Association Between Obesity and Cardiovascular Outcomes: Updated Evidence from Meta-analysis Studies.

PURPOSE OF REVIEW: The prevalence of obesity and cardiovascular disease (CVD) has been increasing worldwide. Studies examining the association between adiposity and CVD outcomes have produced conflicting findings. The interplay between obesity and CVD outcomes in the general population and in specific subpopulations is complex and requires further elucidation. RECENT FINDINGS: We report updated evidence on the association between obesity and CVD events through a review of meta-analysis studies. This review identified that obesity or high body mass index (BMI) was associated with an increased risk of CVD events, including mortality, in the general population and that cardiac respiratory fitness (CRF) and metabolic health status appear to stratify the risk of CVD outcomes. In patients with diabetes, hypertension, or coronary artery disease, mortality displayed a U-shaped association with BMI. This U-shaped association may result from the effect of unintentional weight loss or medication use. By contrast, patients with other severe heart diseases or undergoing cardiac surgery displayed a reverse J-shaped association suggesting the highest mortality associated with low BMI. In these conditions, a prolonged intensive medication use might have attenuated the risk of mortality associated with high BMI. For the general population, a large body of evidence points to the importance of obesity prevention and maintenance of a healthy weight. However, for those with diagnosed cardiovascular diseases or diabetes, the relationship between BMI and cardiovascular outcomes is more complex and varies with the type of disease. More studies are needed to define how heterogeneity in the longitudinal changes in BMI affects mortality, especially in patients with severe heart diseases or going under cardiac surgery, in order to target subgroups for tailored interventions. Interventions for managing body weight, in conjunction with improving CRF and metabolic health status and avoiding unintentional weight loss, should be used to improve CVD outcomes.

Water T2 as an early, global and practical biomarker for metabolic syndrome: an observational cross-sectional study.

BACKGROUND: Metabolic syndrome (MetS) is a highly prevalent condition that identifies individuals at risk for type 2 diabetes mellitus and atherosclerotic cardiovascular disease. Prevention of these diseases relies on early detection and intervention in order to preserve pancreatic ß-cells and arterial wall integrity. Yet, the clinical criteria for MetS are insensitive to the early-stage insulin resistance, inflammation, cholesterol and clotting factor abnormalities that characterize the progression toward type 2 diabetes and atherosclerosis. Here we report the discovery and initial characterization of an atypical new biomarker that detects these early conditions with just one measurement. METHODS: Water T2, measured in a few minutes using benchtop nuclear magnetic resonance relaxometry, is exquisitely sensitive to metabolic shifts in the blood proteome. In an observational cross-sectional study of 72 non-diabetic human subjects, the association of plasma and serum water T2 values with over 130 blood biomarkers was analyzed using bivariate, multivariate and logistic regression. RESULTS: Plasma and serum water T2 exhibited strong bivariate correlations with markers of insulin, lipids, inflammation, coagulation and electrolyte balance. After correcting for confounders, low water T2 values were independently and additively associated with fasting hyperinsulinemia, dyslipidemia and subclinical inflammation. Plasma water T2 exhibited 100% sensitivity and 87% specificity for detecting early insulin resistance in normoglycemic subjects, as defined by the McAuley Index. Sixteen normoglycemic subjects with early metabolic abnormalities (22% of the study population) were identified by low water T2 values. Thirteen of the 16 did not meet the harmonized clinical criteria for metabolic syndrome and would have been missed by conventional screening for diabetes risk. Low water T2 values were associated with increases in the mean concentrations of 6 of the 16 most abundant acute phase proteins and lipoproteins in plasma. CONCLUSIONS: Water T2 detects a constellation of early abnormalities associated with metabolic syndrome, providing a global view of an individual's metabolic health. It circumvents the pitfalls associated with fasting glucose and hemoglobin A1c and the limitations of the current clinical criteria for metabolic syndrome. Water T2 shows promise as an early, global and practical screening tool for the identification of individuals at risk for diabetes and atherosclerosis.

Compact NMR relaxometry of human blood and blood components.

Nuclear magnetic resonance relaxometry is a uniquely practical and versatile implementation of NMR technology. Because it does not depend on chemical shift resolution, it can be performed using low-field compact instruments deployed in atypical settings. Early relaxometry studies of human blood were focused on developing a diagnostic test for cancer. Those efforts were misplaced, as the measurements were not specific to cancer. However, important lessons were learned about the factors that drive the water longitudinal (T1) and transverse (T2) relaxation times. One key factor is the overall distribution of proteins and lipoproteins. Plasma water T2 can detect shifts in the blood proteome resulting from inflammation, insulin resistance and dyslipidemia. In whole blood, T2 is sensitive to hemoglobin content and oxygenation, although the latter can be suppressed by manipulating the static and applied magnetic fields. Current applications of compact NMR relaxometry include blood tests for candidiasis, hemostasis, malaria and insulin resistance.

Nanofluidity of fatty acid hydrocarbon chains as monitored by benchtop time-domain nuclear magnetic resonance.

The functional properties of lipid-rich assemblies such as serum lipoproteins, cell membranes, and intracellular lipid droplets are modulated by the fluidity of the hydrocarbon chain environment. Existing methods for monitoring hydrocarbon chain fluidity include fluorescence, electron spin resonance, and nuclear magnetic resonance (NMR) spectroscopy; each possesses advantages and limitations. Here we introduce a new approach based on benchtop time-domain (1)H NMR relaxometry (TD-NMR). Unlike conventional NMR spectroscopy, TD-NMR does not rely on the chemical shift resolution made possible by homogeneous, high-field magnets and Fourier transforms. Rather, it focuses on a multiexponential analysis of the time decay signal. In this study, we investigated a series of single-phase fatty acid oils, which allowed us to correlate (1)H spin-spin relaxation time constants (T2) with experimental measures of sample fluidity, as obtained using a viscometer. Remarkably, benchtop TD-NMR at 40 MHz was able to resolve two to four T2 components in biologically relevant fatty acids, assigned to nanometer-scale domains in different segments of the hydrocarbon chain. The T2 values for each domain were exquisitely sensitive to hydrocarbon chain structure; the largest values were observed for pure fatty acids or mixtures with the highest cis-double bond content. Moreover, the T2 values for each domain exhibited positive linear correlations with fluidity. The TD-NMR T2 and fluidity measurements appear to be monitoring the same underlying phenomenon: variations in hydrocarbon chain packing. The results from this study validate the use of benchtop TD-NMR T2 as a nanofluidity meter and demonstrate its potential for probing nanofluidity in other systems of biological interest.

Non-Invasive Glucose Monitoring Using Optical Sensor and Machine Learning Techniques for Diabetes Applications.

Diabetes is a major public health challenge affecting more than 451 million people. Physiological and experimental factors influence the accuracy of non-invasive glucose monitoring, and these need to be overcome before replacing the finger prick method. Also, the suitable employment of machine learning techniques can significantly improve the accuracy of glucose predictions. One aim of this study is to use light sources with multiple wavelengths to enhance the sensitivity and selectivity of glucose detection in an aqueous solution. Multiple wavelength measurements have the potential to compensate for errors associated with inter- and intra-individual differences in blood and tissue components. In this study, the transmission measurements of a custom built optical sensor are examined using 18 different wavelengths between 410 and 940 nm. Results show a high correlation value (0.98) between glucose concentration and transmission intensity for four wavelengths (485, 645, 860 and 940 nm). Five machine learning methods are investigated for glucose predictions. When regression methods are used, 9% of glucose predictions fall outside the correct range (normal, hypoglycemic or hyperglycemic). The prediction accuracy is improved by applying classification methods on sets of data arranged into 21 classes. Data within each class corresponds to a discrete 10 mg/dL glucose range. Classification based models outperform regression, and among them, the support vector machine is the most successful with F1-score of 99%. Additionally, Clarke error grid shows that 99.75% of glucose readings fall within the clinically acceptable zones. This is an important step towards critical diagnosis during an emergency patient situation.

NMR structure of a fungal virulence factor reveals structural homology with mammalian saposin B.

The fungal protein CBP (calcium binding protein) is a known virulence factor with an unknown virulence mechanism. The protein was identified based on its ability to bind calcium and its prevalence as Histoplasma capsulatum's most abundant secreted protein. However, CBP has no sequence homology with other CBPs and contains no known calcium binding motifs. Here, the NMR structure of CBP reveals a highly intertwined homodimer and represents the first atomic level NMR model of any fungal virulence factor. Each CBP monomer is comprised of four alpha-helices that adopt the saposin fold, characteristic of a protein family that binds to membranes and lipids. This structural homology suggests that CBP functions as a lipid binding protein, potentially interacting with host glycolipids in the phagolysosome of host cells.

The RXRalpha C-terminus T462 is a NMR sensor for coactivator peptide binding.

The C-terminal activation function-2 (AF-2) helix plays a crucial role in retinoid X receptor alpha (RXRalpha)-mediated gene expression. Here, we report a nuclear magnetic resonance (NMR) study of the RXRalpha ligand-binding domain complexed with 9-cis-retinoic acid and a glucocorticoid receptor-interacting protein 1 peptide. The AF-2 helix and most of the C-terminal residues were undetectable due to a severe line-broadening effect. Due to its outstanding signal-to-noise ratio, the C-terminus residue, threonine 462 (T462) exhibited two distinct crosspeaks during peptide titration, suggesting that peptide binding was in a slow exchange regime on the chemical shift timescale. Consistently, the K(d) derived from T462 intensity decay agreed with that derived from isothermal titration calorimetry. Furthermore, the exchange contribution to the (15)N transverse relaxation rate was measurable in either T462 or the bound peptide. These results suggest that T462 is a sensor for coactivator binding and is a potential probe for AF-2 helix mobility.

Aptamer-based search for correlates of plasma and serum water T2: implications for early metabolic dysregulation and metabolic syndrome.

BACKGROUND: Metabolic syndrome is a cluster of abnormalities that increases the risk for type 2 diabetes and atherosclerosis. Plasma and serum water T2 from benchtop nuclear magnetic resonance relaxometry are early, global and practical biomarkers for metabolic syndrome and its underlying abnormalities. In a prior study, water T2 was analyzed against ~ 130 strategically selected proteins and metabolites to identify associations with insulin resistance, inflammation and dyslipidemia. In the current study, the analysis was broadened ten-fold using a modified aptamer (SOMAmer) library, enabling an unbiased search for new proteins correlated with water T2 and thus, metabolic health. METHODS: Water T2 measurements were recorded using fasting plasma and serum from non-diabetic human subjects. In parallel, plasma samples were analyzed using a SOMAscan assay that employed modified DNA aptamers to determine the relative concentrations of 1310 proteins. A multi-step statistical analysis was performed to identify the biomarkers most predictive of water T2. The steps included Spearman rank correlation, followed by principal components analysis with variable clustering, random forests for biomarker selection, and regression trees for biomarker ranking. RESULTS: The multi-step analysis unveiled five new proteins most predictive of water T2: hepatocyte growth factor, receptor tyrosine kinase FLT3, bone sialoprotein 2, glucokinase regulatory protein and endothelial cell-specific molecule 1. Three of the five strongest predictors of water T2 have been previously implicated in cardiometabolic diseases. Hepatocyte growth factor has been associated with incident type 2 diabetes, and endothelial cell specific molecule 1, with atherosclerosis in subjects with diabetes. Glucokinase regulatory protein plays a critical role in hepatic glucose uptake and metabolism and is a drug target for type 2 diabetes. By contrast, receptor tyrosine kinase FLT3 and bone sialoprotein 2 have not been previously associated with metabolic conditions. In addition to the five most predictive biomarkers, the analysis unveiled other strong correlates of water T2 that would not have been identified in a hypothesis-driven biomarker search. CONCLUSIONS: The identification of new proteins associated with water T2 demonstrates the value of this approach to biomarker discovery. It provides new insights into the metabolic significance of water T2 and the pathophysiology of metabolic syndrome.

Early detection of metabolic dysregulation using water T2 analysis of biobanked samples.

BACKGROUND: The ability to use frozen biobanked samples from cohort studies and clinical trials is critically important for biomarker discovery and validation. Here we investigated whether plasma and serum water transverse relaxation times (T2) from frozen biobanked samples could be used as biomarkers for metabolic syndrome (MetS) and its underlying conditions, specifically insulin resistance, dyslipidemia, and subclinical inflammation. METHODS: Plasma and serum aliquots from 44 asymptomatic, non-diabetic human subjects were biobanked at -80°C for 7-9 months. Water T2 measurements were recorded at 37°C on 50 µL of unmodified plasma or serum using benchtop nuclear magnetic resonance relaxometry. The T2 values for freshly drawn and once-frozen-thawed ("frozen") samples were compared using Huber M-values (M), Lin concordance correlation coefficients (ρc), and Bland-Altman plots. Water T2 values from frozen plasma and serum samples were compared with >130 metabolic biomarkers and analyzed using multi-variable linear/logistic regression and ROC curves. RESULTS: Frozen plasma water T2 values were highly correlated with fresh (M=0.94, 95% CI 0.89, 0.97) but showed a lower level of agreement (ρc=0.74, 95% CI 0.62, 0.82) because of an average offset of -5.6% (-7.1% for serum). Despite the offset, frozen plasma water T2 was strongly correlated with markers of hyperinsulinemia, dyslipidemia, and inflammation and detected these conditions with 89% sensitivity and 91% specificity (100%/63% for serum). Using optimized cut points, frozen plasma and serum water T2 detected hyperinsulinemia, dyslipidemia, and inflammation in 23 of 44 subjects, including nine with an early stage of metabolic dysregulation that did not meet the clinical thresholds for prediabetes or MetS. CONCLUSION: Plasma and serum water T2 values from once-frozen-thawed biobanked samples detect metabolic dysregulation with high sensitivity and specificity. However, the cut points for frozen biobanked samples must be calibrated independent of those for freshly drawn plasma and serum.

Structural determinants of ligand binding in the ternary complex of human ileal bile acid binding protein with glycocholate and glycochenodeoxycholate obtained from solution NMR.

Besides aiding digestion, bile salts are important signal molecules exhibiting a regulatory role in metabolic processes. Human ileal bile acid binding protein (I-BABP) is an intracellular carrier of bile salts in the epithelial cells of the distal small intestine and has a key role in the enterohepatic circulation of bile salts. Positive binding cooperativity combined with site selectivity of glycocholate and glycochenodeoxycholate, the two most abundant bile salts in the human body, make human I-BABP a unique member of the family of intracellular lipid binding proteins. Solution NMR structure of the ternary complex of human I-BABP with glycocholate and glycochenodeoxycholate reveals an extensive network of hydrogen bonds and hydrophobic interactions stabilizing the bound bile salts. Conformational changes accompanying bile salt binding affects four major regions in the protein including the C/D, E/F and G/H loops as well as the helical segment. Most of these protein regions coincide with a previously described network of millisecond time scale fluctuations in the apo protein, a motion absent in the bound state. Comparison of the heterotypic doubly ligated complex with the unligated form provides further evidence of a conformation selection mechanism of ligand entry. Structural and dynamic aspects of human I-BABP-bile salt interaction are discussed and compared with characteristics of ligand binding in other members of the intracellular lipid binding protein family. PROTEIN DATA BANK ACCESSION NUMBERS: The coordinates of the 10 lowest energy structures of the human I-BABP : GCDA : GCA complex as well as the distance restraints used to calculate the final ensemble have been deposited in the Brookhaven Protein Data Bank with accession number 2MM3.

Two homologous rat cellular retinol-binding proteins differ in local conformational flexibility.

Cellular retinol-binding protein I (CRBP I) and cellular retinol-binding protein II (CRBP II) are closely homologous proteins that play distinct roles in the maintenance of vitamin A homeostasis. The solution structure and dynamics of CRBP I and CRBP II were compared by multidimensional NMR techniques. These studies indicated that differences in the mean backbone structures of CRBP I and CRBP II were localized primarily to the alphaII helix. Intraligand NOE cross-peaks were detected for the hydroxyl proton in the NOESY spectrum of CRBP I-bound retinol, but not for CRBP II-bound retinol, indicating that the conformational dynamics of retinol binding are different for these two proteins. As determined by Lipari-Szabo formalism, both the apo and holo forms of CRBP I and CRBP II are conformationally rigid on the pico- to nanosecond timescale. transverse relaxation optimized spectroscopy-Carr-Purcell-Meiboom-Gill -based 15N relaxation dispersion experiments at both 500 MHz and 600 MHz magnetic fields revealed that 84 and 62 residues for apo-CRBP I and II, respectively, showed detectable conformational exchange on a micro- to millisecond timescale, in contrast to three and seven residues for holo-CRBP I and II, respectively. Thus binding of retinol markedly reduced conformational flexibility in both CRBP I and CRBP II on the micro- to millisecond timescale. The 15N relaxation dispersion curves of apo-CRBP I and II were fit to a two-state conformational exchange model by a global iterative fitting process and by an individual (residue) fitting process. In the process of carrying out the global fit, more than half of the residue sites were eliminated. The individual chemical exchange rates k(ex), and chemical shift differences, Deltadelta, were increased in the putative portal region (alphaII helix and betaC-betaD turn) of apo-CRBP II compared to apo-CRBP I. These differences in conformational flexibility likely contribute to differences in how CRBP I and CRBP II interact with ligands, membranes and retinoid metabolizing enzymes.

The NMR structure of a stable and compact all-beta-sheet variant of intestinal fatty acid-binding protein.

Intestinal fatty acid-binding protein (I-FABP) has a clam-shaped structure that may serve as a scaffold for the design of artificial enzymes and drug carriers. In an attempt to optimize the scaffold for increased access to the interior-binding cavity, several helix-less variants of I-FABP have been engineered. The solution-state NMR structure of the first generation helix-less variant, known as Delta17-SG, revealed a larger-than-expected and structurally ill-defined loop flanking the deletion site. We hypothesized that the presence of this loop, on balance, was energetically unfavorable for the stability of the protein. The structure exhibited no favorable pairwise or nonpolar interactions in the loop that could offset the loss of configurational entropy associated with the folding of this region of the protein. As an attempt to generate a more stable protein, we engineered a second-generation helix-less variant of I-FABP (Delta27-GG) by deleting 27 contiguous residues of the wild-type protein and replacing them with a G-G linker. The deletion site of this variant (D9 through N35) includes the 10 residues spanning the unstructured loop of Delta17-SG. Chemical denaturation experiments using steady-state fluorescence spectroscopy showed that the second-generation helix-less variant is energetically more stable than Delta17-SG. The three-dimensional structure of apo-Delta27-GG was solved using triple-resonance NMR spectroscopy along with the structure calculation and refinement protocols contained in the program package ARIA/CNS. In spite of the deletion of 27 residues, the structure assumes a compact all-beta-sheet fold with no unstructured loops and open access to the interior cavity.

An embryo-associated fatty acid-binding protein in the filarial nematode Brugia malayi.

Brugia malayi is a filarial nematode parasite that causes lymphatic filariasis, a disease that affects millions of people in the tropics. Sexual reproduction of filarial worms occurs within the lymphatic vessels of the human host and is crucial for transmission of the parasite to the mosquito vector. We have previously identified several B. malayi genes that exhibit apparent gender-specific expression. One of these had significant sequence similarity to the Ascaris suum embryo-associated fatty acid binding protein, As-p18. The full length cDNA for the B. malayi female-associated fatty acid binding protein (Bm-FAB-1) encodes a 17.8 kDa protein (excluding a signal peptide) with 70% sequence identity with mature As-p18 and significant similarity to Caenorhabditis elegans and mammalian fatty acid-binding proteins (FABPs). Antibodies raised to Bm-FAB-1 bound to developing embryos within female worms, especially around early embryo cells and the surfaces of immature worms within eggs. Functional studies showed that recombinant Bm-FAB-1 binds to several long chain fatty acids including oleate, but not retinol. Taken together, these results demonstrate that Bm-FAB-1 is a member of an unusual nematode-specific, secreted lipid binding protein family. The existence of a novel class of lipid binding proteins in nematode embryos raises the possibility that drugs targeting these proteins could be developed with broad activity against nematode parasites of medical and veterinary importance.

Structural features responsible for the biological stability of Histoplasma's virulence factor CBP.

The virulence factor CBP is the most abundant protein secreted by Histoplasma capsulatum, a pathogenic fungus that causes histoplasmosis. Although the biochemical function and pathogenic mechanism of CBP are unknown, quantitative Ca (2+) binding measurements indicate that CBP has a strong affinity for calcium ( K D = 6.45 +/- 0.4 nM). However, no change in structure was observed upon binding of calcium, prompting a more thorough investigation of the molecular properties of CBP with respect to self-association, secondary structure, and stability. Over a wide range of pH values and salt concentrations, CBP exists predominantly as a stable, noncovalent homodimer in both its calcium-free and -bound states. Solution-state NMR and circular dichroism (CD) measurements indicated that the protein is largely alpha-helical, and its secondary structure content changes little over the range of pH values encountered physiologically. ESI-MS revealed that the six cysteine residues of CBP are involved in three intramolecular disulfide bonds that help maintain a highly protease resistant structure. Thermally and chemically induced denaturation studies indicated that unfolding of disulfide-intact CBP is reversible and provided quantitative measurements of protein stability. This disulfide-linked, protease resistant, homodimeric alpha-helical structure of CBP is likely to be advantageous for a virulence factor that must survive the harsh environment within the phagolysosomes of host macrophages.

Kinetic mechanism of ligand binding in human ileal bile acid binding protein as determined by stopped-flow fluorescence analysis.

Cooperative ligand binding to human ileal bile acid binding protein (I-BABP) was studied using the stopped-flow fluorescence technique. The kinetic data obtained for wild-type protein are in agreement with a four-step mechanism where after a fast conformational change on the millisecond time scale, the ligands bind in a sequential manner, followed by another, slow conformational change on the time scale of seconds. This last step is more pronounced in the case of glycocholate (GCA), the bile salt that binds with high positive cooperativity and is absent in mutant I-BABP proteins that lack positive cooperativity in their bile salt binding. These results suggest that positive cooperativity in human I-BABP is related to a slow conformational change of the protein, which occurs after the second binding step. Analogous to that in the intestinal fatty acid binding protein (I-FABP), we hypothesize that ligand binding in I-BABP is linked to a disorder-order transition between an open and a closed form of the protein.

Analysis of ligand binding and protein dynamics of human retinoid X receptor alpha ligand-binding domain by nuclear magnetic resonance.

Retinoid X receptors (RXRs) are nuclear receptors that can activate transcription as homodimers or as obligate heterodimeric partners of other nuclear receptors. While the crystal structures of the RXR ligand-binding domains (LBD) have been previously determined, the dynamics of activation is less well characterized at an atomic level. To probe the effect of ligand binding on RXR LBD dynamics, we initiated nuclear magnetic resonance studies of recombinant human RXRalpha LBD (T223-T462) with and without bound 9-cis-retinoic acid (9cRA). The 1HN, 15N, 13C(alpha), 13CO, and 13C(beta) resonance assignments were established for 164 of 240 residues in apo-RXRalpha LBD. Resonances corresponding to an additional 47 residues emerged upon 9cRA binding. These additional residues included those located in the vicinity of the ligand-binding pocket (helices H3, H5, and strands S1, S2), as well as residues located at the dimerization interface (helices H9 and H10). Thus 9cRA binding stabilized the ligand-binding pocket and had allosteric effects on the dimerization interface. Ligand-induced chemical shift perturbations outside the binding cavity were mapped to helix H3 and the AF-2 helix H12, indicating conformational changes in these regions. However, helix H11, a component of the tetramerization interface, and a large part of helix H10, a component of the dimerization interface, remained undetectable even after 9cRA binding. Although apo- and holo-hRXRalpha LBD existed predominantly as homodimers in solution, exchange between monomeric, dimeric, and tetrameric forms of the protein could have contributed to line broadening of cross-peaks corresponding to helices H10 and H11. 15N T1, T2, and steady-state {1H}-15N NOE data collected at 500 and 700 MHz static magnetic fields showed that the internal motions for the residues in the H1-H3 loop (K245-D263) were much less restricted than those in the protein core for both apo- and holo-forms. Significant exchange R(ex) contributions to the transverse relaxation rate were detected for most of the residues measured in both apo- and holo-RXRalpha LBDs by transverse relaxation optimized spectroscopy-Carr-Purcell-Meiboom-Gill (CPMG) experiments at two B1 field strengths. Taken together these results suggest that the RXRalpha LBD exists as a dynamic ensemble of conformations, even after binding its cognate ligand. Such dynamic characteristics may allow RXRalpha to partner with multiple nuclear receptors.

Alternate binding mode of C-terminal phenethylamine analogs of G(t)alpha(340-350) to photoactivated rhodopsin.

The C-terminus of the Galpha-subunit of transducin plays an important role in receptor recognition. Synthetic peptides corresponding to the last 11 residues of the subunit have been shown to stabilize the photoactivated form of rhodopsin, Rh*. The Rh*-bound structure of the G(t)alpha(340-350) peptide has been determined using transferred nuclear overhauser effect NMR. In that structure, we observed two interactions between Lys341 and Phe350, a cation-pi interaction between the epsilon-amine and the aromatic ring of Phe350 and a salt-bridge between the epsilon-amine and the C-terminal carboxylate. A series of C-terminal phenethylamine analogs of the G(t)alpha(340-350) peptide were synthesized, lacking the C-terminal carboxylate group, to investigate the forces that contribute to the stability of the Rh*-bound conformation of the peptide. Rh*-stabilization assay data suggest that the C-terminal carboxylate is not necessary to maintain binding affinity. Transferred nuclear overhauser effect NMR experiments reveal that these C-terminal phenethylamine peptides adopt an Rh*-bound structure that is similar overall, but lacking some of the intramolecular interactions observed in the native Rh*-bound G(t)alpha(340-350) structure. These studies suggest that the binding site for G(t)alpha(340-350) on Rh* is adaptable, and we propose that the charged carboxylate of Phe350 does not play a significant role in the interaction with Rh*, but helps stabilize the Rh*-bound confirmation of the native peptide.

Determinants of cooperativity and site selectivity in human ileal bile acid binding protein.

Human ileal bile acid binding protein (I-BABP) is a member of the family of intracellular lipid-binding proteins and is thought to play a role in the enterohepatic circulation of bile salts. Our group has previously shown that human I-BABP binds two molecules of glycocholate (GCA) with low intrinsic affinity but an extraordinary high degree of positive cooperativity. Besides the strong positive cooperativity, human I-BABP exhibits a high degree of site selectivity in its interactions with GCA and glycochenodeoxycholate (GCDA), the two major bile salts in humans. In this study, on the basis of our first generation nuclear magnetic resonance (NMR) structure of the ternary complex of human I-BABP with GCA and GCDA, we introduced single-residue mutations at certain key positions in the binding pocket that might disrupt a hydrogen-bonding network, a likely way of energetic communication between the two sites. Macroscopic binding parameters were determined using isothermal titration calorimetry, and site selectivity was monitored by NMR spectroscopy of isotopically enriched bile salts. According to our results, cooperativity and site selectivity are not linked in human I-BABP. While cooperativity is governed by a subtle interplay of entropic and enthalpic contributions, site selectivity appears to be determined by more localized enthalpic effects. Possible communication pathways between the two binding sites are discussed.

A faster migrating variant masquerades as NICD when performing in vitro gamma-secretase assays with bacterially expressed Notch substrates.

Intramembrane proteolysis is a new and rapidly growing field. In vitro assays utilizing recombinant substrates for gamma-secretase, an intramembrane-cleaving enzyme, are critically important in order to characterize the biochemical properties of this unusual enzyme. Several recombinant Notch proteins of varying length are commonly used as in vitro substrates for CHAPSO-solubilized gamma-secretase. Here we report that several recombinant Notch constructs undergo limited or no proteolysis in vitro. Instead, upon incubation with or without gamma-secretase, variants of the intact protein migrate during SDS-PAGE at the location expected for the gamma-secretase specific cleavage products. In addition, we show that addition of aspartyl- and gamma-secretase specific protease inhibitors are able to retard the formation of these variants independent of gamma-secretase, which could lead to the erroneous conclusion that Notch cleavage by solubilized gamma-secretase was achieved in vitro even when no proteolysis occurred. In contrast, substrates produced in mammalian or insect cells are cleaved efficiently in vitro. These observations suggest that in vitro studies reliant on recombinant, bacterially produced Notch TMD should be performed with the inclusion of additional controls able to differentiate between actual cleavage and this potential artifact.

Relative strength of cation-pi vs salt-bridge interactions: the Gtalpha(340-350) peptide/rhodopsin system.

Interactions between cationic and aromatic side chains of amino acid residues, the so-called cation-pi interaction, are thought to contribute to the overall stability of the folded structure of peptides and proteins. The transferred NOE NMR structure of the G(t)alpha(340-350) peptide bound to photoactivated rhodopsin (R*) geometrically suggests a cation-pi interaction stabilizing the structure between the epsilon-amine of Lys341 and the aromatic ring of the C-terminal residue, Phe350. This interaction has been explored by varying substituents on the phenyl ring to alter the electron density of the aromatic ring of Phe350 and observing the impact on binding of the peptide to R*. The results suggest that while a cation-pi interaction geometrically exists in the G(t)alpha(340-350) peptide when bound to R*, its energetic contribution to the stability of the receptor-bound structure is relatively insignificant, as it was not observed experimentally. The presence of an adjacent and competing salt-bridge interaction between the epsilon-amine of Lys341 and the C-terminal carboxylate of Phe350 effectively shields the charge of the ammonium group. Experimental data supporting a significant cation-pi interaction can be regained through a series of Phe350 analogues where the C-terminal carboxyl has been converted to the neutral carboxamide, thus eliminating the shielding salt-bridge. TrNOE NMR experiments confirmed the existence of the cation-pi interaction in the carboxamide analogues. Various literature estimates of the strength of cation-pi interactions, including some that estimate strengths in excess of salt-bridges, are compromised by omission of the relevant anion in the calculations.