4D prediction of protein <Superscript>1</Superscript>H chemical shifts

A 4D approach for protein ¹H chemical shift prediction was explored. The 4th dimension is the molecular flexibility, mapped using molecular dynamics simulations. The chemical shifts were predicted with a principal component model based on atom coordinates from a database of 40 protein structures. When compared to the corresponding non-dynamic (3D) model, the 4th dimension improved prediction by 6–7%. The prediction method achieved RMS errors of 0.29 and 0.50 ppm for Hα and HN shifts, respectively. However, for individual proteins the RMS errors were 0.17–0.34 and 0.34–0.65 ppm for the Hα and HN shifts, respectively. X-ray structures gave better predictions than the corresponding NMR structures, indicating that chemical shifts contain invaluable information about local structures. The ¹H chemical shift prediction tool 4DSPOT is available from .     

Optimizing <Superscript>19</Superscript>F NMR protein spectroscopy by fractional biosynthetic labeling

In protein NMR experiments which employ nonnative labeling, incomplete enrichment is often associated with inhomogeneous line broadening due to the presence of multiple labeled species. We investigate the merits of fractional enrichment strategies using a monofluorinated phenylalanine species, where resolution is dramatically improved over that achieved by complete enrichment. In NMR studies of calmodulin, a 148 residue calcium binding protein, ¹⁹F and ¹H-¹⁵N HSQC spectra reveal a significant extent of line broadening and the appearance of minor conformers in the presence of complete (>95%) 3-fluorophenylalanine labeling. The effects of varying levels of enrichment of 3-fluorophenylalanine (i.e. between 3 and >95%) were further studied by ¹⁹F and ¹H-¹⁵N HSQC spectra,¹⁵N T₁ and T₂ relaxation measurements, ¹⁹F T₂ relaxation, translational diffusion and heat denaturation experiments via circular dichroism. Our results show that while several properties, including translational diffusion and thermal stability show little variation between non-fluorinated and >95% ¹⁹F labeled samples, ¹⁹F and ¹H-¹⁵N HSQC spectra show significant improvements in line widths and resolution at or below 76% enrichment. Moreover, high levels of fluorination (>80%) appear to increase protein disorder as evidenced by backbone ¹⁵N dynamics. In this study, reasonable signal to noise can be achieved between 60–76% ¹⁹F enrichment, without any detectable perturbations from labeling.     

<Superscript>1</Superscript>H, <Superscript>15</Superscript>N, <Superscript>13</Superscript>C resonance assignment of plant dehydrin early response to dehydration 10 (ERD10)

Early response to dehydration 10 protein (ERD10) is an intrinsically disordered protein from Arabidopsis thaliana. The protein is upregulated during stress however its mechanism of action at atomic level is not well understood. In the present work multidimensional NMR methodologies are used in order to facilitate the process of chemical shift assignment. The information provided here supports further NMR spectroscopy experiments aimed at elucidation of ERD10 behaviour during molecular recognition events with other proteins.     

Refinement of protein structure against non-redundant carbonyl <Superscript>13</Superscript>C NMR relaxation

Carbonyl ¹³C′ relaxation is dominated by the contribution from the ¹³C′ chemical shift anisotropy (CSA). The relaxation rates provide useful and non-redundant structural information in addition to dynamic parameters. It is straightforward to acquire, and offers complimentary structural information to the ¹⁵N relaxation data. Furthermore, the non-axial nature of the ¹³C′ CSA tensor results in a T₁/T₂ value that depends on an additional angular variable even when the diffusion tensor of the protein molecule is axially symmetric. This dependence on an extra degree of freedom provides new geometrical information that is not available from the NH dipolar relaxation. A protocol that incorporates such structural restraints into NMR structure calculation was developed within the program Xplor-NIH. Its application was illustrated with the yeast Fis1 NMR structure. Refinement against the ¹³C′ T₁/T₂ improved the overall quality of the structure, as evaluated by cross-validation against the residual dipolar coupling as well as the ¹⁵N relaxation data. In addition, possible variations of the CSA tensor were addressed. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Residual backbone and side-chain <Superscript>13</Superscript>C and <Superscript>15</Superscript>N resonance assignments of the intrinsic transmembrane light-harvesting 2 protein complex by solid-state Magic Angle Spinning NMR spectroscopy

This study reports the sequence specific chemical shifts assignments for 76 residues of the 94 residues containing monomeric unit of the photosynthetic light-harvesting 2 transmembrane protein complex from Rhodopseudomonas acidophila strain 10050, using Magic Angle Spinning (MAS) NMR in combination with extensive and selective biosynthetic isotope labeling methods. The sequence specific chemical shifts assignment is an essential step for structure determination by MAS NMR. Assignments have been performed on the basis of 2-dimensional proton-driven spin diffusion ¹³C–¹³C correlation experiments with mixing times of 20 and 500 ms and band selective ¹³C–¹⁵N correlation spectroscopy on a series of site-specific biosynthetically labeled samples. The decreased line width and the reduced number of correlation signals of the selectively labeled samples with respect to the uniformly labeled samples enable to resolve the narrowly distributed correlation signals of the backbone carbons and nitrogens involved in the long -helical transmembrane segments. Inter-space correlations between nearby residues and between residues and the labeled BChl a cofactors, provided by the ¹³C–¹³C correlation experiments using a 500 ms spin diffusion period, are used to arrive at sequence specific chemical shift assignments for many residues in the protein complex. In this way it is demonstrated that MAS NMR methods combined with site-specific biosynthetic isotope labeling can be used for sequence specific assignment of the NMR response of transmembrane proteins.     

<Superscript>15</Superscript>N and <Superscript>13</Superscript>C- SOFAST-HMQC editing enhances 3D-NOESY sensitivity in highly deuterated,selectively [<Superscript>1</Superscript>H,<Superscript>13</Superscript>C]-labeled proteins

The ongoing NMR method development effort strives for high quality multidimensional data with reduced collection time. Here, we apply ‘SOFAST-HMQC’ to frequency editing in 3D NOESY experiments and demonstrate the sensitivity benefits using highly deuterated and ¹⁵N, methyl labeled samples in H₂O. The experiments benefit from a combination of selective T ₁ relaxation (or L-optimized effect), from Ernst angle optimization and, in certain types of experiments, from using the mixing time for both NOE buildup and magnetization recovery. This effect enhances sensitivity by up to 2.4× at fast pulsing versus reference HMQC sequences of same overall length and water suppression characteristics. Representative experiments designed to address interesting protein NMR challenges are detailed. Editing capabilities are exploited with heteronuclear ¹⁵N,¹³C-edited, or with diagonal-free ¹³C aromatic/methyl-resolved 3D-SOFAST-HMQC–NOESY–HMQC. The latter experiment is used here to elucidate the methyl-aromatic NOE network in the hydrophobic core of the 19 kDa FliT-FliJ flagellar protein complex. Incorporation of fast pulsing to reference experiments such as 3D-NOESY–HMQC boosts digital resolution, simplifies the process of NOE assignment and helps to automate protein structure determination.     

Automated amino acid side-chain NMR assignment of proteins using <Superscript>13</Superscript>C- and <Superscript>15</Superscript>N-resolved 3D [<Superscript>1</Superscript>H,<Superscript>1</Superscript>H]-NOESY

ASCAN is a new algorithm for automatic sequence-specific NMR assignment of amino acid side-chains in proteins, which uses as input the primary structure of the protein, chemical shift lists of (1)H(N), (15)N, (13)C(alpha), (13)C(beta) and possibly (1)H(alpha) from the previous polypeptide backbone assignment, and one or several 3D (13)C- or (15)N-resolved [(1)H,(1)H]-NOESY spectra. ASCAN has also been laid out for the use of TOCSY-type data sets as supplementary input. The program assigns new resonances based on comparison of the NMR signals expected from the chemical structure with the experimentally observed NOESY peak patterns. The core parts of the algorithm are a procedure for generating expected peak positions, which is based on variable combinations of assigned and unassigned resonances that arise for the different amino acid types during the assignment procedure, and a corresponding set of acceptance criteria for assignments based on the NMR experiments used. Expected patterns of NOESY cross peaks involving unassigned resonances are generated using the list of previously assigned resonances, and tentative chemical shift values for the unassigned signals taken from the BMRB statistics for globular proteins. Use of this approach with the 101-amino acid residue protein FimD(25-125) resulted in 84% of the hydrogen atoms and their covalently bound heavy atoms being assigned with a correctness rate of 90%. Use of these side-chain assignments as input for automated NOE assignment and structure calculation with the ATNOS/CANDID/DYANA program suite yielded structure bundles of comparable quality, in terms of precision and accuracy of the atomic coordinates, as those of a reference structure determined with interactive assignment procedures. A rationale for the high quality of the ASCAN-based structure determination results from an analysis of the distribution of the assigned side chains, which revealed near-complete assignments in the core of the protein, with most of the incompletely assigned residues located at or near the protein surface.     

Sequence-specific <Superscript>1</Superscript>H, <Superscript>15</Superscript>N,and <Superscript>13</Superscript>C resonance assignments of the autophagy-related protein LC3C

Autophagy is a versatile catabolic pathway for lysosomal degradation of cytoplasmic material. While the phenomenological and molecular characteristics of autophagic non-selective (bulk) decomposition have been investigated for decades, the focus of interest is increasingly shifting towards the selective mechanisms of autophagy. Both, selective as well as bulk autophagy critically depend on ubiquitin-like modifiers belonging to the Atg8 (autophagy-related 8) protein family. During evolution, Atg8 has diversified into eight different human genes. While all human homologues participate in the formation of autophagosomal membrane compartments, microtubule-associated protein light chain 3C (LC3C) additionally plays a unique role in selective autophagic clearance of intracellular pathogens (xenophagy), which relies on specific protein–protein recognition events mediated by conserved motifs. The sequence-specific ¹H, ¹⁵N, and ¹³C resonance assignments presented here form the stepping stone to investigate the high-resolution structure and dynamics of LC3C and to delineate LC3C’s complex network of molecular interactions with the autophagic machinery by NMR spectroscopy.     

The spacing of S-domains on HS glycosaminoglycans determines whether the chain is a substrate for intracellular heparanases

Heparanases are mammalian endoglucuronidases that degrade heparan sulfate (HS) glycosaminoglycans to short 5-6 kDa pieces. In the Golgi, HS glycosaminoglycans are modified by a series of interdependent reactions which result in chains that have regions rich in N- and O-sulfate groups and iduronate residues (S-domains), separated by regions that are nearly devoid of sulfate. Structural analysis of the short HS chains produced by Chinese hamster ovary (CHO) cell heparanases indicate that the enzymes recognize differences in sulfate content between S-domains and unmodified sequences, and cleave the chain at junctions between these regions. To look more closely at whether the spacing of S-domains on the gly- cosaminoglycan influences its ability to be cleaved by heparanases, we examined the susceptibility of the HS chains synthesized by the proteoglycan synthesis mutant, pgsE-606. PGS:E-606 cells are deficient in the modification enzyme N-deacetylase/N-sulfotransferase I, and synthesize HS chains that have fewer N- and O-sulfate groups and iduronate residues compared to wild-type (Bame et al., (1991), J. Biol. Chem., 266, 10287). HS glycosaminoglycans were isolated from wild-type and pgsE-606 cells and separated into populations based on sulfate content. Compared to wild-type HS, which has 14 S-domains, pgsE-606 cells synthesize three HS species, 606-1, 606-2, and 606-3, with 1, 4, and 8 S-domains, respectively. The spacing of the S-domains on the pgsE-606 HS chains is similar to the spacing the modified sequences on wild-type HS, indicating that each mutant glycosaminoglycan is composed of wild-type-like sequences and sequences devoid of S-domains. When incubated with partially purified CHO heparanases, only the portion of the mutant HS chains that had S-domains were degraded. Structural analysis of the heparanase-products confirmed that both the number and the arrangement of S-domains on the HS glycosaminoglycan are important for heparanase susceptibility. The structure of the different pgsE-606 HS chains also suggests mechanisms for the placement of S-domains when the gly- cosaminoglycan is synthesized.     

<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.     

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>15</Superscript>N, <Superscript>13</Superscript>C resonance assignments for <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> Rad23 UBL domain

Rad23 functions in nucleotide excision repair and proteasome-mediated protein degradation. It has four distinct structural domains that are connected by flexible linker regions, including an N-terminal ubiquitin-like (UBL) domain that binds proteasomes. We report in this NMR study the ¹H, ¹⁵N and ¹³C resonance assignments for the backbone and side chain atoms of the Rad23 UBL domain (Rad23^UBL) with BioMagResBank accession number 25825. We find that a Rad23 proline amino acid (P20) located in a loop undergoes isomerization. The secondary structural elements predicted from the NMR data fit well to that of the Rad23^UBL when complexed with E4 ubiquitin ligase Ufd2, as reported in a crystallographic structure. These complete assignments can be used to study the protein dynamics of the Rad23^UBL and its interaction of with other ubiquitin receptors or proteasome subunits.     

TEDOR with adiabatic inversion pulses: Resonance assignments of <Superscript>13</Superscript>C/<Superscript>15</Superscript>N labelled RNAs

We have examined via numerical simulations the performance characteristics of different 15N RF pulse schemes employed in the transferred echo double resonance (TEDOR) experimental protocol for generating 13C-15N dipolar chemical shift correlation spectra of isotopically labelled biological systems at moderate MAS frequencies (omega(r) approximately 10 kHz). With an 15N field strength of approximately 30-35 kHz that is typically available in 5 mm triple resonance MAS NMR probes, it is shown that a robust TEDOR sequence with significant tolerance to experimental imperfections sa as H1 inhomogeneity and resonance offsets can be effectively implemented using adiabatic heteronuclear dipolar recoupling pulse schemes. TEDOR-based 15N-13C and 15N-13C-13C chemical shift correlation experiments were carried out for obtaining 13C and 15N resonance assignments of an RNA composed of 97 (CUG) repeats which has been implicated in the neuromuscular disease myotonic dystrophy.     

A diverse view of protein dynamics from NMR studies of HIV-1 protease flaps

Internal motion in proteins fulfills a multitude of roles in biological processes. NMR spectroscopy has been applied to elucidate protein dynamics at the atomic level, albeit at a low resolution, and is often complemented by molecular dynamics simulation. However, it is critical to justify the consistency between simulation results and conclusions often drawn from multiple experiments in which uncertainties arising from assumed motional models may not be explicitly evaluated. To understand the role of the flaps of HIV-1 protease dimer in substrate recognition and protease function, many molecular dynamics simulations have been performed. The simulations have resulted in various proposed models of the flap dynamics, some of which are more consistent than others with our working model previously derived from experiments. However, using the working model to discriminate among the simulation results is not straightforward because the working model was derived from a combination of NMR experiments and crystal structure data. In this study, we use the NMR chemical shifts and relaxation data of the protease "monomer" rather than structural data to narrow down the possible conformations of the flaps of the "dimer". For the first time, we show that the tips of the flaps in the unliganded protease dimer interact with each other in solution. Accordingly, we discuss the consistency of the simulations with the model derived from all experimental data.     

<Superscript>1</Superscript>H, <Superscript>15</Superscript>N and <Superscript>13</Superscript>C resonance assignments and secondary structure of PulG,the major pseudopilin from <Emphasis Type="Italic">Klebsiella oxytoca</Emphasis> type 2 secretion system

Bacteria use complex transporters to secrete functionally relevant proteins to the extracellular medium. The type 2 secretion system (T2SS) translocates folded proteins involved in bacterial nutrient acquisition, virulence and adaptation. The T2SS pseudopilus is a periplasmic filament, assembled by the polymerization of PulG subunits, the major pseudopilin. Pseudopilin proteins have a conserved N-terminal hydrophobic segment followed by a more variable C-terminal periplasmic and globular domain. To better understand the mechanism of assembly and function of the T2SS, we have been studying the structure and dynamics of PulG by NMR, as well as its interaction with other components of the secretion machinery. As a first step on this study, here we reported the chemical shift assignments of PulG C-terminal domain and its secondary structure prediction based on NMR data.     

Bivalent and co-operative binding of complement factor H to heparan sulfate and heparin

FH (Factor H) with 20 SCR (short complement regulator) domains is a major serum regulator of complement, and genetic defects in this are associated with inflammatory diseases. Heparan sulfate is a cell-surface glycosaminoglycan composed of sulfated S-domains and unsulfated NA-domains. To elucidate the molecular mechanism of binding of FH to glycosaminoglycans, we performed ultracentrifugation, X-ray scattering and surface plasmon resonance with FH and glycosaminoglycan fragments. Ultracentrifugation showed that FH formed up to 63% of well-defined oligomers with purified heparin fragments (equivalent to S-domains), and indicated a dissociation constant K(d) of approximately 0.5 μM. Unchanged FH structures that are bivalently cross-linked at SCR-7 and SCR-20 with heparin explained the sedimentation coefficients of the FH-heparin oligomers. The X-ray radius of gyration, R(G), of FH in the presence of heparin fragments 18-36 monosaccharide units long increased significantly from 10.4 to 11.7 nm, and the maximum lengths of FH increased from 35 to 40 nm, confirming that large compact oligomers had formed. Surface plasmon resonance of immobilized heparin with full-length FH gave K(d) values of 1-3 μM, and similar but weaker K(d) values of 4-20 μM for the SCR-6/8 and SCR-16/20 fragments, confirming co-operativity between the two binding sites. The use of minimally-sulfated heparan sulfate fragments that correspond largely to NA-domains showed much weaker binding, proving the importance of S-domains for this interaction. This bivalent and co-operative model of FH binding to heparan sulfate provides novel insights on the immune function of FH at host cell surfaces.     

Application of methyl-TROSY to a large paramagnetic membrane protein without perdeuteration: <Superscript>13</Superscript>C-MMTS-labeled NADPH-cytochrome P450 oxidoreductase

NMR spectroscopy of membrane proteins involved in electron transport is difficult due to the presence of both the lipids and paramagnetic centers. Here we report the solution NMR study of the NADPH-cytochrome P450 oxidoreductase (POR) in its reduced and oxidized states. We interrogate POR, first, in its truncated soluble form (70 kDa), which is followed by experiments with the full-length protein incorporated in a lipid nanodisc (240 kDa). To overcome paramagnetic relaxation in the reduced state of POR as well as the signal broadening due to its high molecular weight, we utilized the methyl-TROSY approach. Extrinsic ¹³C-methyl groups were introduced by modifying the engineered surface-exposed cysteines with methyl-methanethiosulfonate. Chemical shift dispersion of the resonances from different sites in POR was sufficient to monitor differential effects of the reduction–oxidation process and conformation changes in the POR structure related to its function. Despite the high molecular weight of the POR-nanodisc complex, the surface-localized ¹³C-methyl probes were sufficiently mobile to allow for signal detection at 600 MHz without perdeuteration. This work demonstrates a potential of the solution methyl-TROSY in analysis of structure, dynamics, and function of POR, which may also be applicable to similar paramagnetic and flexible membrane proteins.     

<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.