Understanding interactions of gastric inhibitory polypeptide (GIP) with its G-protein coupled receptor through NMR and molecular modeling.

Gastric inhibitory polypeptide (GIP, or glucose-dependent insulinotropic polypeptide) is a 42-amino acid incretin hormone moderating glucose-induced insulin secretion. Antidiabetic therapy based on GIP holds great promise because of the fact that its insulinotropic action is highly dependent on the level of glucose, overcoming the sideeffects of hypoglycemia associated with the current therapy of Type 2 diabetes. The truncated peptide, GIP(1-30)NH2, has the same activity as the full length native peptide. We have studied the structure of GIP(1-30)NH2 and built a model of its G-protein coupled receptor (GPCR). The structure of GIP(1-30)NH2 in DMSO-d6 and H2O has been studied using 2D NMR (total correlation spectroscopy (TOCSY), nuclear overhauser effect spectroscopy (NOESY), double quantum filtered-COSY (DQF-COSY), 13C-heteronuclear single quantum correlation (HSQC) experiments, and its conformation built by MD simulations with the NMR data as constraints. The peptide in DMSO-d6 exhibits an alpha-helix between residues Ile12 and Lys30 with a discontinuity at residues Gln19 and Gln20. In H2O, the alpha-helix starts at Ile7, breaks off at Gln19, and then continues right through to Lys30. GIP(1-30)NH2 has all the structural features of peptides belonging to family B1 GPCRs, which are characterized by a coil at the N-terminal and a long C-terminal alpha-helix with or without a break. A model of the seven transmembrane (TM) helices of the GIP receptor (GIPR) has been built on the principles of comparative protein modeling, using the crystal structure of bovine rhodopsin as a template. The N-terminal domain of GIPR has been constructed from the NMR structure of the N-terminal of corticoptropin releasing factor receptor (CRFR), a family B1 GCPR. The intra and extra cellular loops and the C-terminal have been modeled from fragments retrieved from the PDB. On the basis of the experimental data available for some members of family B1 GPCRs, four pairs of constraints between GIP(1-30)NH2 and its receptor were used in the FTDOCK program, to build the complete model of the GIP(1-30)NH2:GIPR complex. The model can rationalize the various experimental observations including the potency of the truncated GIP peptide. This work is the first complete model at the atomic level of GIP(1-30)NH2 and of the complex with its GPCR.     

Characterization and biological actions of N-terminal truncated forms of glucose-dependent insulinotropic polypeptide

The N-terminal domain of glucose-dependent insulinotropic polypeptide (GIP) plays an important role in regulating biological activity. This study examined biological properties of several N-terminal truncated forms of GIP and two novel forms with substitutions at Phe position-6 with Arg or Val. GIP(6-42), GIP(R6-42), GIP(V6-42), GIP(7-42) and GIP(9-42) stimulated cAMP production in BRIN-BD11 cells similar to native GIP, whereas responses to GIP(3-42), GIP(4-42), GIP(5-42) and GIP(8-42) were reduced (P < 0.01 to P < 0.001). GIP-induced cyclic AMP production was significantly inhibited by GIP(3-42), GIP(4-42), GIP(5-42), GIP(6-42), GIP(R6-42), GIP(7-42) and GIP(8-42) (P < 0.001). Compared with native GIP, in vitro insulinotropic activity of GIP(3-42), GIP(4-42), GIP(5-42), GIP(7-42) and GIP(8-42) was reduced (P < 0.05 to P < 0.001), with GIP(4-42), GIP(5-42), GIP(7-42) and GIP(8-42) also potently inhibiting GIP-stimulated insulin secretion (P < 0.001). In ob/ob mice, GIP(4-42) and GIP(8-42) increased (P < 0.05 to P < 0.01) plasma glucose concentrations compared to the glucose-lowering action of native GIP. When GIP(8-42) was co-administered with native GIP it countered the ability of the native peptide to lower plasma glucose and increase circulating insulin concentrations. These data confirm the importance of the N-terminal region of GIP in regulating bioactivity and reveal that sequential truncation of the peptide yields novel GIP receptor antagonists which may have functional significance.     

Tyr1 and Ile7 of glucose-dependent insulinotropic polypeptide (GIP) confer differential ligand selectivity toward GIP and glucagon-like peptide-1 receptors

Glucagon like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are incretin hormones released in response to food intake and potentiate insulin secretion from pancreatic β cells through their distinct yet related G protein-coupled receptors, GLP1R and GIPR. While GLP-1 and GIP exhibit similarity in their N-terminal sequence and overall α-helical structure, GLP-1 does not bind to GIPR and vice versa. To determine which amino acid residues of these peptide ligands are responsible for specific interaction with their respective receptors, we generated mutant GIP in which several GLP-1-specific amino acid residues were substituted for the original amino acids. The potency of the mutant ligands was examined using HEK293 cells transfected with GLP1R or GIPR expression plasmids together with a cAMP-responsive element-driven luciferase (CRE-luc) reporter plasmid. A mutated GIP peptide in which Tyr¹, Ile⁷, Asp¹⁵, and His¹⁸ were replaced by His, Thr, Glu, and Ala, respectively, was able to activate both GLP1R and GIPR with moderate potency. Replacing the original Tyr¹ and/or Ile⁷ in the N-terminal moiety of this mutant peptide allowed full activation of GIPR but not of GLP1R. However, reintroducing Asp¹⁵ and/or His¹⁸ in the central α-helical region did not significantly alter the ligand potency. These results suggest that Tyr/His¹ and Ile/Thr⁷ of GIP/GLP-1 peptides confer differential ligand selectivity toward GIPR and GLP1R.     

Evolutionarily conserved residues at glucagon-like peptide-1 (GLP-1) receptor core confer ligand-induced receptor activation

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) play important roles in insulin secretion through their receptors, GLP1R and GIPR. Although GLP-1 and GIP are attractive candidates for treatment of type 2 diabetes and obesity, little is known regarding the molecular interaction of these peptides with the heptahelical core domain of their receptors. These core domains are important not only for specific ligand binding but also for ligand-induced receptor activation. Here, using chimeric and point-mutated GLP1R/GIPR, we determined that evolutionarily conserved amino acid residues such as Ile(196) at transmembrane helix 2, Leu(232) and Met(233) at extracellular loop 1, and Asn(302) at extracellular loop 2 of GLP1R are responsible for interaction with ligand and receptor activation. Application of chimeric GLP-1/GIP peptides together with molecular modeling suggests that His(1) of GLP-1 interacts with Asn(302) of GLP1R and that Thr(7) of GLP-1 has close contact with a binding pocket formed by Ile(196), Leu(232), and Met(233) of GLP1R. This study may provide critical clues for the development of peptide and/or nonpeptide agonists acting at GLP1R.     

A novel acylated form of (d-Ala)GIP with improved antidiabetic potential,lacking effect on body fat stores

Background

Rapid enzymatic degradation of the incretin hormone, glucose-dependent insulinotropic polypeptide (GIP), limits therapeutic use of the native peptide for diabetes. However, enzymatically stable analogues of GIP, such as (d-Ala²)GIP, have been generated, but are still susceptible to renal filtration.

Methods

The present study examines the in vitro and in vivo biological actions of a novel, acylated GIP analogue, (d-Ala²)GIP[Lys³⁷PAL].

Results

In BRIN-BD11 cells, (d-Ala²)GIP[Lys³⁷PAL] concentration-dependently stimulated (p < 0.05 to p < 0.001) insulin secretion at 5.6 and 16.7 mM glucose. Intraperitoneal administration of (d-Ala²)GIP[Lys³⁷PAL] to normal mice 8 h prior to a glucose load significantly reduced (p < 0.05) the overall glycaemic excursion compared to controls, and increased (p < 0.001) the insulinotropic response compared to (d-Ala²)GIP and saline treated high fat control mice. Once daily administration of (d-Ala²)GIP[Lys³⁷PAL] for 21 days in high fat fed mice did not affect energy intake, body weight or fat deposition. However, circulating blood glucose was significantly lower (p < 0.05) accompanied by increased (p < 0.05) insulin concentrations by day 21. In addition, (d-Ala²)GIP[Lys³⁷PAL] treatment significantly (p < 0.01) reduced the overall glycaemic excursion and increased pancreatic insulin content (p < 0.05) and the insulinotropic response (p < 0.01) to an exogenous glucose challenge on day 21. Chronic treatment with (d-Ala²)GIP[Lys³⁷PAL] did not result in resistance to the metabolic effects of a bolus injection of native GIP. Finally, insulin sensitivity was significantly improved (p < 0.001) in (d-Ala²)GIP[Lys³⁷PAL] treated mice compared to high fat controls.

Conclusions

These data confirm that (d-Ala²)GIP[Lys³⁷PAL] is a stable, long-acting potent GIP agonist.

General significance

(d-Ala²)GIP[Lys³⁷PAL] may be suitable for further evaluation and future clinical development.     

Actions of incretin metabolites on locomotor activity,cognitive function and in vivo hippocampal synaptic plasticity in high fat fed mice

The incretin hormones glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) improve markers of cognitive function in obesity–diabetes, however, both are rapidly degraded to their major metabolites, GLP-1(9-36)amide and GIP(3-42), respectively. Therefore, the present study investigated effects of GLP-1(9-36)amide and GIP(3-42) on locomotor activity, cognitive function and hippocampal synaptic plasticity in mice with diet-induced obesity and insulin resistance. High-fat fed Swiss TO mice treated with GLP-1(9-36)amide, GIP(3-42) or exendin(9-39)amide (twice-daily for 60 days) did not exhibit any changes in bodyweight, non-fasting plasma glucose and plasma insulin concentrations or glucose tolerance compared with high-fat saline controls. Similarly, locomotor and feeding activity, O₂ consumption, CO₂ production, respiratory exchange ratio and energy expenditure were not altered by chronic treatment with incretin metabolites. Administration of the truncated metabolites did not alter general behavior in an open field test or learning and memory ability as recorded during an object recognition test. High-fat mice exhibited a significant impairment in hippocampal long-term potentiation (LTP) which was not affected by treatment with incretin metabolites. These data indicate that incretin metabolites do not influence locomotor activity, cognitive function and hippocampal synaptic plasticity when administered at pharmacological doses to mice fed a high-fat diet.     

Site‐specific labeling of synthetic peptide using the chemoselective reaction between N‐methoxyamino acid and isothiocyanate

Site‐specific labeling of synthetic peptides carrying N‐methoxyglycine (MeOGly) by isothiocyanate is demonstrated. A nonapeptide having MeOGly at its N‐terminus was synthesized by the solid‐phase method and reacted with phenylisothiocyanate under various conditions. In acidic solution, the reaction specifically gave a peptide having phenylthiourea structure at its N‐terminus, leaving side chain amino group intact. The synthetic human β‐defensin‐2 carrying MeOGly at its N‐terminus or the side chain amino group of Lys¹⁰ reacted with phenylisothiocyanate or fluorescein isothiocyanate also at the N‐methoxyamino group under the same conditions, demonstrating that this method is generally useful for the site‐specific labeling of linear synthetic peptides as well as disulfide‐containing peptides. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

The bioactive conformation of glucose-dependent insulinotropic polypeptide by NMR and CD spectroscopy

Glucose-dependent insulinotropic polypeptide (GIP) is a gastrointestinal incretin hormone, which modulates physiological insulin secretion. Because of its glucose-sensitive insulinotropic activity, there has been a considerable interest in utilizing the hormone as a potential treatment for type 2 diabetes. Structural parameters obtained from NMR spectroscopy combined with molecular modeling techniques play a vital role in the design of new therapeutic drugs. Therefore, to understand the structural requirements for the biological activity of GIP, the solution structure of GIP was investigated by circular dichroism (CD) followed by proton nuclear magnetic resonance (NMR) spectroscopy. CD studies showed an increase in the helical character of the peptide with increasing concentration of trifluoroethanol (TFE) up to 50%. Therefore, the solution structure of GIP in 50% TFE was determined. It was found that there was an alpha-helix between residues 6 and 29, which tends to extend further up to residue 36. The implications of the C-terminal extended helical segment in the inhibitory properties of GIP on gastric acid secretion are discussed. It is shown that the adoption by GIP of an alpha-helical secondary structure is a requirement for its biological activity. Knowledge of the solution structure of GIP will help in the understanding of how the peptide interacts with its receptor and aids in the design of new therapeutic agents useful for the treatment of diabetes.     

Structural and Pharmacological Characterization of Novel Potent and Selective Monoclonal Antibody Antagonists of Glucose-dependent Insulinotropic Polypeptide Receptor

Glucose-dependent insulinotropic polypeptide (GIP) is an endogenous hormonal factor (incretin) that, upon binding to its receptor (GIPr; a class B G-protein-coupled receptor), stimulates insulin secretion by beta cells in the pancreas. There has been a lack of potent inhibitors of the GIPr with prolonged in vivo exposure to support studies on GIP biology. Here we describe the generation of an antagonizing antibody to the GIPr, using phage and ribosome display libraries. Gipg013 is a specific competitive antagonist with equally high potencies to mouse, rat, dog, and human GIP receptors with a K_i of 7 nm for the human GIPr. Gipg013 antagonizes the GIP receptor and inhibits GIP-induced insulin secretion in vitro and in vivo. A crystal structure of Gipg013 Fab in complex with the human GIPr extracellular domain (ECD) shows that the antibody binds through a series of hydrogen bonds from the complementarity-determining regions of Gipg013 Fab to the N-terminal α-helix of GIPr ECD as well as to residues around its highly conserved glucagon receptor subfamily recognition fold. The antibody epitope overlaps with the GIP binding site on the GIPr ECD, ensuring competitive antagonism of the receptor. This well characterized antagonizing antibody to the GIPr will be useful as a tool to further understand the biological roles of GIP.     

Total synthesis and stereochemical reassignment of tasiamide

The total synthesis of a marine acyclic peptide tasiamide and three diastereomers was reported for the first time. The synthesis has led to a reassignment of the N^α‐Me‐L ‐Gln of tasiamide to be N^α‐Me‐D ‐Gln, which was supported by ¹H NMR, ¹³C NMR, COSY, HMQC, HMBC, and optical rotation. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Assembly of binding loops on aromatic templates as VCAM‐1 mimetics

The design and synthesis of cyclic mimetics of VCAM‐1 protein that reproduce the integrin‐binding domain are presented. The unprotected peptide precursor 37 – 43 , Thr‐Gln‐Ile‐Asp‐Ser‐Pro‐Leu, was grafted onto functional templates of type naphthalene, biphenyl and benzyl through the chemoselective formation of C‐ and N‐terminal oximes resulting in a mixture of four isomeric forms due to syn–anti isomerism of the oxime bonds. Some isomers could be monitored by HPLC and identified by NMR. The molecule containing a naphthalene‐derived template was found to inhibit the VCAM‐1/VLA‐4 interaction more efficiently than previously reported for sulfur‐bridged cyclic peptides containing similar sequences. The finding confirms the importance of incorporating conformational constraints between the terminal ends of the peptide loop 37 – 43 in the design of synthetic inhibitors of the VCAM‐1/integrin interaction. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Two incretin hormones GLP-1 and GIP: comparison of their actions in insulin secretion and β cell preservation

Gastric inhibitory polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are the two primary incretin hormones secreted from the intestine upon ingestion of glucose or nutrients to stimulate insulin secretion from pancreatic β cells. GIP and GLP-1 exert their effects by binding to their specific receptors, the GIP receptor (GIPR) and the GLP-1 receptor (GLP-1R), which belong to the G-protein coupled receptor family. Receptor binding activates and increases the level of intracellular cAMP in pancreatic β cells, thereby stimulating insulin secretion glucose-dependently. In addition to their insulinotropic effects, GIP and GLP-1 have been shown to preserve pancreatic β cell mass by inhibiting apoptosis of β cells and enhancing their proliferation. Due to such characteristics, incretin hormones have been gaining mush attention as attractive targets for treatment of type 2 diabetes, and indeed incretin-based therapeutics have been rapidly disseminated worldwide. However, despites of plethora of rigorous studies, molecular mechanisms underlying how GIPR and GLP-1R activation leads to enhancement of glucose-dependent insulin secretion are still largely unknown. Here, we summarize the similarities and differences of these two incretin hormones in secretion and metabolism, their insulinotropic actions and their effects on pancreatic β cell preservation. We then try to discuss potential of GLP-1 and GIP in treatment of type 2 diabetes.     

Adipocyte expression of the glucose-dependent insulinotropic polypeptide receptor involves gene regulation by PPARγ and histone acetylation

Glucose-dependent insulinotropic polypeptide (GIP) is a gastrointestinal hormone that exerts insulinotropic and growth and survival effects on pancreatic β-cells. Additionally, there is increasing evidence supporting an important role for GIP in the regulation of adipocyte metabolism. In the current study we examined the molecular mechanisms involved in the regulation of GIP receptor (GIPR) expression in 3T3-L1 cells. GIP acted synergistically with insulin to increase neutral lipid accumulation during progression of 3T3-L1 preadipocytes to the adipocyte phenotype. Both GIPR protein and mRNA expression increased during 3T3-L1 cell differentiation, and this increase was associated with upregulation of nuclear levels of sterol response element binding protein 1c (SREBP-1c) and peroxisome proliferator-activated receptor γ (PPARγ), as well as acetylation of histones H3/H4. The PPARγ receptor agonists LY171883 and rosiglitazone increased GIPR expression in differentiated 3T3-L1 adipocytes, whereas the antagonist GW9662 ablated expression. Additionally, both PPARγ and acetylated histones H3/H4 were shown to bind to a region of the GIPR promoter containing the peroxisome proliferator response element (PPRE). Knockdown of PPARγ in differentiated 3T3-L1 adipocytes, using RNA interference, reduced GIPR expression, supporting a functional regulatory role. Taken together, these studies show that GIP and insulin act in a synergistic manner on 3T3-L1 cell development and that adipocyte GIPR expression is upregulated through a mechanism involving interactions between PPARγ and a GIPR promoter region containing an acetylated histone region.     

NMR and alanine scan studies of glucose-dependent insulinotropic polypeptide in water

Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone that stimulates the secretion of insulin after ingestion of food. GIP also promotes the synthesis of fatty acids in adipose tissue. Therefore, it is not surprising that numerous literature reports have shown that GIP is linked to diabetes and obesity-related diseases. In this study, we present the solution structure of GIP in water determined by NMR spectroscopy. The calculated structure is characterized by the presence of an alpha-helical motif between residues Ser(11) and Gln(29). The helical conformation of GIP is further supported by CD spectroscopic studies. Six GIP-(1-42)Ala(1-7) analogues were synthesized by replacing individual N-terminal residues with alanine. Alanine scan studies of these N-terminal residues showed that the GIP-(1-42)Ala(6) was the only analogue to show insulin-secreting activity similar to that of the native GIP. However, when compared with glucose, its insulinotropic ability was reduced. For the first time, these NMR and modeling results contribute to the understanding of the structural requirements for the biological activity of GIP.     

Covalent structure and some pharmacological features of native and cleaved α‐KTx12−1, a four disulfide‐bridged toxin from Tityus serrulatus venom

A toxin with four disulfide bridges from Tityus serrulatus venom was able to compete with ¹²⁵I‐kaliotoxin on rat brain synaptosomal preparations, with an IC₅₀ of 46 nM . The obtained amino acid sequence and molecular mass are identical to the previously described butantoxin. Enzymatic cleavages in the native peptide followed by mass spectrometry peptide mapping analysis were used to determine the disulfide bridge pattern of α‐KTx_12?1. Also, after the cleavage of the first six N‐terminal residues, including the unusual disulfide bridge which forms an N‐terminus ring, the potency of the cleaved peptide was found to decrease about 100 fold compared with the native protein. Copyright © 2003 European Peptide Society and John Wiley & Sons, Ltd.     

The GIP Receptor Displays Higher Basal Activity than the GLP-1 Receptor but Does Not Recruit GRK2 or Arrestin3 Effectively

Background and Objectives

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are important regulators of insulin secretion, and their functional loss is an early characteristic of type 2 diabetes mellitus (T2DM). Pharmacological levels of GLP-1, but not GIP, can overcome this loss. GLP-1 and GIP exert their insulinotropic effects through their respective receptors expressed on pancreatic β-cells. Both the GLP-1 receptor (GLP-1R) and the GIP receptor (GIPR) are members of the secretin family of G protein-coupled receptors (GPCRs) and couple positively to adenylate cyclase. We compared the signalling properties of these two receptors to gain further insight into why GLP-1, but not GIP, remains insulinotropic in T2DM patients.

Methods

GLP-1R and GIPR were transiently expressed in HEK-293 cells, and basal and ligand-induced cAMP production were investigated using a cAMP-responsive luciferase reporter gene assay. Arrestin3 (Arr3) recruitment to the two receptors was investigated using enzyme fragment complementation, confocal microscopy and fluorescence resonance energy transfer (FRET).

Results

GIPR displayed significantly higher (P<0.05) ligand-independent activity than GLP-1R. Arr3 displayed a robust translocation to agonist-stimulated GLP-1R but not to GIPR. These observations were confirmed in FRET experiments, in which GLP-1 stimulated the recruitment of both GPCR kinase 2 (GRK2) and Arr3 to GLP-1R. These interactions were not reversed upon agonist washout. In contrast, GIP did not stimulate recruitment of either GRK2 or Arr3 to its receptor. Interestingly, arrestin remained at the plasma membrane even after prolonged (30 min) stimulation with GLP-1. Although the GLP-1R/arrestin interaction could not be reversed by agonist washout, GLP-1R and arrestin did not co-internalise, suggesting that GLP-1R is a class A receptor with regard to arrestin binding.

Conclusions

GIPR displays higher basal activity than GLP-1R but does not effectively recruit GRK2 or Arr3.     

Lipid interactions of acylated tryptophan‐methylated lactoferricin peptides by solid‐state NMR

Lactoferricin (LfB) is a 25‐residue innate immunity peptide released by pepsin from the N‐terminal region of bovine lactoferrin. A smaller amidated peptide, LfB6 (RRWQWR‐NH₂) retains antimicrobial activity and is thought to constitute the “antimicrobial active‐site” (Tomita, Acta Paediatr Jpn. 1994; 36 : 585–91). Here we report on N‐acylation of 1‐Me‐Trp⁵‐LfB6, Cn‐RRWQ[1‐Me‐W]R‐NH₂, where Cn is an acyl chain having n = 0, 2, 4, 6 or 12 carbons. Tryptophan 5 (Trp⁵) was methylated to enhance membrane binding and to allow for selective deuteration at that position. Peptide/lipid interactions of Cn‐RRWQ[1‐Me‐W ]R‐NH₂ (deuterated 1‐Me‐Trp⁵ underlined), were monitored by solid state ³¹P NMR and ²H NMR. The samples consisted of macroscopically oriented bilayers of mixed neutral (dimyristoylphosphatidylcholine, DMPC) and anionic (dimyristoylphosphatidylglycerol, DMPG) lipids in a 3:1 ratio with Cn‐RRWQ[&1‐Me‐W ]R‐NH₂ peptides added at a 1:25 peptide to lipid ratio. ²H‐NMR spectra reveal that the acylated peptides are well aligned in DMPC:DMPG bilayers. The ²H NMR quadrupolar splittings suggest that the 1‐Me‐Trp is located in a motionally restricted environment, indicating partial alignment at the membrane interface. ³¹P‐NMR spectra reveal that the lipids are predominantly in a bilayer configuration, with little perturbation by the peptides. Methylation alone, in C0‐RRWQ[1‐Me‐W ]R‐NH₂, resulted in a 3–4 fold increase in antimicrobial activity against E. coli. N‐acylation with a C12 fatty acid enhanced activity almost 90 fold. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Global fold of human cannabinoid type 2 receptor probed by solid‐state 13C‐, 15N‐MAS NMR and molecular dynamics simulations

The global fold of human cannabinoid type 2 (CB₂) receptor in the agonist‐bound active state in lipid bilayers was investigated by solid‐state ¹³C‐ and ¹⁵N magic‐angle spinning (MAS) NMR, in combination with chemical‐shift prediction from a structural model of the receptor obtained by microsecond‐long molecular dynamics (MD) simulations. Uniformly ¹³C‐ and ¹⁵N‐labeled CB₂ receptor was expressed in milligram quantities by bacterial fermentation, purified, and functionally reconstituted into liposomes. ¹³C MAS NMR spectra were recorded without sensitivity enhancement for direct comparison of C_α, C_β, and C?O bands of superimposed resonances with predictions from protein structures generated by MD. The experimental NMR spectra matched the calculated spectra reasonably well indicating agreement of the global fold of the protein between experiment and simulations. In particular, the ¹³C chemical shift distribution of C_α resonances was shown to be very sensitive to both the primary amino acid sequence and the secondary structure of CB₂. Thus the shape of the C_α band can be used as an indicator of CB₂ global fold. The prediction from MD simulations indicated that upon receptor activation a rather limited number of amino acid residues, mainly located in the extracellular Loop 2 and the second half of intracellular Loop 3, change their chemical shifts significantly (≥1.5 ppm for carbons and ≥5.0 ppm for nitrogens). Simulated two‐dimensional ¹³C_α(i)? ¹³C?O(i) and ¹³C?O(i)? ¹⁵NH(i + 1) dipolar‐interaction correlation spectra provide guidance for selective amino acid labeling and signal assignment schemes to study the molecular mechanism of activation of CB₂ by solid‐state MAS NMR. Proteins 2014; 82:452–465. © 2013 Wiley Periodicals, Inc.     

A triazole‐based fluorescence probe for detecting Hg2+ ions and its biological application

A new compound, ethyl 5‐phenyl‐2‐(p‐tolyl)‐2H‐1,2,3‐triazole‐4‐carboxylate was successfully introduced and synthesized as a novel rhodamine B derivative named REPPC, and characterized by ¹H nuclear magnetic resonance (NMR), ¹³C NMR, and high resolution mass spectrometry (HRMS). It showed an obvious fluorescence and UV–visible light absorption enhancement towards Hg²⁺ ion without interference from common metal ions in N,N‐dimethylformamide–H₂O (pH 7.4). The spirolactam ring moiety of rhodamine in REPPC was converted to the open‐ring form generating a 1:1 complex with the intervention of a mercury ion, verified by electrospray ionization‐mass spectroscopy testing and density functional theory calculation. REPPC was used to visualize the level of mercury ions in living HeLa cells with encouraging results.     

Conformational studies of a bicyclic,lactam‐constrained parathyroid hormone‐related protein‐derived agonist

The N‐terminal 1–34 segments of both parathyroid hormone (PTH) and parathyroid hormone‐related protein (PTHrP) bind and activate the same membrane receptor in spite of major differences in their amino acid sequence. The hypothesis was made that they share the same bioactive conformation when bound to the receptor. A common structural motif in all bioactive fragments of the hormone in water/trifluoroethanol mixtures or in aqueous solution containing detergent micelles is the presence of two helical segments at the N‐ and C‐termini of the sequence. In order to stabilize the helical structures, we have recently synthesized and studied the PTHrP(1–34) analog [(Lys13–As p¹⁷, Lys26–As p³⁰)]PTHrP(1–34)NH₂, which contains lactam‐constrained Lys‐Asp side chains at positions i, i+4. This very potent agonist exhibits enhanced helix stability with respect to the corresponding linear peptide and also two flexible sites at positions 12 and 19 in 1:1 trifluoroethanol/water. These structural elements have been suggested to play a critical role in bioactivity. In the present work we have extended our conformational studies on the bicyclic lactam‐constrained analog to aqueous solution. By CD, 2D‐NMR and structure calculations we have shown that in water two helical segments are present in the region of the lactam bridges (13–18, and 26–31) with high flexibility around Gly¹² and Arg¹⁹. Thus, the essential structural features observed in the aqueous‐organic medium are maintained in water even if, in this solvent, the overall structure is more flexible. Our findings confirm the stabilizing effect of side‐chain lactam constraints on the α‐helical structure. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.