Various Factors that are Causing Difference in Prevalence of Coronary Risk Factors among Siblings.

BACKGROUND AND AIM: Coronary artery disease (CAD) is one of the predominant types of cardiovascular disease (CVD). The aim of present study was to study various factors that are causing difference in prevalence of coronary risk factors among siblings. MATERIALS AND METHODS: This cross-sectional study was conducted in Dayanand Medical College and Hospital, among the healthy individuals (not known CAD) attending regular health care outpatient department (OPD) and their siblings over a period of 1½ years. All individuals coming for regular health checkup (not known CAD) of age more than 30 years or above and their siblings (with or without known CAD). RESULTS: This was a cross-sectional study, conducted among 100 pairs of healthy siblings (not known cases of CAD) who came for health checkup at health center of Dayanand Medical College and Hospital, a tertiary care hospital in North India. Prevalence of obesity was more in siblings living in urban area than their counter siblings living in rural area, but it was statistically insignificant. Six had impaired fasting blood sugar (FBS) and two were diabetic. Among their siblings living in urban area, 21 were nondiabetic, 10 had impaired FBS, and seven were diabetic. This correlation was statistically significant with p-value of 0.02. Among the CAD negative, out of 23 subjects, two subjects (9.0%) had heavy stress level, while remaining four subjects (17.0%) and 17 subjects (74.0%) had light and moderate stress levels, respectively. Among the CAD negative, out of 23 subjects, 10 subjects (43.0%) had high stress level, while remaining zero subject (0%) and 13 subjects (57.0%) had light and moderate stress levels, respectively. Significant results were obtained while comparing the CAD findings of subjects divided on the basis of stress level. CONCLUSION: In our study, among siblings (CAD positive and CAD negative), significant results were obtained for residence, socioeconomic class, physical activity, stress levels, smoking, waist-to-hip ratio (WHR), and diabetes, that is, all these factors have correlation in increasing CAD among siblings.

Chemoradiation in locally advanced Ca Cx: Effect on NGAL levels.

INTRODUCTION: Neutrophil gelatinase-associated lipocalin (NGAL) has been reported to be unregulated in many cancers and to suppress tumor suppressor genes like p53 leading to cell proliferation. Studies to report its relationship with carcinoma cervix (Ca Cx) are still scant. MATERIALS AND METHODS: Serum NGAL levels were analyzed in 30 patients of histopathologically proven locally advanced Ca Cx at the time of diagnosis and 3 weeks after standard chemoradiation by enzyme-linked immunosorbent assay. These patients underwent either brachytherapy or supplementary external beam radiotherapy (EBRT) depending on the response of treatment. The results were analyzed statistically by applying Student's paired t-test. RESULTS: No statistically significant difference (P > 0.05) was observed in patients of Ca Cx before and after treatment or when compared stage wise, histopathological grade wise, or response wise. But the levels were found to increase when duration of treatment was ≥8 weeks (P = 0.040) and to decrease significantly when duration of treatment was <8 weeks (P = 0.0052). The NGAL levels also increased significantly after treatment in patients who received EBRT and supplementary radiotherapy (P = 0.019) while the pre- and post-treatment difference in NGAL levels was not statistically significant in patients who received EBRT + intracavitary brachytherapy (P > 0.05). CONCLUSION: As the duration as well as modality of treatment is quite important in Ca Cx, shorter duration associated with better results and lower NGAL levels, NGAL might prove to be a useful biomarker although further studies are needed to support the claim.

CDK4/6 Inhibition Augments Antitumor Immunity by Enhancing T-cell Activation.

Immune checkpoint blockade, exemplified by antibodies targeting the PD-1 receptor, can induce durable tumor regressions in some patients. To enhance the efficacy of existing immunotherapies, we screened for small molecules capable of increasing the activity of T cells suppressed by PD-1. Here, we show that short-term exposure to small-molecule inhibitors of cyclin-dependent kinases 4 and 6 (CDK4/6) significantly enhances T-cell activation, contributing to antitumor effects in vivo, due in part to the derepression of NFAT family proteins and their target genes, critical regulators of T-cell function. Although CDK4/6 inhibitors decrease T-cell proliferation, they increase tumor infiltration and activation of effector T cells. Moreover, CDK4/6 inhibition augments the response to PD-1 blockade in a novel ex vivo organotypic tumor spheroid culture system and in multiple in vivo murine syngeneic models, thereby providing a rationale for combining CDK4/6 inhibitors and immunotherapies.Significance: Our results define previously unrecognized immunomodulatory functions of CDK4/6 and suggest that combining CDK4/6 inhibitors with immune checkpoint blockade may increase treatment efficacy in patients. Furthermore, our study highlights the critical importance of identifying complementary strategies to improve the efficacy of immunotherapy for patients with cancer. Cancer Discov; 8(2); 216-33. ©2017 AACR.See related commentary by Balko and Sosman, p. 143See related article by Jenkins et al., p. 196This article is highlighted in the In This Issue feature, p. 127.

Distribution and kinetics of the Kv1.3-blocking peptide HsTX1[R14A] in experimental rats.

The peptide HsTX1[R14A] is a potent and selective blocker of the voltage-gated potassium channel Kv1.3, which is a highly promising target for the treatment of autoimmune diseases and other conditions. In order to assess the biodistribution of this peptide, it was conjugated with NOTA and radiolabelled with copper-64. [64Cu]Cu-NOTA-HsTX1[R14A] was synthesised in high radiochemical purity and yield. The radiotracer was evaluated in vitro and in vivo. The biodistribution and PET studies after intravenous and subcutaneous injections showed similar patterns and kinetics. The hydrophilic peptide was rapidly distributed, showed low accumulation in most of the organs and tissues, and demonstrated high molecular stability in vitro and in vivo. The most prominent accumulation occurred in the epiphyseal plates of trabecular bones. The high stability and bioavailability, low normal-tissue uptake of [64Cu]Cu-NOTA-HsTX1[R14A], and accumulation in regions of up-regulated Kv channels both in vitro and in vivo demonstrate that HsTX1[R14A] represents a valuable lead for conditions treatable by blockade of the voltage-gated potassium channel Kv1.3. The pharmacokinetics shows that both intravenous and subcutaneous applications are viable routes for the delivery of this potent peptide.

Reinvestigation of the biological activity of d-allo-ShK protein.

ShK toxin from the sea anemone Stichodactyla helianthus is a 35-residue protein that binds to the Kv1.3 ion channel with high affinity. Recently we determined the X-ray structure of ShK toxin by racemic crystallography, in the course of which we discovered that d-ShK has a near-background IC50 value ∼50,000 times lower than that of the l-ShK toxin. This lack of activity was at odds with previously reported results for an ShK diastereomer designated d-allo-ShK, for which significant biological activity had been observed in a similar receptor-blocking assay. As reported, d-allo-ShK was made up of d-amino acids, but with retention of the natural stereochemistry of the chiral side chains of the Ile and Thr residues, i.e. containing d-allo-Ile and d-allo-Thr along with d-amino acids and glycine. To understand its apparent biological activity, we set out to chemically synthesize d-allo-ShK and determine its X-ray structure by racemic crystallography. Using validated allo-Thr and allo-Ile, both l-allo-ShK and d-allo-ShK polypeptide chains were prepared by total chemical synthesis. Neither the l-allo-ShK nor the d-allo-ShK polypeptides folded, whereas both l-ShK and d-ShK folded smoothly under the same conditions. Re-examination of NMR spectra of the previously reported d-allo-ShK protein revealed that diagnostic Thr and Ile signals were the same as for authentic d-ShK. On the basis of these results, we conclude that the previously reported d-allo-ShK was in fact d-ShK, the true enantiomer of natural l-ShK toxin, and that the apparent biological activity may have arisen from inadvertent contamination with trace amounts of l-ShK toxin.

Stereoselective synthesis and structural elucidation of dicarba peptides.

A facile stereoselective synthesis of cis and trans unsaturated dicarba peptides has been established using preformed diaminosuberic acid derivatives as bridging units. In addition, characteristic spectral differences in the (13)C-NMR spectra of the cis- and trans-isomers show that the chemical shift of carbons in the Δ4,5-diaminosuberic acid residue can be used to assign stereochemistry in unsaturated dicarba peptides formed from ring closing metathesis of linear peptide sequences.

Structural Basis for the Inhibition of Voltage-gated Sodium Channels by Conotoxin µO§-GVIIJ.

Cone snail toxins are well known blockers of voltage-gated sodium channels, a property that is of broad interest in biology and therapeutically in treating neuropathic pain and neurological disorders. Although most conotoxin channel blockers function by direct binding to a channel and disrupting its normal ion movement, conotoxin µO§-GVIIJ channel blocking is unique, using both favorable binding interactions with the channel and a direct tether via an intermolecular disulfide bond. Disulfide exchange is possible because conotoxin µO§-GVIIJ contains anS-cysteinylated Cys-24 residue that is capable of exchanging with a free cysteine thiol on the channel surface. Here, we present the solution structure of an analog of µO§-GVIIJ (GVIIJ[C24S]) and the results of structure-activity studies with synthetic µO§-GVIIJ variants. GVIIJ[C24S] adopts an inhibitor cystine knot structure, with two antiparallel ß-strands stabilized by three disulfide bridges. The loop region linking the ß-strands (loop 4) presents residue 24 in a configuration where it could bind to the proposed free cysteine of the channel (Cys-910, rat NaV1.2 numbering; at site 8). The structure-activity study shows that three residues (Lys-12, Arg-14, and Tyr-16) located in loop 2 and spatially close to residue 24 were also important for functional activity. We propose that the interaction of µO§-GVIIJ with the channel depends on not only disulfide tethering via Cys-24 to a free cysteine at site 8 on the channel but also the participation of key residues of µO§-GVIIJ on a distinct surface of the peptide.

A Naturally Occurring Peptide with an Elementary Single Disulfide-Directed ß-Hairpin Fold.

Certain peptide folds, owing to a combination of intrinsic stability and resilience to amino acid substitutions, are particularly effective for the display of diverse functional groups. Such "privileged scaffolds" are valuable as starting points for the engineering of new bioactive molecules. We have identified a precursor peptide expressed in the venom gland of the marine snail Conus victoriae, which appears to belong to a hitherto undescribed class of molluscan neuropeptides. Mass spectrometry matching with the venom confirmed the complete mature peptide sequence as a 31-residue peptide with a single disulfide bond. Solution structure determination revealed a unique peptide fold that we have designated the single disulfide-directed ß hairpin (SDH). The SDH fold is highly resistant to thermal denaturation and forms the core of several other multiple disulfide-containing peptide folds, including the inhibitor cystine knot. This elementary fold may offer a valuable starting point for the design and engineering of new bioactive peptides.

Installation of a Rigid EDTA-Like Motif into a Protein α-Helix for Paramagnetic NMR Spectroscopy with Cobalt(II) Ions.

Coupling two copies of an iminodiacetic acid-cysteine hybrid ligand to a pair of cysteine residues positioned in an i, i+4 arrangement within a protein α-helix leads to generation of an EDTA-like metal ion-binding motif. Rigid binding of a Co(II) ion by this motif produces pseudo-contact shifts suitable for paramagnetic NMR structural studies.

Elevated baseline plasma phospholipid protein (PLTP) levels are an independent predictor of long-term all-cause mortality in patients with diabetes mellitus and known or suspected coronary artery disease.

OBJECTIVES: To investigate the long-term prognostic significance of baseline plasma PLTP levels in a group of well-characterized male patients with diabetes mellitus and known or suspected coronary artery disease referred for coronary angiography. BACKGROUND: PLTP is a plasma protein that mediates the net transfer and exchange of phospholipids between lipoproteins. It has been implicated in the pathogenesis of atherosclerosis and elevated plasma levels have been reported in patients with diabetes mellitus. METHODS: Baseline plasma PLTP levels were measured in 154 male patients with diabetes mellitus who were referred for coronary angiography and followed prospectively for 5 years for the development of all-cause mortality. RESULTS: After adjustment for a variety of baseline clinical, angiographic and laboratory parameters, plasma PLTP levels (analyzed as a continuous variable) were an independent predictor of all-cause mortality at 5 years (HR, 1.55; 95% CI, 1.22-2.00; P = 0.0009). Furthermore, in 3 additional multivariate models that also included a wide variety of contemporary biomarkers with established prognostic efficacy (i.e., ST2, GDF-15, Cystatin C, Fibrinogen, and NT-proBNP), PLTP remained an independent predictor of all-cause mortality at 5 years. CONCLUSIONS: Elevated baseline plasma levels of PLTP are associated with an increased risk of long-term all-cause mortality in patients with diabetes and known or suspected coronary disease. Furthermore, this association is independent of a variety of clinical, angiographic, and laboratory variables, including a whole host of contemporary biomarkers with established prognostic efficacy.

Development of highly selective Kv1.3-blocking peptides based on the sea anemone peptide ShK.

ShK, from the sea anemone Stichodactyla helianthus, is a 35-residue disulfide-rich peptide that blocks the voltage-gated potassium channel Kv1.3 at ca. 10 pM and the related channel Kv1.1 at ca. 16 pM. We developed an analog of this peptide, ShK-186, which is currently in Phase 1b-2a clinical trials for the treatment of autoimmune diseases such as multiple sclerosis and rheumatoid arthritis. While ShK-186 displays a >100-fold improvement in selectivity for Kv1.3 over Kv1.1 compared with ShK, there is considerable interest in developing peptides with an even greater selectivity ratio. In this report, we describe several variants of ShK that incorporate p-phophono-phenylalanine at the N-terminus coupled with internal substitutions at Gln16 and Met21. In addition, we also explored the combinatorial effects of these internal substitutions with an alanine extension at the C-terminus. Their selectivity was determined by patch-clamp electrophysiology on Kv1.3 and Kv1.1 channels stably expressed in mouse fibroblasts. The peptides with an alanine extension blocked Kv1.3 at low pM concentrations and exhibited up to 2250-fold selectivity for Kv1.3 over Kv1.1. Analogs that incorporates p-phosphono-phenylalanine at the N-terminus blocked Kv1.3 with IC50s in the low pM range and did not affect Kv1.1 at concentrations up to 100 nM, displaying a selectivity enhancement of >10,000-fold for Kv1.3 over Kv1.1. Other potentially important Kv channels such as Kv1.4 and Kv1.6 were only partially blocked at 100 nM concentrations of each of the ShK analogs.

Structure-based design and development of functionalized Mercaptoguanine derivatives as inhibitors of the folate biosynthesis pathway enzyme 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase from Staphylococcus aureus.

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK), an enzyme from the folate biosynthesis pathway, catalyzes the pyrophosphoryl transfer from ATP to 6-hydroxymethyl-7,8-dihydropterin and is a yet-to-be-drugged antimicrobial target. Building on our previous discovery that 8-mercaptoguanine (8MG) is an inhibitor of Staphylococcus aureus HPPK (SaHPPK), we have identified and characterized the binding of an S8-functionalized derivative (3). X-ray structures of both the SaHPPK/3/cofactor analogue ternary and the SaHPPK/cofactor analogue binary complexes have provided insight into cofactor recognition and key residues that move over 30 Å upon binding of 3, whereas NMR measurements reveal a partially plastic ternary complex active site. Synthesis and binding analysis of a set of analogues of 3 have identified an advanced new lead compound (11) displaying >20-fold higher affinity for SaHPPK than 8MG. A number of these exhibited low micromolar affinity for dihydropteroate synthase (DHPS), the adjacent, downstream enzyme to HPPK, and may thus represent promising new leads to bienzyme inhibitors.

Kikuchi's disease--a rare cause of lymphadenopathy and fever.

Kikuchi's disease is a rare, benign, self-limited disorder, characterised clinically by fever and tender regional lymphadenopathy. It has been reported worldwide and is particularly common in people of Asian descent. The cause of Kikuchi's disease is unknown. It predominantly affects young females and can closely mimic several infectious and immunological conditions. Histopathologic features of lymph nodes in Kikuchi's disease are characteristic and permit differentiation of this benign condition from lymphomas, systemic lupus erythematosus and infectious lymphadenopathies. We report a female patient presenting with fever and tender cervical lymphadenopathy. She was being treated for tubercular lymphadenitis and was referred after she developed a transient hepatitis and a skin rash following treatment with anti-tubercular drugs. An excisional biopsy of the lymph node revealed histiocytic necrotising lymphadenitis, consistent with Kikuchi's disease. A brief review of the pathogenesis and differential diagnosis of Kikuchi's disease is presented.

Conformational flexibility in the binding surface of the potassium channel blocker ShK.

ShK is a 35-residue peptide that binds with high affinity to human voltage-gated potassium channels through a conserved K-Y dyad. Here we have employed NMR measurements of backbone-amide (15)N spin-relaxation rates to investigate motions of the ShK backbone. Although ShK is rigid on the ps to ns timescale, increased linewidths observed for 11 backbone-amide (15)N resonances identify chemical or conformational exchange contributions to the spin relaxation. Relaxation dispersion profiles indicate that exchange between major and minor conformers occurs on the sub-millisecond timescale. Affected residues are mostly clustered around the central helix-kink-helix structure and the critical K22-Y23 motif. We suggest that the less structured minor conformer increases the exposure of Y23, known to contribute to binding affinity and selectivity, thereby facilitating its interaction with potassium channels. These findings have potential implications for the design of new channel blockers based on ShK.

A potent cyclic peptide targeting SPSB2 protein as a potential anti-infective agent.

The protein SPSB2 mediates proteosomal degradation of inducible nitric oxide synthase (iNOS). Inhibitors of SPSB2-iNOS interaction may prolong the lifetime of iNOS and thereby enhance the killing of persistent pathogens. We have designed a cyclic peptide, Ac-c[CVDINNNC]-NH2, containing the key sequence motif mediating the SPSB2-iNOS interaction, which binds to the iNOS binding site on SPSB2 with a Kd of 4.4 nM, as shown by SPR, [(1)H,(15)N]-HSQC, and (19)F NMR. An in vitro assay on macrophage cell lysates showed complete inhibition of SPSB2-iNOS interactions by the cyclic peptide. Furthermore, its solution structure closely matched (backbone rmsd 1.21 Å) that of the SPSB2-bound linear DINNN peptide. The designed peptide was resistant to degradation by the proteases pepsin, trypsin, and chymotrypsin and stable in human plasma. This cyclic peptide exemplifies potentially a new class of anti-infective agents that acts on the host innate response, thereby avoiding the development of pathogen resistance.

Interactions of disulfide-deficient selenocysteine analogs of µ-conotoxin BuIIIB with the α-subunit of the voltage-gated sodium channel subtype 1.3.

Inhibitors of the α-subunit of the voltage-gated sodium channel subtype 1.3 (NaV 1.3) are of interest as pharmacological tools for the study of neuropathic pain associated with spinal cord injury and have potential therapeutic applications. The recently described µ-conotoxin BuIIIB (µ-BuIIIB) from Conus bullatus was shown to block NaV 1.3 with submicromolar potency (Kd = 0.2 µm), making it one of the most potent peptidic inhibitors of this subtype described to date. However, oxidative folding of µ-BuIIIB results in numerous folding isoforms, making it difficult to obtain sufficient quantities of the active form of the peptide for detailed structure-activity studies. In the present study, we report the synthesis and characterization of µ-BuIIIB analogs incorporating a disulfide-deficient, diselenide-containing scaffold designed to simplify synthesis and facilitate structure-activity studies directed at identifying amino acid residues involved in NaV 1.3 blockade. Our results indicate that, similar to other µ-conotoxins, the C-terminal residues (Trp16, Arg18 and His20) are most crucial for NaV 1 blockade. At the N-terminus, replacement of Glu3 by Ala resulted in an analog with an increased potency for NaV 1.3 (Kd = 0.07 µm), implicating this position as a potential site for modification for increased potency and/or selectivity. Further examination of this position showed that increased negative charge, through γ-carboxyglutamate replacement, decreased potency (Kd = 0.33 µm), whereas replacement with positively-charged 2,4-diamonobutyric acid increased potency (Kd = 0.036 µm). These results provide a foundation for the design and synthesis of µ-BuIIIB-based analogs with increased potency against NaV 1.3.

A potent and selective peptide blocker of the Kv1.3 channel: prediction from free-energy simulations and experimental confirmation.

The voltage-gated potassium channel Kv1.3 is a well-established target for treatment of autoimmune diseases. ShK peptide from a sea anemone is one of the most potent blockers of Kv1.3 but its application as a therapeutic agent for autoimmune diseases is limited by its lack of selectivity against other Kv channels, in particular Kv1.1. Accurate models of Kv1.x-ShK complexes suggest that specific charge mutations on ShK could considerably enhance its specificity for Kv1.3. Here we evaluate the K18A mutation on ShK, and calculate the change in binding free energy associated with this mutation using the path-independent free energy perturbation and thermodynamic integration methods, with a novel implementation that avoids convergence problems. To check the accuracy of the results, the binding free energy differences were also determined from path-dependent potential of mean force calculations. The two methods yield consistent results for the K18A mutation in ShK and predict a 2 kcal/mol gain in Kv1.3/Kv1.1 selectivity free energy relative to wild-type peptide. Functional assays confirm the predicted selectivity gain for ShK[K18A] and suggest that it will be a valuable lead in the development of therapeutics for autoimmune diseases.

A novel inhibitor of α9α10 nicotinic acetylcholine receptors from Conus vexillum delineates a new conotoxin superfamily.

Conotoxins (CTxs) selectively target a range of ion channels and receptors, making them widely used tools for probing nervous system function. Conotoxins have been previously grouped into superfamilies according to signal sequence and into families based on their cysteine framework and biological target. Here we describe the cloning and characterization of a new conotoxin, from Conus vexillum, named αB-conotoxin VxXXIVA. The peptide does not belong to any previously described conotoxin superfamily and its arrangement of Cys residues is unique among conopeptides. Moreover, in contrast to previously characterized conopeptide toxins, which are expressed initially as prepropeptide precursors with a signal sequence, a ''pro'' region, and the toxin-encoding region, the precursor sequence of αB-VxXXIVA lacks a ''pro'' region. The predicted 40-residue mature peptide, which contains four Cys, was synthesized in each of the three possible disulfide arrangements. Investigation of the mechanism of action of αB-VxXXIVA revealed that the peptide is a nicotinic acetylcholine receptor (nAChR) antagonist with greatest potency against the α9α10 subtype. (1)H nuclear magnetic resonance (NMR) spectra indicated that all three αB-VxXXIVA isomers were poorly structured in aqueous solution. This was consistent with circular dichroism (CD) results which showed that the peptides were unstructured in buffer, but adopted partially helical conformations in aqueous trifluoroethanol (TFE) solution. The α9α10 nAChR is an important target for the development of analgesics and cancer chemotherapeutics, and αB-VxXXIVA represents a novel ligand with which to probe the structure and function of this protein.

Structure of S. aureus HPPK and the discovery of a new substrate site inhibitor.

The first structural and biophysical data on the folate biosynthesis pathway enzyme and drug target, 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (SaHPPK), from the pathogen Staphylococcus aureus is presented. HPPK is the second essential enzyme in the pathway catalysing the pyrophosphoryl transfer from cofactor (ATP) to the substrate (6-hydroxymethyl-7,8-dihydropterin, HMDP). In-silico screening identified 8-mercaptoguanine which was shown to bind with an equilibrium dissociation constant, K(d), of ∼13 µM as measured by isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR). An IC(50) of ∼41 µM was determined by means of a luminescent kinase assay. In contrast to the biological substrate, the inhibitor has no requirement for magnesium or the ATP cofactor for competitive binding to the substrate site. The 1.65 Å resolution crystal structure of the inhibited complex showed that it binds in the pterin site and shares many of the key intermolecular interactions of the substrate. Chemical shift and (15)N heteronuclear NMR measurements reveal that the fast motion of the pterin-binding loop (L2) is partially dampened in the SaHPPK/HMDP/α,ß-methylene adenosine 5'-triphosphate (AMPCPP) ternary complex, but the ATP loop (L3) remains mobile on the µs-ms timescale. In contrast, for the SaHPPK/8-mercaptoguanine/AMPCPP ternary complex, the loop L2 becomes rigid on the fast timescale and the L3 loop also becomes more ordered--an observation that correlates with the large entropic penalty associated with inhibitor binding as revealed by ITC. NMR data, including (15)N-(1)H residual dipolar coupling measurements, indicate that the sulfur atom in the inhibitor is important for stabilizing and restricting important motions of the L2 and L3 catalytic loops in the inhibited ternary complex. This work describes a comprehensive analysis of a new HPPK inhibitor, and may provide a foundation for the development of novel antimicrobials targeting the folate biosynthetic pathway.

DOTA-amide lanthanide tag for reliable generation of pseudocontact shifts in protein NMR spectra.

Structural studies of proteins and protein-ligand complexes by nuclear magnetic resonance (NMR) spectroscopy can be greatly enhanced by site-specific attachment of lanthanide ions to create paramagnetic centers. In particular, pseudocontact shifts (PCS) generated by paramagnetic lanthanides contain important and unique long-range structure information. Here, we present a high-affinity lanthanide binding tag that can be attached to single cysteine residues of proteins. The new tag has many advantageous features that are not available in this combination from previously published tags: (i) it binds lanthanide ions very tightly, minimizing the generation of nonspecific effects, (ii) it produces PCSs with high reliability as its bulkiness prevents complete motional averaging of PCSs, (iii) it can be attached to single cysteine residues, alleviating the need of detailed prior knowledge of the 3D structure of the target protein, and (iv) it does not display conformational exchange phenomena that would increase the number of signals in the NMR spectrum. The performance of the tag is demonstrated with the N-terminal domain of the E. coli arginine repressor and the A28C mutant of human ubiquitin.