offers New Dimension of Structure Refinement in Largescale Molecular Dynamics Simulation

Semi-empirical chemical shift prediction, where molecular coordinates and empirical data are reduced to backbone and sidechain chemical shifts through input to differentiable functions, offers a unique opportunity to validate and refine protein structure during largescale molecular dynamics (MD) simulation but poses the challenge of optimizing performance to levels comparable to modern GPU-enabled MD engines. The software PPM_One predicts backbone and sidechain chemical shifts with competitive accuracy to purely data-driven counterparts while obviating the need for deep network processing and homology assignment. To poise PPM_One for practical application in largescale biomolecular modeling, a newly optimized GPU-accelerated version of PPM_One was developed. Code refactoring and parallel GPU-processing through the directive-based OpenACC API provide a 67-fold speedup between unaccelerated and Nvidia V100 accelerated PPM_One test-cases, comparatively, in a system of 21 million protein atoms. The core functions that comprise the prediction model and compute perturbation due to Hydrogen bonding, magnetic anisotropy and ring currents were profiled at 271x, 322x, and 211x speedups, respectively. Remaining components of the predictive model saw similar individual speedups through profiling. Implementing the newly optimized functional core into NAMD is NMRForces, a global force module that enables users to compute backbone and sidechain chemical shifts at each timestep and force atoms along the prediction model's gradient with a magnitude proportional to deviance from experimental chemical shift data.

PH-Driven Conformational Reorganization of Proteins: NMR Spectroscopy Study with Buried Lys Residues

The pH sensitivity of proteins is essential for biological energy transduction and for cellular and physiological homeostasis. The thermodynamic basis of pH effects in proteins is well understood but detailed mechanistic understanding of how changes in pH drive conformational reorganization in proteins is not. This was addressed systematically using NMR spectroscopy to characterize pH-dependent conformational changes coupled to the ionization of residues buried in the hydrophobic core of a protein. The study used 25 variants of staphylococcal nuclease with buried Lys residues. The pKa values of 19 of 25 of these internal Lys residues are depressed relative to the normal pKa of Lys in water, some by as much as 5 pH units. This shift in pKa ensures that the ionizable moiety stays neutral at pH values below 10 when it is buried, consistent with what is expected from the unfavorable transfer of a charge from water to a hydrophobic environment. In most cases, ionization of the buried Lys shifts the conformation of the protein into a state where the charged Lys can contact water. This study contributes a detailed description of the location, amplitude and time scale of reorganization coupled to the ionization of buried Lys residues. The structural details of the conformational response were characterized with backbone chemical shift perturbation analysis, titrations of the buried Nζ atom, and in some cases even X-ray crystallography. At least in this protein, the anomalous pKa values of buried groups are governed by the propensity of the protein to reorganize. These results demonstrate that computational modeling of pH dependent processes in proteins will require prediction of alternative conformational states and accurate calculation of free energies; this remains a significant challenge even under the most favorable circumstances.

1H, 15N and 13C backbone assignment of apo TDP-43 RNA recognition motifs.

TAR DNA-binding protein 43 (TDP-43) is a ubiquitously expressed nuclear protein that influences diverse cellular processes by regulating alternative splicing of RNA and microRNA biogenesis. It is also a pathological protein found in sporadic ALS and in the most common subtype of frontotemporal lobar degeneration with ubiquitinated inclusions (FLTD-U). TDP-43 has two tandem RNA-binding domains, RRM1 and RRM2. The NMR structure of TDP-43 was solved in the presence of UG-rich RNA sequences bound to the RRM1 and RRM2 domains. Here we report the backbone assignment of apo TDP-43. The chemical shift (HN, N, C, Cα and Cβ) analysis shows the predicted regions of secondary structure are in good agreement with those observed for TDP-43 in complex with RNA. However, our data show that the apo structure of TPD-43 has increased flexibility in the regions that would normally have been used to anchor the RNA bases. The backbone chemical shifts assignments will prove useful in the study of TDP-43 interaction with non-canonical RNA and RRM-binding proteins.

Complete NMR chemical shift assignments of odorant binding protein 22 from the yellow fever mosquito, Aedes aegypti, bound to arachidonic acid

Aedes aegypti mosquitoes are the vector for transmission of Dengue, Zika and chikungunya viruses. These mosquitos feed exclusively on human hosts for a blood meal. Previous studies have established that Dengue virus infection of the mosquito results in increased expression of the odorant binding proteins 22 and 10 within the mosquito salivary gland and silencing of these genes dramatically reduces blood-feeding behaviors. Odorant binding proteins are implicated in modulating the chemosensory perception of external stimuli that regulate behaviors such as host location, feeding and reproduction. However, the role that AeOBP22 plays in the salivary gland is unclear. Here, as a first step to a more complete understanding of the function of AeOBP22, we present the complete backbone and side chain chemical shift assignments of the protein in the complex it forms with arachidonic acid. These assignments reveal that the protein consists of seven α-helices, and that the arachidonic acid is bound tightly to the protein. Comparison with the chemical shift assignments of the apo-form of the protein reveals that binding of the fatty acid is accompanied by a large conformational change in the C-terminal helix, which appears disordered in the absence of lipid. This NMR data provides the basis for determining the structure of AeOBP22 and understanding the nature of the conformational changes that occur upon ligand binding. This information will provide a path to discover novel compounds that can interfere with AeOBP22 function and impact blood feeding by this mosquito.