Side Chain Chemical Shift Research Articles

Backbone and side chain chemical shift assignment of diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris, an organophosphorus-degrading enzyme.

NMR chemical shift assignments are reported for backbone (15N, 1H) and partial side chain (13Cα and β, side chain 1H) atoms of diisopropyl fluorophosphatase (DFPase), a calcium-dependent phosphotriesterase capable of hydrolyzing phosphorus - fluorine bonds in a variety of toxic organophosphorus compounds. Analysis of residues lining the active site of DFPase highlight a number of residues whose chemical shifts can be used as a diagnostic of binding and detection of organophosphorus compounds.

Evaluation of implicit solvent models in molecular dynamics simulation of α-Synuclein

We report conventional and accelerated molecular dynamics simulations of α-Synuclein, designed to assess performance of using different starting conformation, solvation environment and force field combination. Backbone and sidechain chemical shifts, radius of gyration, presence of β-hairpin structures in KTK(E/Q)GV repeats and secondary structure percentages were used to evaluate how variations in forcefield, solvation model and simulation protocol provide results that correlate with experimental findings. We show that with suitable choice of forcefield and solvent, ff03ws and OBC implicit model, respectively, acceptable reproduction of experimental data on size and secondary structure is obtained by both conventional and accelerated MD. In contrast to the implicit solvent model, simulations in explicit TIP4P/2005 solvent do not properly represent size or secondary structure of α-Synuclein. Communicated by Ramaswamy H. Sarma

Secondary structure and 1H, 15 N & 13C resonance assignments of the periplasmic domain of OutG, major pseudopilin from Dickeya dadantii type II secretion system

The ability to interact and adapt to the surrounding environment is vital for bacteria that colonise various niches and organisms. One strategy developed by Gram-negative bacteria is to secrete exoprotein substrates via the type II secretion system (T2SS). The T2SS is a proteinaceous complex spanning the bacterial envelope that translocates folded proteins such as toxins and enzymes from the periplasm to the extracellular milieu. In the T2SS, a cytoplasmic ATPase elongates in the periplasm the pseudopilus, a non-covalent polymer composed of protein subunits named pseudopilins, and anchored in the inner membrane by a transmembrane helix. The pseudopilus polymerisation is coupled to the secretion of substrates. The T2SS of Dickeya dadantii secretes more than 15 substrates, essentially plant cell wall degrading enzymes. In D. dadantii, the major pseudopilin or the major subunit of the pseudopilus is called OutG. To better understand the mechanism of secretion of these numerous substrates via the pseudopilus, we have been studying the structure of OutG by NMR. Here, as the first part of this study, we report the 1H, 15N and 13C backbone and sidechain chemical shift assignment of the periplasmic domain of OutG and its NMR derived secondary structure.

Backbone NMR resonance assignment of the intrinsically disordered UBact protein from Nitrospira nitrosa.

Ubiquitin signaling in eukaryotes is responsible for a variety of cellular outcomes, most notably proteasomal degradation. A recent bioinformatic study has revealed the existence of a new proteasomal operon in certain gram-negative bacteria phyla. This operon contains genes similar to those included in the prokaryotic ubiquitin-like protein (Pup) proteasomal operon, but do not themselves contain Pup. Instead, they encode for a protein termed UBact with 30% sequence similarity to Pup. Here, we report the near-complete NMR assignment of the backbone and partial assignment of the side chain chemical shifts of the UBact protein from Nitrospira nitrosa. The 1H-15N HSQC spectrum shows a narrow spread of proton NMR signals, characteristic of an intrinsically disordered protein. This chemical shift assignment will facilitate further NMR studies to explore the role of UBact in this new putative proteasomal operon.

Backbone and nearly complete side-chain chemical shift assignments of the human Uncharacterized protein CXorf51A

Backbone and nearly complete side-chain chemical shift assignments of the human Uncharacterized protein CXorf51A

Backbone and nearly complete side-chain chemical shift assignments reveal the human uncharacterized protein CXorf51A as intrinsically disordered

Even though the human genome project showed that our DNA contains a mere 20,000 to 25,000 protein coding genes, an unexpectedly large number of these proteins remain functionally uncharacterized. A structural characterization of these “unknown” proteins may help to identify possible cellular tasks. We therefore used a combination of bioinformatics and nuclear magnetic resonance spectroscopy to structurally de-orphanize one of these gene products, the 108 amino acid human uncharacterized protein CXorf51A. Both our bioinformatics analysis as well as the ^1H, ^{13}C, ^{15}N backbone and near-complete side-chain chemical shift assignments indicate that it is an intrinsically disordered protein.

1H, 15N and 13C resonance assignments of the C-terminal domain of PulL, a component of the Klebsiella oxytoca type II secretion system.

Type II secretion systems (T2SS) allow Gram-negative bacteria to transport toxins and enzymes from the periplasm to the external milieu, and are thus important for the pathogenicity of bacteria. To drive secretion, T2SS assemble filaments called pseudopili closely related to bacterial type IV pili. These filaments are non-covalent polymers of proteins that are assembled by an inner membrane complex called the assembly platform connected to a cytoplasmic ATPase motor. In the Klebsiella oxytoca T2SS, the PulL protein from the assembly platform is essential for pseudopilus assembly and protein secretion. However, its role in these processes is not well understood. To decipher the molecular basis of PulL function, we used solution NMR to study its structure and interactions with other components of the machinery. Here as a first step, we report the 1H, 15N and 13C backbone and side-chain chemical shift assignments of the C-terminal periplasmic domain of PulL and its secondary structure based on NMR data.

Investigating Atomic Level Structure in Pyriform Silk Proteins

Spider silks are biomaterials used for many diverse adaptations by spiders, with mechanical properties comparable to Kevlar and high strength steel. Orb-weaver spiders create up to seven distinct types of silk, including pyriform silk. Pyriform silk is a major constituent of spider attachment discs, which connect web silks to each other and also to disparate materials using glue-coated fibres. Although filling a critical role in web formation, prior to our work neither the structural nor mechanical properties of pyriform silk had been widely investigated. Based on the central pyriform silk repetitive domain from Argiope argentata, we successfully engineered recombinant pyriform silk-based proteins. Using these proteins, we showed that, in contrast to mechanical extremes of strength at the expense of extensibility or vice versa that are seen in most silks, recombinant pyriform silk is both strong and extensible. To understand the structure-function relationships for this distinctive class of silk, we are performing solution and fibre-state structural studies. Our initial studies have implied distinct regions of order and disorder in the repetitive unit based on the observation of differing degrees of NMR spectroscopy chemical shift dispersion that correlate to the sign of heteronuclear 1H-15N nuclear Overhauser effect enhancements, consistent with a sequence that has segregated regions of disordered and ordered tertiary structuring. Through backbone and side chain chemical shift assignment, we expanded on this, with current data suggesting a central 5-6 helix bundle with long disordered linkers at each end of the bundle that shows no structural perturbation upon the addition of extra repeat blocks, indicating that this protein is likely to behave in a “beads-on-a-string” manner. Current investigation into structural transitions upon fibrillogenesis have shown an anticipated shift from predominantly α-helix in solution to β-sheet in fibres, with further studies ongoing.

NMR assignments for the C-terminal domain of human TDP-43.

Transactive response DNA-binding protein of 43kDa (TDP-43) is a 414-residue protein whose aberrant aggregation is implicated in neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) or frontotemporal lobar degeneration (FTLD). Intriguingly, TDP-43 has also been shown to functionally oligomerize to carry out physiological functions. TDP-43 also exists in mixed condensates or granules with other proteins (e.g. neuronal or stress granules), and its large C-terminal domain (CTD, residues 267-414) seems responsible for TDP-43 both homo- and heterotypic interactions underlying such diverse functional and pathological aggregation events. A myriad of distinct triggers may drive TDP-43 oligomerization, including interaction partners or changes in pH or salinity. In this Assignment Note, we report the complete backbone and a wealth of side chain chemical shift assignments for the CTD of TDP-43at pH 4. The assignments presented here provide a solid starting point to study the aggregation pathway of TDP-43at pH values below those considered physiological but relevant in pathological settings, and to contrast the aggregation behaviour under distinct conditions and in the presence of interacting partners.

Resonance assignments and secondary structure of thermophile single-stranded DNA binding protein from Sulfolobus solfataricus at 323K.

Single-stranded DNA (ssDNA)-binding proteins (SSBs) are essential for DNA replication, recombination, and repair processes in all organisms. Sulfolobus solfataricus (S. solfataricus), a hyperthermophilic species, overexpresses its SSB (S. solfataricus SSB (SsoSSB)) to protect ssDNA during DNA metabolisms. Even though the crystal structure of apo SsoSSB and its ssDNA-bound solution structure have been reported at room temperature, structural information at high temperature is not yet available. To find out how SsoSSB maintains its structure and ssDNA binding affinity at high temperatures, we performed multidimensional NMR experiments for SsoSSB at 323K. In this study, we present the backbone and side chain chemical shifts and predict the secondary structure of SsoSSB from the chemical shifts. We found that SsoSSB is ordered, even at high temperatures, and has the same fold at high temperature as at room temperature. Our data will help improve structural analyses and our understanding of the features of thermophilic proteins.

Backbone and nearly complete side-chain chemical shift assignments of the human death-associated protein 1 (DAP1)

Death-associated protein 1 (DAP1) is a proline-rich cytoplasmatic protein highly conserved in most eukaryotes. It has been reported to be involved in controlling cell growth and migration, autophagy and apoptosis. The presence of human DAP1 is associated to a favourable prognosis in different types of cancer. Here we describe the almost complete {{^{1}}text {H}}, {{^{13}}text {C}}, and {{^{15}}text {N}} chemical shift assignments of the human DAP1. The limited spectral dispersion, mainly in the {{^{1}}text {H}{^{text{N}}}} region, and the lack of defined secondary structure elements, predicted based on chemical shifts, identifies human DAP1 as an intrinsically disordered protein (IDP). This work lays the foundation for further structural investigations, dynamic studies, mapping of potential interaction partners or drug screening and development.

1H, 13C, and 15N backbone and side chain chemical shift assignments of the SARS-CoV-2 non-structural protein 7.

The SARS-CoV-2 genome encodes for approximately 30 proteins. Within the international project covid19-nmr, we distribute the spectroscopic analysis of the viral proteins and RNA. Here, we report NMR chemical shift assignments for the protein nsp7. The 83 amino acid nsp7 protein is an essential cofactor in the RNA-dependent RNA polymerase. The polymerase activity and processivity of nsp12 are greatly enhanced by binding 1 copy of nsp7 and 2 copies of nsp8 to form a 160 kD complex. A separate hexadecameric complex of nsp7 and nsp8 (8 copies of each) forms a large ring-like structure. Thus, nsp7 is an important component of several large protein complexes that are required for replication of the large and complex coronavirus genome. We here report the near-complete NMR backbone and sidechain resonance assignment (1H,13C,15N) of isolated nsp7 from SARS-CoV-2 in solution. Further, we derive the secondary structure and compare it to the previously reported assignments and structure of the SARS-CoV nsp7.

Nearly complete 1H, 13C and 15N chemical shift assignment of monomeric form of N-terminal domain of Nephila clavipes major ampullate spidroin 2.

Spider dragline silk is well recognized due to its excellent mechanical properties. Dragline silk protein mainly consists of two proteins, namely, major ampullate spidroin 1 (MaSp1) and major ampullate spidroin 2 (MaSp2). The MaSp N-terminal domain (NTD) conformation displays a strong dependence on ion and pH gradients, which is crucial for the self-assembly behavior of spider silk. In the spider major ampullate gland, where the pH is neutral and concentration of NaCl is high, the NTD forms a monomer. In contrast, within the spinning duct, where pH becomes more acidic (to pH ~ 5) and the concentration of salt is low, NTD forms a dimer in antiparallel orientation. In this study, we report near-complete backbone and side chain chemical shift assignment of the monomeric form of NTD of MaSp2 from Nephila clavipes at pH 7 in the presence of 300mM NaCl. Our NMR data demonstrate that secondary structure of monomeric form of NTD MaSp2 consists of five helix regions.

Understanding and solving abnormal peak splitting in 3D HCCH-TOCSY and HCC(CO)NH-TOCSY.

The 3D HCCH-TOCSY and HCC(CO)NH-TOCSY experiments provide through bond connectivity and are used for side-chain chemical shift assignment by solution-state NMR. Careful design and implementation of the pulse sequence are key to the successful application of the technique particularly when trying to extract the maximum information out of challenging biomolecules. Here we investigate the source of and propose solutions for abnormal peak splitting ranging from 152 to 80Hz and below that were found in three popular TOCSY-based experiment types: H(F1)-C(F2)-DIPSI-H(F3), C(F1)-DIPSI-C(F2)-H(F3), and C(F1)-DIPSI-N(F2)-HN(F3). Peak splitting occurs in the indirect C(F1) or C(F2) dimension before DIPSI and analyses indicate that the artifacts are resulted mainly from the DIPSI transforming a double spin order [Formula: see text] from 13C-13C scalar 1JCC coupling during t1 into observable megnetization. The splitting is recapitulated by numerical simulation and approaches are proposed to remove it. Adding a pure delay of 3.7ms immediately before DIPSI is a simple and effective strategy to achieve 3D HCCH-TOCSY and HCC(CO)NH-TOCSY spectra free of splitting with full crosspeak intensity.

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.

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 .

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.

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.