TROSY Experiments Research Articles

Affordable amino acid α/β-deuteration and specific labeling for NMR signal enhancement: Evaluation on the kinase p38α

Although very effective in decreasing NMR relaxation of large proteins, homogeneous deuteration can be costly, and anyway unsuitable for recombinant production in metazoan systems. We sought to explore other deuteration schemes, which would be adapted to protein expression in mammalian cells. Here, we evaluate the benefits of the deuteration on alpha- and beta-positions of amino acids for a typical middle size protein domain, namely the model 40 kDa-large kinase p38α. We report the position-specific deuteration of free amino acids by using enzyme-assisted H/D exchange, executed by the cystathionine gamma-synthase and a newly designed high-performance mutant E325A. Then, we used cell-free expression in bacterial extracts to avoid any scrambling and back-protonation of the tested isotopically labelled amino acids (Ala, Leu, Lys, Ser, Asp, Glu, Gly). Our results show signal enhancements up to three in 1H-15N spectra when these α/β-deuterated amino acids are integrated. Because our approach relies on single 2Hα/β-15N-amino acid labeling, an additional three-fold increase in sensitivity is obtained by the possible use of moderate resolution SOFAST-HMQC instead of the classical HSQC or TROSY experiments. This allows recording residue-resolved solution 1H-15N NMR spectra of 100 μg of p38α in one hour with S/N∼10.

Investigating the Structure and Dynamics of RNAs that Distinguish between Human and Chimpanzee by NMR using Selective Isotopically Labeled RNAs

Long non-coding RNAs (lncRNAs) are known to play significant roles in the human genome. One example is the human accelerated region (HAR), a group of lncRNAs that function in brain development. HAR1 encodes a 118 nucleotide (nt) RNA. Biophysical techniques that characterize RNA structure and dynamics at atomic resolution are necessary to understand the function of HAR1. One such technique is nuclear magnetic resonance (NMR) spectroscopy. Large RNAs (>30 nts) limit the efficacy of NMR for two reasons: (1) extensive overlap in chemical shifts and (2) fast signal relaxation. In 2012, Ziegeler et al., used a ‘divide and conquer’ method to study HAR1 in human and chimpanzees by NMR and determined that human and chimpanzee HAR1s are structurally similar, implying that their functional difference must be explained by something other than the 3D structure. We hypothesize RNA dynamics. Our research group developed a chemo-enzymatic labeling strategy to effectively study large RNAs by NMR. This approach uses recombinant enzymes to couple chemically synthesized selective isotopically labeled nucleobases to selectively labeled ribose. Presently, we are collecting preliminary NMR data on HAR1 fragments to validate the results of Ziegeler et al., Ultimately, we will generate customized 13C-labeled HAR1 RNAs with atom and position specificity for1H, 2D NOESY, HSQC, TROSY, and TOCSY experiments. This can enable us to determine the specific nucleotides responsible for the altered RNA dynamics, and thus their function.

Nitrogen detected TROSY at high field yields high resolution and sensitivity for protein NMR.

Detection of (15)N in multidimensional NMR experiments of proteins has sparsely been utilized because of the low gyromagnetic ratio (γ) of nitrogen and the presumed low sensitivity of such experiments. Here we show that selecting the TROSY components of proton-attached (15)N nuclei (TROSY (15)NH) yields high quality spectra in high field magnets (>600MHz) by taking advantage of the slow (15)N transverse relaxation and compensating for the inherently low (15)N sensitivity. The (15)N TROSY transverse relaxation rates increase modestly with molecular weight but the TROSY gain in peak heights depends strongly on the magnetic field strength. Theoretical simulations predict that the narrowest line width for the TROSY (15)NH component can be obtained at 900MHz, but sensitivity reaches its maximum around 1.2GHz. Based on these considerations, a (15)N-detected 2D (1)H-(15)N TROSY-HSQC ((15)N-detected TROSY-HSQC) experiment was developed and high-quality 2D spectra were recorded at 800MHz in 2h for 1mM maltose-binding protein at 278K (τc~40ns). Unlike for (1)H detected TROSY, deuteration is not mandatory to benefit (15)N detected TROSY due to reduced dipolar broadening, which facilitates studies of proteins that cannot be deuterated, especially in cases where production requires eukaryotic expression systems. The option of recording (15)N TROSY of proteins expressed in H2O media also alleviates the problem of incomplete amide proton back exchange, which often hampers the detection of amide groups in the core of large molecular weight proteins that are expressed in D2O culture media and cannot be refolded for amide back exchange. These results illustrate the potential of (15)NH-detected TROSY experiments as a means to exploit the high resolution offered by high field magnets near and above 1GHz.

Increased resolution of aromatic cross peaks using alternate 13C labeling and TROSY.

For typical globular proteins, contacts involving aromatic side chains would constitute the largest number of distance constraints that could be used to define the structure of proteins and protein complexes based on NOE contacts. However, the (1)H NMR signals of aromatic side chains are often heavily overlapped, which hampers extensive use of aromatic NOE cross peaks. Some of this overlap can be overcome by recording (13)C-dispersed NOESY spectra. However, the resolution in the carbon dimension is rather low due to the narrow dispersion of the carbon signals, large one-bond carbon-carbon (C-C) couplings, and line broadening due to chemical shift anisotropy (CSA). Although it has been noted that the CSA of aromatic carbons could be used in TROSY experiments for enhancing resolution, this has not been used much in practice because of complications arising from large aromatic one-bond C-C couplings, and 3D or 4D carbon dispersed NOESY are typically recorded at low resolution hampering straightforward peak assignments. Here we show that the aromatic TROSY effect can optimally be used when employing alternate (13)C labeling using 2-(13)C glycerol, 2-(13)C pyruvate, or 3-(13)C pyruvate as the carbon source. With the elimination of the strong one-bond C-C coupling, the TROSY effect can easily be exploited. We show that (1)H-(13)C TROSY spectra of alternately (13)C labeled samples can be recorded at high resolution, and we employ 3D NOESY aromatic-TROSY spectra to obtain valuable intramolecular and intermolecular cross peaks on a protein complex.

Multiplet-filtered and gradient-selected zero-quantum TROSY experiments for 13C1H3 methyl groups in proteins

Multiplet-filtered and gradient-selected heteronuclear zero-quantum coherence (gsHZQC) TROSY experiments are described for measuring (1)H-(13)C correlations for (13)CH(3) methyl groups in proteins. These experiments provide improved suppression of undesirable, broad outer components of the heteronuclear zero-quantum multiplet in medium-sized proteins, or in flexible sites of larger proteins, compared to previously described HZQC sequences (Tugarinov et al. in J Am Chem Soc 126:4921-4925, 2004; Ollerenshaw et al. in J Biomol NMR 33:25-41, 2005). Hahn-echo versions of the gsHZQC experiment also are described for measuring zero- and double-quantum transverse relaxation rate constants for identification of chemical exchange broadening. Application of the proposed pulse sequences to Escherichia coli ribonuclease HI, with a molecular mass of 18 kD, indicates that improved multiplet suppression is obtained without substantial loss of sensitivity.

A tribute to Lewis E Kay on his 50th birthday

It is a great pleasure for us to introduce this special issue of the Journal of Biomolecular NMR on the occasion of Prof. Lewis E. Kay’s 50th birthday. We are grateful to the Editors of the Journal of Biomolecular NMR for accepting our proposal. In particular, we thank Prof. Martin Billeter for editing this issue, and Dr. Ad Bax for advice and support. We believe that Lewis merits this special issue of Journal of Biomolecular NMR for his innovative contributions to biomolecular NMR spectroscopy, as well as for his role as inspirer for a large group of young research professionals. The following short list of examples serves to illustrate how the work of Lewis Kay has helped to advance our field: Lewis has consistently been a pioneer in the development of novel methods essential to increasing the size of proteins that may be studied by NMR. In the days when assigning chemical shifts of a protein with 150 amino acids was a major endeavour, the Kay lab––in collaboration with Prof. Cheryl Arrowsmith––combined advances in gradientenhanced spectroscopy, protein deuteration and pulse sequence design to establish the resonance assignments for a 37 kDa complex. As these techniques became more broadly used, his lab forged ahead with clever biochemical tricks for incorporating protonated methyl groups in otherwise deuterated proteins, and developed methyl TROSY experiments that permit well-resolved spectra to be obtained for high molecular weight complexes, extending to 1 MDa. Lewis has been a leader in using NMR to measure fast timescale macromolecular dynamics, starting with his contributions to inverse-detected N relaxation experiments. His deuterium spin relaxation methodologies have had a huge impact on our ability to characterize side chain dynamics in proteins. In addition, the Kay lab has extensively developed and applied new methodologies for studying low-populated states of proteins and their roles in biology. Building on transverse relaxation dispersion experiments, the Kay lab has introduced approaches to elucidate the threeK. H. Gardner (&) Department of Biochemistry, UT Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-8816, USA e-mail: Kevin.Gardner@UTSouthwestern.edu

Interaction of Actin with Carcinoembryonic Antigen-related Cell Adhesion Molecule 1 (CEACAM1) Receptor in Liposomes Is Ca2+- and Phospholipid-dependent

The regulation of binding of G-actin to cytoplasmic domains of cell surface receptors is a common mechanism to control diverse biological processes. To model the regulation of G-actin binding to a cell surface receptor we used the cell-cell adhesion molecule carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1-S) in which G-actin binds to its short cytoplasmic domain (12 amino acids; Chen, C. J., Kirshner, J., Sherman, M. A., Hu, W., Nguyen, T., and Shively, J. E. (2007) J. Biol. Chem. 282, 5749-5760). A liposome model system demonstrates that G-actin binds to the cytosolic domain peptide of CEACAM1-S in the presence of negatively charged palmitoyl-oleoyl phosphatidylserine (POPS) liposomes and Ca(2+). In contrast, no binding of G-actin was observed in palmitoyl-oleoyl phosphatidylcholine (POPC) liposomes or when a key residue in the peptide, Phe-454, is replaced with Ala. Molecular Dynamics simulations on CEACAM1-S in an asymmetric phospholipid bilayer show migration of Ca(2+) ions to the lipid leaflet containing POPS and reveal two conformations for Phe-454 explaining the reversible availability of this residue for G-actin binding. NMR transverse relaxation optimized spectroscopic analysis of (13)C-labeled Phe-454 CEACAM1-S peptide in liposomes plus actin further confirmed the existence of two peptide conformers and the Ca(2+) dependence of actin binding. These findings explain how a receptor with a short cytoplasmic domain can recruit a cytosolic protein in a phospholipid and Ca(2+)-specific manner. In addition, this model system provides a powerful approach that can be applied to study other membrane protein interactions with their cytosolic targets.

Selective 13C labeling of nucleotides for large RNA NMR spectroscopy using an E. coli strain disabled in the TCA cycle

Escherichia coli (E. coli) is an ideal organism to tailor-make labeled nucleotides for biophysical studies of RNA. Recently, we showed that adding labeled formate enhanced the isotopic enrichment at protonated carbon sites in nucleotides. In this paper, we show that growth of a mutant E. coli strain DL323 (lacking succinate and malate dehydrogenases) on 13C-2-glycerol and 13C-1,3-glycerol enables selective labeling at many useful sites for RNA NMR spectroscopy. For DL323 E. coli grown in 13C-2-glycerol without labeled formate, all the ribose carbon atoms are labeled except the C3′ and C5′ carbon positions. Consequently the C1′, C2′ and C4′ positions remain singlet. In addition, only the pyrimidine base C6 atoms are substantially labeled to ~96% whereas the C2 and C8 atoms of purine are labeled to ~5%. Supplementing the growth media with 13C-formate increases the labeling at C8 to ~88%, but not C2. Not unexpectedly, addition of exogenous formate is unnecessary for attaining the high enrichment levels of ~88% for the C2 and C8 purine positions in a 13C-1,3-glycerol based growth. Furthermore, the ribose ring is labeled in all but the C4′ carbon position, such that the C2′ and C3′ positions suffer from multiplet splitting but the C5′ position remains singlet and the C1′ position shows a small amount of residual C1′–C2′ coupling. As expected, all the protonated base atoms, except C6, are labeled to ~90%. In addition, labeling with 13C-1,3-glycerol affords an isolated methylene ribose with high enrichment at the C5′ position (~90%) that makes it particularly attractive for NMR applications involving CH2-TROSY modules without the need for decoupling the C4′ carbon. To simulate the tumbling of large RNA molecules, perdeuterated glycerol was added to a mixture of the four nucleotides, and the methylene TROSY experiment recorded at various temperatures. Even under conditions of slow tumbling, all the expected carbon correlations were observed, which indicates this approach of using nucleotides obtained from DL323 E. coli will be applicable to high molecular weight RNA systems.

1H, 15N chemical shift assignments of the imino groups in the base pairs of Escherichia coli tRNALeu (CAG)

tRNA molecules are the adaptors in ribosome-based protein biosynthesis and are stabilized by Mg(2+). However, the detailed mechanism for the Mg(2+) mediated stability is not fully understood. To study the effects of Mg(2+) on conformational flexibility of Escherichia coli tRNA(Leu) (CAG) at millisecond timescale, we applied NMR spectroscopic approach to measure proton exchange rates of imino groups in the presence of different concentration of Mg(2+) and correlated them with the corresponding aminoacylation activity of tRNA(Leu). Here, we report the first part of the above mentioned study, the (1)H, (15)N chemical shift assignments of the imino groups in all base pairs of Escherichia coli tRNA(Leu) (CAG) based on 2D (1)H-(15)N TROSY, 2D NOESY and 3D NOESY-HMQC experiments. This work laid the foundation for the NMR study of tRNA(Leu) (BMRB deposits with accession number 17078).

New Insights into the Interfacial Activation of Secreted Phospholipase A2

Despite numerous studies towards elucidation of the structural basis of activation of secreted PLA2s upon membrane binding (interfacial activation), no consistent or clear picture has emerged thus far. Previously we have reported significant changes in the secondary and dynamic structures of human group IB and IIA PLA2s, as well as changes in their mode of membrane binding during activation. Here we have conducted atomic resolution NMR studies on free and phospholipid micelle-bound human group IIA PLA2 (hIIAPLA2) to detect more detailed molecular events underlying interfacial activation. Two-dimensional 1H,15N-HSQC spectra have been obtained at 600 MHz on Ca2+-free and Ca2+-loaded hIIAPLA2 in the presence of dodecylphosphocholine (DPC) micelles. Upon complex formation with the micelles, signals from arginine side chain -NH2 groups of Ca2+-loaded hIIAPLA2 are observed, whereas for Ca2+-free PLA2 these signals are absent because of fast H/D exchange with the solvent. This suggests that the Ca2+-loaded hIIAPLA2 tightly binds to the micelles so these groups are sequestered at the PLA2-micellar interface and shielded from the solvent, or that they are otherwise stabilized by strong hydrogen bonding in the micelle-bound state. TROSY experiments (900 MHz) on Ca2+-loaded, 15N,13C-labeled hIIAPLA2 in the absence and presence of DPC micelles (1:600 protein-to-DPC molar ratio) identify substantial conformational changes in PLA2 upon binding to the micelles. Based on the assigned chemical shifts, important structural changes occur throughout the protein. The molecular mechanism of the strong increase in activity of hIIAPLA2 upon phospholipid surface binding is likely to involve a widening of the substrate binding pocket, mediated by a rigid-body movement of the N-terminal helix via interactions of the cationic residues (e.g., Arg7) with lipid phosphate groups. This mechanism will be tested in further studies and may be shared by other secreted PLA2 isoforms.

Identification of Allosteric PIF‐Pocket Ligands for PDK1 using NMR‐Based Fragment Screening and 1H‐15N TROSY Experiments

Aberrant activation of the phosphoinositide 3-kinase pathway because of genetic mutations of essential signalling proteins has been associated with human diseases including cancer and diabetes. The pivotal role of 3-phosphoinositide-dependent kinase-1 in the PI3K signalling cascade has made it an attractive target for therapeutic intervention. The N-terminal lobe of the 3-phosphoinositide-dependent kinase-1 catalytic domain contains a docking site which recognizes the non-catalytic C-terminal hydrophobic motifs of certain substrate kinases. The binding of substrate in this so-called PDK1 Interacting Fragment pocket allows interaction with 3-phosphoinositide-dependent kinase-1 and enhanced phosphorylation of downstream kinases. NMR spectroscopy was used to a screen 3-phosphoinositide-dependent kinase-1 domain construct against a library of chemically diverse fragments in order to identify small, ligand-efficient fragments that might interact at either the ATP site or the allosteric PDK1 Interacting Fragment pocket. While majority of the fragment hits were determined to be ATP-site binders, several fragments appeared to interact with the PDK1 Interacting Fragment pocket. Ligand-induced changes in 1H-15N TROSY spectra acquired using uniformly 15N-enriched PDK1 provided evidence to distinguish ATP-site from PDK1 Interacting Fragment-site binding. Caliper assay data and 19F NMR assay data on the PDK1 Interacting Fragment pocket fragments and structurally related compounds identified them as potential allosteric activators of PDK1 function.

An efficient protocol for the complete incorporation of methyl-protonated alanine in perdeuterated protein

A strategy for the introduction of ((1)H,(13)C-methyl)-alanine into perdeuterated proteins is described. Specific protonation of alanine methyl groups to a level of 95% can be achieved by overexpressing proteins in M9/D(2)O based bacterial growth medium supplemented with 800 mg/l of 2-[(2)H], 3-[(13)C] L: -alanine. However, though simple, this approach results in undesired, non-specific background labeling due to isotope scrambling via different amino acid metabolic pathways. Following a careful analysis of known metabolic pathways we found that co-addition of perdeuterated forms of alpha-ketoisovalerate-d(7), succinate-d(4) and L: -isoleucine-d(10) with labeled L: -alanine, reduces undesired background labeling to <1%. When combined with recently developed methyl TROSY experiments, this methyl-specific labeling protocol permits the acquisition of excellent quality correlation spectra of alanine methyl groups in high molecular weight proteins. Our cost effective strategy offers a significant enhancement in the level of incorporation of methyl-labeled alanine in overexpressed proteins over previously reported methods.

NMR-based homology model for the solution structure of the C-terminal globular domain of EMILIN1

EMILIN1 is a glycoprotein of elastic tissues that has been recently linked to the pathogenesis of hypertension. The protein is formed by different independently folded structural domains whose role has been partially elucidated. In this paper the solution structure, inferred from NMR-based homology modelling of the C-terminal trimeric globular C1q domain (gC1q) of EMILIN1, is reported. The high molecular weight and the homotrimeric structure of the protein required the combined use of highly deuterated (15)N, (13)C-labelled samples and TROSY experiments. Starting from a homology model, the protein structure was refined using heteronuclear residual dipolar couplings, chemical shift patterns, NOEs and H-exchange data. Analysis of the gC1q domain structure of EMILIN1 shows that each protomer of the trimer adopts a nine-stranded beta sandwich folding topology which is related to the conformation observed for other proteins of the family. Distinguishing features, however, include a missing edge-strand and an unstructured 19-residue loop. Although the current data do not allow this loop to be precisely defined, the available evidence is consistent with a flexible segment that protrudes from each subunit of the globular trimeric assembly and plays a key role in inter-molecular interactions between the EMILIN1 gC1q homotrimer and its integrin receptor alpha4beta1.

Solution State NMR Structure and Dynamics of KpOmpA, a 210 Residue Transmembrane Domain Possessing a High Potential for Immunological Applications

The three-dimensional structure of the outer membrane protein A from Klebsiella pneumoniae transmembrane domain was determined by NMR. This protein induces specific humoral and cytotoxic responses, and is a potent carrier protein. This is one of the largest integral membrane proteins (210 residues) for which nearly complete resonance assignment, including side chains, has been achieved so far. The methodology rested on the use of 900 MHz 3D and 4D TROSY experiments recorded on a uniformly 15N, 13C, 2H-labeled sample and on a perdeuterated methyl protonated sample. The structure was refined from 920 experimental constraints, giving an ensemble of 20 best structures with an r.m.s. deviation of 0.54 Å for the main chain atoms in the core eight-stranded β-barrel. The protein dynamics was assessed, in a residue-specific manner, by 1H- 15N NOEs (pico- to nanosecond timescale), exchange broadening (millisecond to second) and 1H- 2H chemical exchange (hour-weeks).

Solution NMR Structure of Selenium-binding Protein from Methanococcus vannielii

Selenium is an important nutrient. The lack of selenium will suppress expression of various enzymes that will lead to cell abnormality and diseases. However, high concentrations of free selenium are toxic to the cell because it adversely affects numerous cell metabolic pathways. In Methanococcus vannielii, selenium transport in the cell is established by the selenium-binding protein, SeBP. SeBP sequesters selenium during transport, thus regulating the level of free selenium in the cell, and delivers it specifically to the selenophosphate synthase enzyme. In solution, SeBP is an oligomer of 8.8-kDa subunits. It is a symmetric pentamer. The solution structure of SeBP was determined by NMR spectroscopy. Each subunit of SeBP is composed of an alpha-helix on top of a 4-stranded twisted beta-sheet. The stability of the five subunits stems mainly from hydrophobic interactions and supplemented by hydrogen bond interactions. The loop containing Cys(59) has been shown to be important for selenium binding, is flexible, and adopts multiple conformations. However, the cysteine accessibility is restricted in the structure, reducing the possibility of the binding of free selenium readily. Therefore, a different selenium precursor or other factors might be needed to facilitate opening of this loop to expose Cys(59) for selenium binding.

TROSY-Based NMR Evidence for a Novel Class of 20S Proteasome Inhibitors

The proteasome plays a central role in maintaining cellular homeostasis, in controlling the cell cycle, in removing misfolded proteins that can be toxic, and in regulating the immune system. It is also an important target for novel anticancer drugs, such as bortezomib, a potent inhibitor that has been used successfully in the treatment of multiple myeloma. Here, we show that the antimalaria drug chloroquine inhibits proteasome function in eukaryotic cell extracts and in preparations of purified 20S archaeal proteasome from Thermoplasma acidophilium. Methyl-TROSY-based NMR spectroscopy experiments conducted with the 670 kDa 20S proteasome localize chloroquine binding to regions between the alpha and beta subunits of the alpha-beta-beta-alpha barrel-like structure, approximately 20 A from the proteolytic active sites in this 7-fold symmetric molecule. Complementary amide TROSY experiments that provide further probes of proteasome-inhibitor interactions were performed on a novel 180 kDa single-ring construct containing only alpha subunits, the proper assembly of which was confirmed by electron microscopy. In contrast to the chloroquine-proteasome interaction described here, all previously reported inhibitors of the proteasome, including MG132, bind the catalytic region directly. Consistent with the NMR chemical shift perturbation data reported here that place chloroquine binding distal from sites of proteolysis, we show that MG132 and chloroquine can bind the proteasome simultaneously, further establishing that they exploit two completely separate binding pockets. Our data thus establish a novel class of proteasome inhibitor that functions via a mechanism distinct from binding to active sites.

Physical methods and techniques : NMR spectroscopy

NMR spectroscopy continues to evolve, and publications in 2007 provide an eclectic collection of applications, advances and incremental improvements, with solid-state MAS investigations of bio-systems making a significant contribution. Higher magnetic field spectrometers (or more accurately, some funding to enable the purchase of) have allowed “difficult-to-measure” nuclei such as 17O, for example, to become more mainstream. Fast 2D methods, covariance analysis and multiple acquisition methods allow chemists rapid access to detailed analyses. A 7D protein NMR data set can be recorded in 50 hours. The TROSY experiment has been used extensively to obtain dynamics information from large proteins. The accuracy of calculated and predicted NMR parameters (δ, J, D, CSA, Q) has improved and is becoming more accessible to the bench chemist, such that many solid-state NMR papers contain considerable computational and modelling data. I am indebted to the great and the good whose work has been reported here, all credit is theirs; errors, omissions and blame are all mine.

TROSY of side-chain amides in large proteins

By using the mixed solvent of 50% H 2O/50% D 2O and employing deuterium decoupling, TROSY experiments exclusively detect NMR signals from semideuterated isotopomers of carboxamide groups with high sensitivities for proteins with molecular weights up to 80 kDa. This isotopomer-selective strategy extends TROSY experiments from exclusively detecting backbone to both backbone and side-chain amides, particularly in large proteins. Because of differences in both TROSY effect and dynamics between 15N–H E {D Z } and 15N–H Z {D E } isotopomers of the same carboxamide, the 15N transverse magnetization of the latter relaxes significantly faster than that of the former, which provides a direct and reliable stereospecific distinction between the two configurations. The TROSY effects on the 15N–H E {D Z } isotopomers of side-chain amides are as significant as on backbone amides.

Simultaneous NMR assignment of backbone and side chain amides in large proteins with IS-TROSY

A new strategy for the simultaneous NMR assignment of both backbone and side chain amides in large proteins with isotopomer-selective transverse-relaxation-optimized spectroscopy (IS-TROSY) is reported. The method considers aspects of both the NMR sample preparation and the experimental design. First, the protein is dissolved in a buffer with 50%H2O/50%D2O in order to promote the population of semideuterated NHD isotopomers in side chain amides of Asn/Gln residues. Second, a 13C'-coupled 2D 15N-1H IS-TROSY spectrum provides a stereospecific distinction between the geminal protons in the E and Z configurations of the carboxyamide group. Third, a suite of IS-TROSY-based triple-resonance NMR experiments, e.g. 3D IS-TROSY-HNCA and 3D IS-TROSY-HNCACB, are designed to correlate aliphatic carbon atoms with backbone amides and, for Asn/Gln residues, at the same time with side chain amides. The NMR assignment procedure is similar to that for small proteins using conventional 3D HNCA/3D HNCACB spectra, in which, however, signals from NH2 groups are often very weak or even missing due to the use of broad-band proton decoupling schemes and NOE data have to be used as a remedy. For large proteins, the use of conventional TROSY experiments makes resonances of side chain amides not observable at all. The application of IS-TROSY experiments to the 35-kDa yeast cytosine deaminase has established a complete resonance assignment for the backbone and stereospecific assignment for side chain amides, which otherwise could not be achieved with existing NMR experiments. Thus, the development of IS-TROSY-based method provides new opportunities for the NMR study of important structural and biological roles of carboxyamides and side chain moieties of arginine and lysine residues in large proteins as well as amino moieties in nucleic acids.

The Vinculin Binding Sites of Talin and α-Actinin Are Sufficient to Activate Vinculin

Vinculin regulates both cell-cell and cell-matrix junctions and anchors adhesion complexes to the actin cytoskeleton through its interactions with the vinculin binding sites of alpha-actinin or talin. Activation of vinculin requires a severing of the intramolecular interactions between its N- and C-terminal domains, which is necessary for vinculin to bind to F-actin; yet how this occurs in cells is not resolved. We tested the hypothesis that talin and alpha-actinin activate vinculin through their vinculin binding sites. Indeed, we show that these vinculin binding sites have a high affinity for full-length vinculin, are sufficient to sever the head-tail interactions of vinculin, and they induce conformational changes that allow vinculin to bind to F-actin. Finally, microinjection of these vinculin binding sites specifically targets vinculin in cells, disrupting its interactions with talin and alpha-actinin and disassembling focal adhesions. In their native (inactive) states the vinculin binding sites of talin and alpha-actinin are buried within helical bundles present in their central rod domains. Collectively, these results support a model where the engagement of adhesion receptors first activates talin or alpha-actinin, by provoking structural changes that allow their vinculin binding sites to swing out, which are then sufficient to bind to and activate vinculin.