Side Chain Chemical Shift Research Articles

Backbone and side-chain chemical shift assignments for the C-terminal domain of Tcb2, a cytoskeletal calcium-binding protein from Tetrahymena thermophila.

Tcb2 is a putative calcium-binding protein from the membrane-associated cytoskeleton of the ciliated protozoan Tetrahymena thermophila. It has been hypothesized to participate in several calcium-mediated processes in Tetrahymena, including ciliary movement, cell cortex signaling, and pronuclear exchange. Sequence analysis suggests that the protein belongs to the calmodulin family, with N- and C-terminal domains connected by a central linker, and two helix-loop-helix motifs in each domain. However, its calcium-binding properties, structure and precise biological function remain unknown. Interestingly, Tcb2 is a major component of unique contractile fibers isolated from the Tetrahymena cytoskeleton; in these fibers, addition of calcium triggers an ATP-independent type of contraction. Here we report the (1)H, (13)C and (15)N backbone and side-chain chemical shift assignments of the C-terminal domain of the protein (Tcb2-C) in the absence and presence of calcium ions. (1)H-(15)N HSQC spectra show that the domain is well folded both in the absence and presence of calcium, and undergoes a dramatic conformational change upon calcium addition. Secondary structure prediction from chemical shifts reveals an architecture encountered in other calcium-binding proteins, with paired EF-hand motifs connected by a flexible linker. These studies represent a starting point for the determination of the high-resolution solution structure of Tcb2-C at both low and high calcium levels, and, together with additional structural studies on the full-length protein, will help establish the molecular basis of Tcb2 function and unique contractile properties.

Chemical shift assignment of the alternative scaffold protein IscA.

The IscA protein (11.5 kDa) is an essential component of the iron sulphur cluster biogenesis machine. In bacteria, the machine components are clustered in operons, amongst which the most important is the isc operon. Bacterial IscA has direct homologues also in eukaryotes. Like the protein IscU, IscA is thought to assist cluster formation as an alternative scaffold protein which receives the cluster before transferring it further to the final acceptors. Several crystal structures have been published. They all report an IscA dimeric form, although the packing of the protomers in the dimers differs amongst structures. No solution studies have currently been reported. Here we report the 1H, 13C and 15N backbone and side-chain chemical shift assignments of the cluster-free E. coli IscA as a starting point for further studies of the structure and functions of this still poorly characterized protein. We show that IscA exists in solution as an equilibrium between different species. Spectrum assignment was thus challenging given the heterogeneous nature of the sample but doable through judicious choice of selective labelling and concentration dependent studies.

Access to side-chain carbon information in deuterated solids under fast MAS through non-rotor-synchronized mixing.

We demonstrate the accessibility of aliphatic (13)C side chain chemical shift sets for solid-state NMR despite perdeuteration and fast MAS using isotropic, non-rotor-synchronized (13)C-(13)C mixing. Combined with amide proton detection, we unambiguously and sensitively detect whole side chain to backbone correlations for two proteins using around 1 mg of sample.

Side Chain Conformational Distributions of a Small Protein Derived from Model-Free Analysis of a Large Set of Residual Dipolar Couplings.

Accurate quantitative measurement of structural dispersion in proteins remains a prime challenge to both X-ray crystallography and NMR spectroscopy. Here we use a model-free approach based on measurement of many residual dipolar couplings (RDCs) in differentially orienting aqueous liquid crystalline solutions to obtain the side chain χ1 distribution sampled by each residue in solution. Applied to the small well-ordered model protein GB3, our approach reveals that the RDC data are compatible with a single narrow distribution of side chain χ1 angles for only about 40% of the residues. For more than half of the residues, populations greater than 10% for a second rotamer are observed, and four residues require sampling of three rotameric states to fit the RDC data. In virtually all cases, sampled χ1 values are found to center closely around ideal g–, g+ and t rotameric angles, even though no rotamer restraint is used when deriving the sampled angles. The root-mean-square difference between experimental 3JHαHβ couplings and those predicted by the Haasnoot-parametrized, motion-adjusted Karplus equation reduces from 2.05 to 0.75 Hz when using the new rotamer analysis instead of the 1.1-Å X-ray structure as input for the dihedral angles. A comparison between observed and predicted 3JHαHβ values suggests that the root-mean-square amplitude of χ1 angle fluctuations within a given rotamer well is ca. 20°. The quantitatively defined side chain rotamer equilibria obtained from our study set new benchmarks for evaluating improved molecular dynamics force fields, and also will enable further development of quantitative relations between side chain chemical shift and structure.

Backbone and side chain chemical shift assignments of apolipophorin III from Galleria mellonella.

Apolipophorin III, a 163 residue monomeric protein from the greater wax moth Galleria mellonella (abbreviated as apoLp-IIIGM), has roles in upregulating expression of antimicrobial proteins as well as binding and deforming bacterial membranes. Due to its similarity to vertebrate apolipoproteins there is interest in performing atomic resolution analysis of apoLp-IIIGM as part of an effort to better understand its mechanism of action in innate immunity. In the first step towards structural characterization of apoLp-IIIGM, 99 % of backbone and 88 % of side chain (1)H, (13)C and (15)N chemical shifts were assigned. TALOS+ analysis of the backbone resonances has predicted that the protein is composed of five long helices, which is consistent with the reported structures of apolipophorins from other insect species. The next stage in the characterization of apoLp-III from G. mellonella will be to utilize these resonance assignments in solving the solution structure of this protein.

An approach to NMR assignment of intrinsically disordered proteins.

An efficient approach to NMR assignments in intrinsically disordered proteins is presented, making use of the good dispersion of cross peaks observed in [(15) N,(13) C']- and [(13) C',(1) H(N) ]-correlation spectra. The method involves the simultaneous collection of {3D (H)NCO(CAN)H and 3D (HACA)CON(CA)HA} spectra for backbone assignments via sequential H(N) and H(α) correlations and {3D (H)NCO(CACS)HS and 3D (HS)CS(CA)CO(N)H} spectra for side-chain (1) H and (13) C assignments, employing sequential (1) H data acquisitions with direct detection of both the amide and aliphatic protons. The efficacy of the approach for obtaining resonance assignments with complete backbone and side-chain chemical shifts is demonstrated experimentally for the 61-residue [(13) C,(15) N]-labelled peptide of a voltage-gated potassium channel protein of the Kv1.4 channel subunit. The general applicability of the approach for the characterisation of moderately sized globular proteins is also demonstrated.

Lipid bilayer-bound conformation of an integral membrane beta barrel protein by multidimensional MAS NMR

The human voltage dependent anion channel 1 (VDAC) is a 32 kDa β-barrel integral membrane protein that controls the transport of ions across the outer mitochondrial membrane. Despite the determination of VDAC solution and diffraction structures, a structural basis for the mechanism of its function is not yet fully understood. Biophysical studies suggest VDAC requires a lipid bilayer to achieve full function, motivating the need for atomic resolution structural information of VDAC in a membrane environment. Here we report an essential step toward that goal: extensive assignments of backbone and side chain resonances for VDAC in DMPC lipid bilayers via magic angle spinning nuclear magnetic resonance (MAS NMR). VDAC reconstituted into DMPC lipid bilayers spontaneously forms two-dimensional lipid crystals, showing remarkable spectral resolution (0.5-0.3 ppm for (13)C line widths and <0.5 ppm (15)N line widths at 750 MHz). In addition to the benefits of working in a lipid bilayer, several distinct advantages are observed with the lipid crystalline preparation. First, the strong signals and sharp line widths facilitated extensive NMR resonance assignments for an integral membrane β-barrel protein in lipid bilayers by MAS NMR. Second, a large number of residues in loop regions were readily observed and assigned, which can be challenging in detergent-solubilized membrane proteins where loop regions are often not detected due to line broadening from conformational exchange. Third, complete backbone and side chain chemical shift assignments could be obtained for the first 25 residues, which comprise the functionally important N-terminus. The reported assignments allow us to compare predicted torsion angles for VDAC prepared in DMPC 2D lipid crystals, DMPC liposomes, and LDAO-solubilized samples to address the possible effects of the membrane mimetic environment on the conformation of the protein. Concluding, we discuss the strengths and weaknesses of the reported assignment approach and the great potential for even more complete assignment studies and de novo structure determination via (1)H detected MAS NMR.

Automated resonance assignment of the 21 kDa stereo-array isotope labeled thioldisulfide oxidoreductase DsbA

The automated chemical shift assignment algorithm FLYA has been extended for use with stereo-array isotope labeled (SAIL) proteins to determine the sequence-specific resonance assignments of large proteins. Here we present the assignment of the backbone and sidechain chemical shifts of the 21kDa thioldisulfide oxidoreductase DsbA from Escherichia coli that were determined with the SAIL-FLYA algorithm in conjunction with automated peak picking. No manual corrections of peak lists or assignments were applied. The assignments agreed with manually determined reference assignments in 95.4% of the cases if 16 input spectra were used, 94.1% if only 3D 13C/15N-resolved NOESY, CBCA(CO)NH, and 2D [13C/15N,1H]-HSQC were used, and 86.8% if exclusively 3D 13C/15N-resolved NOESY spectra were used. Considering only the assignments that are classified as reliable by the SAIL-FLYA algorithm, the degrees of agreement increased to 97.5%, 96.5%, and 94.2%, respectively. With our approach it is thus possible to automatically obtain almost complete and correct assignments of proteins larger than 20kDa.

Chemical shift assignments of DRB4 (1-153), a dsRNA binding protein in A. thaliana RNAi pathway.

RNA interference (RNAi) is a conserved biological response to dsRNA and regulates the expression of protein-coding genes to mediate resistance to both endogenous parasitic and exogenous pathogenic nucleic acids. In RNAi pathway, dsRNA binding proteins assists Dicer at various stages of RNAi. In plants, DRB4, is a multidomain protein containing two dsRNA binding domains that recognizes the long exogenous/endogenous dsRNA and presents it to Ribonuclease enzyme, Dicer like 4, resulting in the production of 21nt small interfering RNA. Here, we report nearly complete backbone and sidechain chemical shift assignments of N-terminus of DRB4 (1-153, ~18kDa), containing both double stranded RNA binding domains and the linker.

Influence of NMR Data Completeness on Structure Determinations of Homodimeric Proteins

AbstractThe structure determination of homodimeric proteins by NMR using conventional NOESY experiments is still challenging due to the degeneracy of the chemical shifts in the identical monomers, which causes ambiguity in the NOE assignments. Residues involved in the interface between two monomers provide essential intermolecular NOEs for the structure determinations of homodimeric proteins. Hence NMR data, such as NOE peak lists and chemical shift assignments of these interface residues, play a crucial role for the successful structure determination of homodimeric proteins. This paper extends our previous report (Lin, Y.‐J.; Kirchner, D. K.; Güntert, P. J. Magn. Reson.­ 2012, 222, 96) and investigates the influence of incomplete NOESY peak lists combined with incomplete 1H chemical shift assignments of the interface residues on the structure determination of homodimeric proteins using the program CYANA. Data incompleteness was simulated by random omission of both NOESY cross peaks and interface 1H chemical shifts. Our results for three proteins with different percentages of interface residues reveal that the algorithm can tolerate about 40–50% NOESY peak omission with complete interface chemical shift assignments, which indicates that partial NOESY peak omission does not cause severe problems when the interface chemical shifts are completely assigned. Combining NOESY peak omission with incomplete interface chemical shift assignments, the tolerance for interface chemical shift omission decreases with the extent of omitted NOESY peaks. The tolerance for unassigned interface side chain, methyl and aromatic chemical shifts is affected more strongly by NOESY peak omission than that for the omission of general interface 1H chemical shifts including the backbone. In general about 10–30% peaks omission is tolerated in conjunction with missing chemical shift assignments. If more NOESY peaks are omitted calculations gradually become unstable and tend not to tolerate any missing interface chemical shifts. A large amount of omitted NOESY peaks, for instance 30% omission in our calculations, could decrease the tolerance for missing aromatic or methyl interface 1H chemical shifts to as few as 2–4 missing chemical shifts, suggesting that complete aromatic and methyl 1H chemical shift assignments are important when the NOESY peak data is significantly incomplete. Finally, for homodimeric proteins with a low percentage of interface residues, our results reveal that the omission of NOESY peaks, even at an extent of only 10%, can result in no tolerance against the omission of interface 1H chemical shifts, suggesting that the completeness of both interface 1H chemical shift assignments and NOESY peaks are important for the successful structure determination of proteins with a small homodimer interface.

NMR characterization of the electrostatic interaction of the basic residues in HDGF and FGF2 during heparin binding

Electrostatic interaction is a major driving force in the binding of proteins to highly acidic glycosaminoglycan, such as heparin. Although NMR backbone chemical shifts have generally been used to identify the heparin-binding site on a protein, however, there is no correlation between the binding free energies and the perturbed backbone chemical shifts for individual residues. The binding event occurs at the end of a side chain of basic residue, and does not require causing significant alterations in the backbone environment at a distance of multiple bonds. We used the H2CN NMR pulse sequence to detect heparin binding through the side-chain resonances Hε–Cε–Nζ of Lys and Hδ–Cδ–Nε of Arg in the two proteins of hepatoma-derived growth factor (HDGF) and basic fibroblast growth factor (FGF2). H2CN titration experiments revealed chemical shift perturbations in the side chains, which were correlated with the free energy changes in various mutants. The residues K19 in HDGF and K125 in FGF2 demonstrated the most significant perturbations, consistent with our previous observation that the two residues are crucial for binding. The result suggests that H2CN NMR provides a precise evaluation for the electrostatic interactions. The discrepancy observed between backbone and side chain chemical shifts is correlated to the solvent accessibility of residues that the K19 and K125 backbones are highly buried with the restricted backbone conformation and are not strongly affected by the events at the end of the side chains.

(1)H, (13)C and (15)N chemical shift assignments of the NDH-1 complex subunit CupS.

The cyanobacterial NDH-1 complex is involved in respiratory as well as in cyclic electron transfer around photosystem I. Here, we report both backbone and side chain chemical shift assignments of CupS, a small subunit of the multisubunit membrane protein complex NDH-1 from Thermosynechococcus elongatus. The construct contains 159 amino acids including a Strep-tag and two additional amino acids.

(1)H, (13)C, and (15)N backbone and sidechain chemical shift assignments for the HEAT2 domain of human eIF4GI.

The translation initiation factor eIF4G is required for the translation of many eukaryotic messenger RNAs. Its interaction with the ATP-dependent RNA helicase eIF4A plays an important role in the regulation of translation initiation. eIF4G in humans and other higher eukaryotes contains three HEAT domains, of which HEAT1 and HEAT2 contain binding sites for eIF4A. Here we report the near complete NMR resonance assignment of the 192-residue HEAT2 domain of the human translation initiation factor eIF4GI. The chemical shift data constitute the basis for NMR structural studies aimed at expanding understanding of the role of interactions between the initiation factor eIF4A and eIF4G in translation initiation.

Silica morphogenesis by lysine-leucine peptides with hydrophobic periodicity.

The use of biomimetic approaches in the production of inorganic nanostructures is of great interest to the scientific and industrial community due to the relatively moderate physical conditions needed. In this vein, taking cues from silaffin proteins used by unicellular diatoms, several studies have identified peptide candidates for the production of silica nanostructures. In the current article, we study intensively one such silica-precipitating peptide, LKα14 (Ac-LKKLLKLLKKLLKL-c), an amphiphilic lysine/leucine repeat peptide that self-organizes into an α-helical secondary structure under appropriate concentration and buffer conditions. The suggested mechanism of precipitation is that the sequestration of hydrophilic lysines on one side of this helix allows interaction with the negatively charged surface of silica nanoparticles, which in turn can aggregate further into larger structures. To investigate the process, we carry out 1D and 2D solid-state NMR (ssNMR) studies on samples with one or two uniformly (13)C- and (15)N-labeled residues to determine the backbone and side-chain chemical shifts. We also further study the dynamics of two leucine residues in the sequence through (13)C spin-lattice relaxation times (T1) to determine the impact of silica coprecipitation on their mobility. Our results confirm the α-helical secondary structure in both the neat and silica-complexed states of the peptide, and the patterns of chemical shift and relaxation time changes between the two states suggest possible mechanisms of self-aggregation and silica precipitation.

Backbone and stereospecific 13C methyl Ile (δ1), Leu and Val side-chain chemical shift assignments of Crc

Carbon catabolite repression (CCR) allows bacteria to selectively assimilate a preferred compound among a mixture of several potential carbon sources, thus boosting growth and economizing the cost of adaptability to variable nutrients in the environment. The RNA-binding catabolite repression control (Crc) protein acts as a global post-transcriptional regulator of CCR in Pseudomonas species. Crc triggers repression by inhibiting the expression of genes involved in transport and catabolism of non-preferred substrates, thus indirectly favoring assimilation of preferred one. We report here a nearly complete backbone and stereospecific (13)C methyl side-chain chemical shift assignments of Ile (δ1), Leu and Val of Crc (~31kDa) from Pseudomonas syringae Lz4W.

A Conformational Ensemble Derived Using NMR Methyl Chemical Shifts Reveals a Mechanical Clamping Transition That Gates the Binding of the HU Protein to DNA

Recent improvements in the accuracy of structure-based methods for the prediction of nuclear magnetic resonance chemical shifts have inspired numerous approaches for determining the secondary and tertiary structures of proteins. Such advances also suggest the possibility of using chemical shifts to characterize the conformational fluctuations of these molecules. Here we describe a method of using methyl chemical shifts as restraints in replica-averaged molecular dynamics (MD) simulations, which enables us to determine the conformational ensemble of the HU dimer and characterize the range of motions accessible to its flexible β-arms. Our analysis suggests that the bending action of HU on DNA is mediated by a mechanical clamping mechanism, in which metastable structural intermediates sampled during the hinge motions of the β-arms in the free state are presculpted to bind DNA. These results illustrate that using side-chain chemical shift data in conjunction with MD simulations can provide quantitative information about the free energy landscapes of proteins and yield detailed insights into their functional mechanisms.

Generating NMR chemical shift assignments of intrinsically disordered proteins using carbon-detected NMR methods

There is an extraordinary need to describe the structures of intrinsically disordered proteins (IDPs) due to their role in various biological processes involved in signaling and transcription. However, general study of IDPs by NMR spectroscopy is limited by the poor 1H amide chemical shift dispersion typically observed in their spectra. Recently, 13C direct-detected NMR spectroscopy has been recognized as enabling broad structural study of IDPs. Most notably, multidimensional experiments based on the 15N,13C CON spectrum make complete chemical shift assignment feasible. Here we document a collection of NMR-based tools that efficiently lead to chemical shift assignment of IDPs, motivated by a case study of the C-terminal disordered region from the human pancreatic transcription factor Pdx1. Our strategy builds on the combination of two three-dimensional (3D) experiments, (HN-flip)N(CA)CON and 3D (HN-flip)N(CA)NCO, that enable daisy chain connections to be built along the IDP backbone, facilitated by acquisition of amino acid-specific 15N,13C CON-detected experiments. Assignments are completed through carbon-detected, total correlation spectroscopy (TOCSY)-based side chain chemical shift measurement. Conducting our study required producing valuable modifications to many previously published pulse sequences, motivating us to announce the creation of a database of our pulse programs, which we make freely available through our website.

A Simple Method to Measure Protein Side-Chain Mobility Using NMR Chemical Shifts

Protein side-chain motions are involved in many important biological processes including enzymatic catalysis, allosteric regulation, and the mediation of protein-protein, protein-DNA, protein-RNA, and protein-cofactor interactions. NMR spectroscopy has long been used to provide insights into the motions of side-chain groups. Currently, the method of choice for studying side-chain dynamics by NMR is the measurement of methyl (2)H autorelaxation. Methyl (2)H autorelaxation exhibits simple relaxation mechanisms and can be straightforwardly converted to meaningful dynamic parameters. However, methyl groups can only be found in 6 of 19 side-chain bearing amino acids. Consequently, only a sparse assessment of protein side-chain dynamics is possible. Therefore, there is a significant interest in developing novel methods of studying side-chain motions that can be applied to all types of side-chains. Here, we show how side-chain chemical shifts can be used to determine the magnitude of fast side-chain motions in proteins. The chemical shift method is applicable to all side-chain bearing residues and does not require any additional measurements beyond standard NMR experiments for backbone and side-chain assignments.

Chemical shift assignment of the ataxin-1 AXH domain in complex with a CIC ligand peptide

Ataxin-1 is the protein responsible for the genetically-inherited neurodegenerative disease spinocerebellar ataxia type-1 linked to the expansion of a polyglutamine tract within the protein sequence. The AXH domain of ataxin-1 is essential for the protein to function as a transcriptional co-repressor and mediates the majority of the interactions of ataxin-1 with cellular partners, mainly transcriptional regulators. One of the best characterized ataxin-1 functional partners is Capicua (CIC), a transcriptional repressor involved in signalling pathways that regulate mammalian development, tumorigenesis and, through the interaction with ataxin-1, also neurodegeneration. Complex formation of ataxin-1 with CIC is important both for the function of the wild-type protein and for pathogenesis as transcriptional disregulation is observed since the early stages of the development of the disease. Here we report the 1H, 13C and 15N backbone and side-chain chemical shift assignments of the human ataxin-1 AXH domain in complex with a CIC ligand-peptide.

1H, 13C, and 15N backbone and side-chain chemical shift assignments of the free and bound forms of the human PTPN11 second SH2 domain

Src homology 2 (SH2) domains have an important role in the regulation of protein activity and intracellular signaling processes. They are geared to bind to specific phosphotyrosine (pY) motifs, with a substrate sequence specificity depending on the three amino acids immediately C-terminal to the pY. Here we report for the first time the (1)H, (15)N and (13)C backbone and side-chain chemical shift assignments for the C-terminal SH2 domain of the human protein tyrosine phosphatase PTPN11, both in its free and bound forms, where the ligand in the latter corresponds to a specific sequence of the human erythropoietin receptor.