(3, 2)D <Superscript>1</Superscript>H, <Superscript>13</Superscript>C BIRD<Superscript>r,X</Superscript>-HSQC-TOCSY for NMR structure elucidation of mixtures: application to complex carbohydrates

Overlap of NMR signals is the major cause of difficulties associated with NMR structure elucidation of molecules contained in complex mixtures. A 2D homonuclear correlation spectroscopy in particular suffers from low dispersion of ¹H chemical shifts; larger dispersion of ¹³C chemical shifts is often used to reduce this overlap, while still providing the proton–proton correlation information e.g. in the form of a 2D ¹H, ¹³C HSQC-TOCSY experiment. For this methodology to work, ¹³C chemical shift must be resolved. In case of ¹³C chemical shifts overlap, ¹H chemical shifts can be used to achieve the desired resolution. The proposed (3, 2)D ¹H, ¹³C BIRD^r,X-HSQC-TOCSY experiment achieves this while preserving singlet character of cross peaks in the F₁ dimension. The required high-resolution in the ¹³C dimension is thus retained, while the cross peak overlap occurring in a regular HSQC-TOCSY experiment is eliminated. The method is illustrated on the analysis of a complex carbohydrate mixture obtained by depolymerisation of a fucosylated chondroitin sulfate isolated from the body wall of the sea cucumber Holothuria forskali.     

Direct amide <Superscript>15</Superscript>N to <Superscript>13</Superscript>C transfers for solid-state assignment experiments in deuterated proteins

The assignment of protein backbone and side-chain NMR chemical shifts is the first step towards the characterization of protein structure. The recent introduction of proton detection in combination with fast MAS has opened up novel opportunities for assignment experiments. However, typical 3D sequential-assignment experiments using proton detection under fast MAS lead to signal intensities much smaller than the theoretically expected ones due to the low transfer efficiency of some of the steps. Here, we present a selective 3D experiment for deuterated and (amide) proton back-exchanged proteins where polarization is directly transferred from backbone nitrogen to selected backbone or sidechain carbons. The proposed pulse sequence uses only ¹H–¹⁵N cross-polarization (CP) transfers, which are, for deuterated proteins, about 30% more efficient than ¹H–¹³C CP transfers, and employs a dipolar version of the INEPT experiment for N–C transfer. By avoiding H^N–C (H^N stands for amide protons) and C–C CP transfers, we could achieve higher selectivity and increased signal intensities compared to other pulse sequences containing long-range CP transfers. The REDOR transfer is designed with an additional selective π pulse, which enables the selective transfer of the polarization to the desired ¹³C spins.     

<Superscript>1</Superscript>H, <Superscript>15</Superscript>N, <Superscript>13</Superscript>C backbone resonance assignment of the C-terminal domain of enzyme I from <Emphasis Type="Italic">Thermoanaerobacter tengcongensis</Emphasis>

Phosphoenolpyruvate binding to the C-terminal domain (EIC) of enzyme I of the bacterial phosphotransferase system (PTS) initiates a phosphorylation cascade that results in sugar translocation across the cell membrane and controls a large number of essential pathways in bacterial metabolism. EIC undergoes an expanded to compact conformational equilibrium that is regulated by ligand binding and determines the phosphorylation state of the overall PTS. Here, we report the backbone ¹H, ¹⁵N and ¹³C chemical shift assignments of the 70 kDa EIC dimer from the thermophilic bacterium Thermoanaerobacter tengcongensis. Assignments were obtained at 70 °C by heteronuclear multidimensional NMR spectroscopy. In total, 90% of all backbone resonances were assigned, with 264 out of a possible 299 residues assigned in the ¹H–¹⁵N TROSY spectrum. The secondary structure predicted from the assigned backbone resonance using the program TALOS+ is in good agreement with the X-ray crystal structure of T. tengcongensis EIC. The reported assignments will allow detailed structural and thermodynamic investigations on the coupling between ligand binding and conformational dynamics in EIC.     

Near-complete <Superscript>1</Superscript>H, <Superscript>13</Superscript>C, <Superscript>15</Superscript>N resonance assignments of dimethylsulfoxide-denatured TGFBIp FAS1-4 A546T

The transforming growth factor beta induced protein (TGFBIp) is a major protein component of the human cornea. Mutations occurring in TGFBIp may cause corneal dystrophies, which ultimately lead to loss of vision. The majority of the disease-causing mutations are located in the C-terminal domain of TGFBIp, referred as the fourth fascilin-1 (FAS1-4) domain. In the present study the FAS1-4 Ala546Thr, a mutation that causes lattice corneal dystrophy, was investigated in dimethylsulfoxide using liquid-state NMR spectroscopy, to enable H/D exchange strategies for identification of the core formed in mature fibrils. Isotope-labeled fibrillated FAS1-4 A546T was dissolved in a ternary mixture 95/4/1 v/v/v% dimethylsulfoxide/water/trifluoroacetic acid, to obtain and assign a reference 2D ¹H–¹⁵N HSQC spectrum for the H/D exchange analysis. Here, we report the near-complete assignments of backbone and aliphatic side chain ¹H, ¹³C and ¹⁵N resonances for unfolded FAS1-4 A546T at 25 °C.     

Measurement of protein backbone <Superscript>13</Superscript>CO and <Superscript>15</Superscript>N relaxation dispersion at high resolution

The ³¹P NMR pressure response of guanine nucleotides bound to proteins has been studied in the past for characterizing the pressure perturbation of conformational equilibria. The pressure response of the ³¹P NMR chemical shifts of the phosphate groups of GMP, GDP, and GTP as well as the commonly used GTP analogs GppNHp, GppCH₂p and GTPγS was measured in the absence and presence of Mg²⁺-ions within a pressure range up to 200 MPa. The pressure dependence of chemical shifts is clearly non-linear. For all nucleotides a negative first order pressure coefficient B ₁ was determined indicating an upfield shift of the resonances with pressure. With exception of the α-phosphate group of Mg²⁺·GMP and Mg²⁺·GppNHp the second order pressure coefficients are positive. To describe the data of Mg²⁺·GppCH₂p and GTPγS a Taylor expansion of 3rd order is required. For distinguishing pH effects from pressure effects a complete pH titration set is presented for GMP, as well as GDP and GTP in absence and presence of Mg²⁺ ions using indirect referencing to DSS under identical experimental conditions. By a comparison between high pressure ³¹P NMR data on free Mg²⁺-GDP and Mg²⁺-GDP in complex with the proto-oncogene Ras we demonstrate that pressure induced changes in chemical shift are clearly different between both forms.     

Critical test of some computational methods for prediction of NMR <Superscript>1</Superscript>H and <Superscript>13</Superscript>C chemical shifts

Performance of 18 DFT functionals (B1B95, B3LYP, B3PW91, B97D, BHandHLYP, BMK, CAM-B3LYP, HSEh1PBE, M06-L, mPW1PW91, O3LYP, OLYP, OPBE, PBE1PBE, tHCTHhyb, TPSSh, wB97xD, VSXC) in combinations with six basis sets (cc-pVDZ, aug-cc-pVDZ, cc-pVTZ, aug-cc-pVTZ, IGLO-II, and IGLO-III) and three methods for calculating magnetic shieldings (GIAO, CSGT, IGAIM) was tested for predicting ¹H and ¹³C chemical shifts for 25 organic compounds, for altogether 86 H and 88 C atoms. Proton shifts varied between 1.03 ppm to 12.00 ppm and carbon shifts between 7.87 ppm to 209.28 ppm. It was found that the best method for calculating ¹³C shifts is PBE1PBE/aug-cc-pVDZ with CSGT or IGAIM approaches (mae?=?1.66 ppm), for ¹H the best results were obtained with HSEh1PBE, mPW1PW91, PBE1PBE, CAM-B3LYP, and B3PW91 functionals with cc-pVTZ basis set and with CSGT or IGAIM approaches (mae?=?0.28 ppm). We found that often larger basis sets do not give better results for chemical shifts. The best basis sets for calculating ¹H and ¹³C chemical shifts were cc-pVTZ and aug-cc-pVDZ, respectively. CSGT and IGAIM NMR approaches can perform really well and are in most cases better than popular GIAO approach.

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Graphical Abstract Mean absolute errors for ¹H and ¹³C chemical shifts and computational times of neutral toluene molecule with aug-cc-pVDZ basis set and CSGT approach

     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N chemical shift assignments of Saccharomyces cerevisiae type 1 thioredoxin in the oxidized state by solution NMR spectroscopy

Thioredoxins (Trx) are ubiquitous proteins that regulate several biochemical processes inside the cell. Trx is an important player, displaying oxidoreductase activity and helping to keep and regulate the oxidative state of the cellular environment. Trx also participates in the regulation of many cellular functions, such as DNA synthesis, protection against oxidative stress, cell cycle and signal transduction. The oxidized Trx is the target for another set of proteins, such as thioredoxin reductase (TrR), which used the reductive potential of NADPH. The oxidized state of Trx also plays important role in regulation of redox state in the cells. In this regard, the oxidized form of Trx is a putative conformer that contributes to the cellular redox environment. Here we report the chemical shift assignments (¹H, ¹³C and ¹⁵N) in solution at 15 °C. We also showed the secondary structure analysis of the oxidized form of yeast thioredoxin (yTrx1) as basis for future NMR studies of protein–target interactions and dynamics. The assignment was done at low concentration (200 µM) because it is important to keep intact the water cavity.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N backbone resonance assignment of the lamin C-terminal region specific to prelamin A

Lamins are the main components of the nucleoskeleton. They form a protein meshwork that underlies the inner nuclear membrane. Mutations in the LMNA gene coding for A-type lamins (lamins A and C) cause a large panel of human diseases, referred to as laminopathies. These diseases include muscular dystrophies, lipodystrophies and premature aging diseases. Lamin A exhibits a C-terminal region that is different from lamin C and is post-translationally modified. It is produced as prelamin A and it is then farnesylated, cleaved, carboxymethylated and cleaved again in order to become mature lamin A. In patients with the severe Hutchinson–Gilford progeria syndrome, a specific single point mutation in LMNA leads to an aberrant splicing of the LMNA gene preventing the post-translational processing of prelamin A. This leads to the accumulation of a permanently farnesylated lamin A mutant lacking 50 amino acids named progerin. We here report the NMR ¹H, ¹⁵N, ¹³CO, ¹³Cα and ¹³Cβ chemical shift assignment of the C-terminal region that is specific to prelamin A, from amino acid 567 to amino acid 664. We also report the NMR ¹H, ¹⁵N, ¹³CO, ¹³Cα and ¹³Cβ chemical shift assignment of the C-terminal region of the progerin variant, from amino acid 567 to amino acid 614. Analysis of these chemical shift data confirms that both prelamin A and progerin C-terminal domains are largely disordered and identifies a common partially populated α-helix from amino acid 576 to amino acid 585. This helix is well conserved from fishes to mammals.     

<Superscript>1</Superscript>H, <Superscript>15</Superscript>N,and <Superscript>13</Superscript>C chemical shift assignments of the regulatory domain of human calcineurin

Calcineurin (CaN) plays an important role in T-cell activation, cardiac system development and nervous system function. Previous studies have demonstrated that the regulatory domain (RD) of CaN binds calmodulin (CaM) towards the N-terminal end. Calcium-loaded CaM activates the serine/threonine phosphatase activity of CaN by binding to the RD, although the mechanistic details of this interaction remain unclear. It is thought that CaM binding at the RD displaces the auto-inhibitory domain (AID) from the active site of CaN, activating phosphatase activity. In the absence of calcium-loaded CaM, the RD is disordered, and binding of CaM induces folding in the RD. In order to provide mechanistic detail about the CaM–CaN interaction, we have undertaken an NMR study of the RD of CaN. Complete ¹³C, ¹⁵N and ¹H assignments of the RD of CaN were obtained using solution NMR spectroscopy. The backbone of RD has been assigned using a combination of ¹³C-detected CON-IPAP experiments as well as traditional HNCO, HNCA, HNCOCA and HNCACB-based 3D NMR spectroscopy. A ¹⁵N-resolved TOCSY experiment has been used to assign Hα and Hβ chemical shifts.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N resonance assignment of human guanylate kinase

Human guanylate kinase (hGMPK) is a critical enzyme that, in addition to phosphorylating its physiological substrate (d)GMP, catalyzes the second phosphorylation step in the conversion of anti-viral and anti-cancer nucleoside analogs to their corresponding active nucleoside analog triphosphates. Until now, a high-resolution structure of hGMPK is unavailable and thus, we studied free hGMPK by NMR and assigned the chemical shift resonances of backbone and side chain ¹H, ¹³C, and ¹⁵N nuclei as a first step towards the enzyme’s structural and mechanistic analysis with atomic resolution.     

Automatic <Superscript>13</Superscript>C chemical shift reference correction for unassigned protein NMR spectra

Poor chemical shift referencing, especially for ¹³C in protein Nuclear Magnetic Resonance (NMR) experiments, fundamentally limits and even prevents effective study of biomacromolecules via NMR, including protein structure determination and analysis of protein dynamics. To solve this problem, we constructed a Bayesian probabilistic framework that circumvents the limitations of previous reference correction methods that required protein resonance assignment and/or three-dimensional protein structure. Our algorithm named Bayesian Model Optimized Reference Correction (BaMORC) can detect and correct ¹³C chemical shift referencing errors before the protein resonance assignment step of analysis and without three-dimensional structure. By combining the BaMORC methodology with a new intra-peaklist grouping algorithm, we created a combined method called Unassigned BaMORC that utilizes only unassigned experimental peak lists and the amino acid sequence. Unassigned BaMORC kept all experimental three-dimensional HN(CO)CACB-type peak lists tested within ±?0.4 ppm of the correct ¹³C reference value. On a much larger unassigned chemical shift test set, the base method kept ¹³C chemical shift referencing errors to within ±?0.45 ppm at a 90% confidence interval. With chemical shift assignments, Assigned BaMORC can detect and correct ¹³C chemical shift referencing errors to within ±?0.22 at a 90% confidence interval. Therefore, Unassigned BaMORC can correct ¹³C chemical shift referencing errors when it will have the most impact, right before protein resonance assignment and other downstream analyses are started. After assignment, chemical shift reference correction can be further refined with Assigned BaMORC. These new methods will allow non-NMR experts to detect and correct ¹³C referencing error at critical early data analysis steps, lowering the bar of NMR expertise required for effective protein NMR analysis.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N NMR assignments of a bacterial immunoglobulin-like domain (group 2) of a protein of a bacterium <Emphasis Type="Italic">Paenarthrobacter aurescens</Emphasis> TC1

The bacterial immunoglobulin-like (Big) domain is one of the prevalent domain types, which facilitates cell–cell adhesion by assembling into multi-domain architectures. We selected a four Big_2 domain protein (named ‘Arig’) from a Gram positive, Paenarthrobacter aurescens TC1 (known earlier as Arthrobacter aurescens TC1). In an attempt to characterize structural and ligand-binding features of individual Big_2 domains, we have cloned, overexpressed, isolated and purified the second Big_2 domain of Arig along with a few of its adjacent Big_2 domain residues (residue 143 to 269) referred to as ‘Arig2’. The ¹³C/¹⁵N-doubly-labeled His-tagged Arig2 (133 residues long) showed an ordered conformation as revealed by the well dispersed 2D [¹⁵N-¹H]-HSQC spectrum. Subsequently, a suite of heteronuclear 3D NMR experiments has enabled almost complete ¹H, ¹³C and ¹⁵N NMR resonance assignments of Arig2.     

<Superscript>13</Superscript>C-direct detected NMR experiments for the sequential J-based resonance assignment of RNA oligonucleotides

We present here a set of ¹³C-direct detected NMR experiments to facilitate the resonance assignment of RNA oligonucleotides. Three experiments have been developed: (1) the (H)CC-TOCSY-experiment utilizing a virtual decoupling scheme to assign the intraresidual ribose ¹³C-spins, (2) the (H)CPC-experiment that correlates each phosphorus with the C4′ nuclei of adjacent nucleotides via J(C,P) couplings and (3) the (H)CPC-CCH-TOCSY-experiment that correlates the phosphorus nuclei with the respective C1′,H1′ ribose signals. The experiments were applied to two RNA hairpin structures. The current set of ¹³C-direct detected experiments allows direct and unambiguous assignment of the majority of the hetero nuclei and the identification of the individual ribose moieties following their sequential assignment. Thus, ¹³C-direct detected NMR methods constitute useful complements to the conventional ¹H-detected approach for the resonance assignment of oligonucleotides that is often hindered by the limited chemical shift dispersion. The developed methods can also be applied to large deuterated RNAs.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N backbone NMR chemical shift assignments of the C-terminal P4 domain of Ahnak

Ahnak is a ~?700 kDa polypeptide that was originally identified as a tumour-related nuclear phosphoprotein, but later recognized to play a variety of diverse physiological roles related to cell architecture and migration. A critical function of Ahnak is modulation of Ca²⁺ signaling in cardiomyocytes by interacting with the β subunit of the L-type Ca²⁺ channel (Ca_V1.2). Previous studies have identified the C-terminal region of Ahnak, designated as P3 and P4 domains, as a key mediator of its functional activity. We report here the nearly complete ¹H, ¹³C and ¹⁵N backbone NMR chemical shift assignments of the 11 kDa C-terminal P4 domain of Ahnak. This study lays the foundations for future investigations of functional dynamics, structure determination and interaction site mapping of the Ca_V1.2-Ahnak complex.     

Selective <Superscript>1</Superscript>H-<Superscript>13</Superscript>C NMR spectroscopy of methyl groups in residually protonated samples of large proteins

Methyl ¹³CHD₂ isotopomers of all methyl-containing amino-acids can be observed in residually protonated samples of large proteins obtained from [U-¹³C,¹H]-glucose/D₂O-based bacterial media, with sensitivity sufficient for a number of NMR applications. Selective detection of some subsets of methyl groups (Ala^β, Thr^γ2) is possible using simple ‘out-and-back’ NMR methodology. Such selective methyl-detected ‘out-and-back’ NMR experiments allow complete assignments of threonine γ2 methyls in residually protonated, [U-¹³C,¹H]-glucose/D₂O-derived samples of an 82-kDa enzyme Malate Synthase G. [U-¹³C,¹H]-glucose/D₂O-derived protein samples are relatively inexpensive and are usually available at very early stages of any NMR study of high-molecular-weight systems.     

3-O-Methyl-<Emphasis Type="SmallCaps">d</Emphasis>-glucose mutarotation and proton exchange rates assessed by <Superscript>13</Superscript>C, <Superscript>1</Superscript>H NMR and by chemical exchange saturation transfer and spin lock measurements

3-O-Methyl-d-glucose (3OMG) was recently suggested as an agent to image tumors using chemical exchange saturation transfer (CEST) MRI. To characterize the properties of 3OMG in solution, the anomeric equilibrium and the mutarotation rates of 3OMG were studied by ¹H and ¹³C NMR. This information is essential in designing the in vivo CEST experiments. At room temperature, the ratio of α and β 3OMG anomers at equilibrium was 1:1.4, and the time to reach 95% equilibrium was 6 h. The chemical exchange rates between the hydroxyl protons of 3OMG and water, measured by CEST and spin lock at pH 6.14 and a temperature of 4 °C, were in the range of 360–670 s^?1.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N resonance assignments for a chemokine receptor-binding domain of FROUNT,a cytoplasmic regulator of chemotaxis

FROUNT is a cytoplasmic protein that interacts with the membrane-proximal C-terminal regions (Pro-Cs) of the CCR2 and CCR5 chemokine receptors. The interactions between FROUNT and the chemokine receptors play an important role in the migration of inflammatory immune cells. Therefore, FROUNT is a potential drug target for inflammatory diseases. However, the structural basis of the interactions between FROUNT and the chemokine receptors remains to be elucidated. We previously identified the C-terminal region (residues 532–656) of FROUNT as the structural domain responsible for the Pro-C binding, referred to as the chemokine receptor-binding domain (CRBD), and then constructed its mutant, bearing L538E/P612S mutations, with improved NMR spectral quality, referred to as CRBD_LEPS. We now report the main-chain and side-chain ¹H, ¹³C, and ¹⁵N resonance assignments of CRBD_LEPS. The NMR signals of CRBD_LEPS were well dispersed and their intensities were uniform on the ¹H–¹⁵N HSQC spectrum, and thus almost all of the main-chain and side-chain resonances were assigned. This assignment information provides the foundation for NMR studies of the three-dimensional structure of CRBD_LEPS in solution and its interactions with chemokine receptors.     

Backbone <Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N chemical shift assignment of full-length human uracil DNA glycosylase UNG2

Human uracil N-glycosylase isoform 2—UNG2 consists of an N-terminal intrinsically disordered regulatory domain (UNG2 residues 1–92, 9.3 kDa) and a C-terminal structured catalytic domain (UNG2 residues 93–313, 25.1 kDa). Here, we report the backbone ¹H, ¹³C, and ¹⁵N chemical shift assignment as well as secondary structure analysis of the N-and C-terminal domains of UNG2 representing the full-length UNG2 protein.     

<Superscript>1</Superscript>H, <Superscript>15</Superscript>N and <Superscript>13</Superscript>C backbone resonance assignments of pentaerythritol tetranitrate reductase from <Emphasis Type="Italic">Enterobacter cloacae</Emphasis> PB2

Pentaerythritol tetranitrate reductase (PETNR) is a flavoenzyme possessing a broad substrate specificity and is a member of the Old Yellow Enzyme family of oxidoreductases. As well as having high potential as an industrial biocatalyst, PETNR is an excellent model system for studying hydrogen transfer reactions. Mechanistic studies performed with PETNR using stopped-flow methods have shown that tunneling contributes towards hydride transfer from the NAD(P)H coenzyme to the flavin mononucleotide (FMN) cofactor and fast protein dynamics have been inferred to facilitate this catalytic step. Herein, we report the near-complete ¹H, ¹⁵N and ¹³C backbone resonance assignments of PETNR in a stoichiometric complex with the FMN cofactor in its native oxidized form, which were obtained using heteronuclear multidimensional NMR spectroscopy. A total of 97% of all backbone resonances were assigned, with 333 out of a possible 344 residues assigned in the ¹H–¹⁵N TROSY spectrum. This is the first report of an NMR structural study of a flavoenzyme from the Old Yellow Enzyme family and it lays the foundation for future investigations of functional dynamics in hydride transfer catalytic mechanism.