Plasma water T2 detects age-stratified differences in cardiometabolic health among familial CCM patients with Hispanic CCM1 mutation.

Cerebral cavernous malformations (CCMs) are abnormal clusters of capillaries in the nervous system. This pilot study analyzed the cardiometabolic health status of individuals with familial CCMs caused by a rare mutation in the CCM1 gene (fCCM1). The aim was to compare plasma water T2 values from individuals with fCCM1 with values from metabolically unhealthy and healthy individuals with no known CCM mutations. This observational, cross-sectional study included 75 participants: 11 fCCM1 patients, 24 metabolically unhealthy and 40 metabolically healthy individuals. Plasma water T2, an early, global and practical marker of cardiometabolic health, was measured in the time domain using benchtop magnetic resonance relaxometry. The results were stratified by age (equal to or less than 45 vs. older than 45 years). Group means were compared using Welch's one-way ANOVA and post hoc Tukey-Kramer tests. Multivariable linear regression, with T2 as the outcome variable, was used to explore associations with age, gender, Hispanic ethnicity and fCCM1 status. In the younger age stratum, the fCCM1 group had a mean plasma water T2 value comparable to the metabolically healthy group (p = 0.6388), but higher than the unhealthy group (p < 0.0001). By contrast, in the older stratum, the mean plasma water T2 value for the fCCM1 group was comparable to the metabolically unhealthy group (p = 0.7819) and lower than the healthy group (p = 0.0005). Multivariable linear regression revealed that age and the interaction between age and fCCM1 status were significant predictors of T2, even after adjusting for gender and Hispanic ethnicity. Plasma water T2 shows potential as a biomarker for assessing the health status of individuals with fCCM1. Further research is needed to validate these preliminary observations and elucidate the association between CCMs and cardiometabolic health.

Plasma water T2 detects age-stratified differences in cardiometabolic health among familial CCM patients with Hispanic CCM1 mutation.

Introduction: Cerebral cavernous malformations (CCMs) are abnormal clusters of capillaries in the nervous system. This pilot study analyzed the cardiometabolic health status of individuals with familial CCMs caused by a rare mutation in the CCM1 gene (fCCM1). The aim was to compare plasma water T2 values from individuals with fCCM1 with values from metabolically unhealthy and healthy individuals with no known CCM mutations. Design: This observational, cross-sectional study included 75 participants: 11 fCCM1 patients, 24 metabolically unhealthy and 40 metabolically healthy individuals. Plasma water T2, an early, global and practical marker of cardiometabolic health, was measured in the time domain using benchtop magnetic resonance relaxometry. The results were stratified by age (equal to or less than 45 vs. older than 45 years). Group means were compared using Welch's one-way ANOVA and post hoc Tukey-Kramer tests. Multivariable linear regression, with T2 as the outcome variable, was used to explore associations with age, gender, Hispanic ethnicity and fCCM1 status. Results: In the younger age stratum, the fCCM1 group had a mean plasma water T2 value comparable to the metabolically healthy group (p=0.6388), but higher than the unhealthy group (p<0.0001). By contrast, in the older stratum, the mean plasma water T2 value for the fCCM1 group was comparable to the metabolically unhealthy group (p=0.7819) and lower than the healthy group (p=0.0005). Multivariable linear regression revealed that age and the interaction between age and fCCM1 status were significant predictors of T2, even after adjusting for gender and Hispanic ethnicity. Conclusion: Plasma water T2 shows potential as a biomarker for assessing the health status of individuals with fCCM1. Further research is needed to validate these preliminary observations and elucidate the association between CCMs and cardiometabolic health.

Plasma water T2 detects age-stratified differences in cardiometabolic health among familial CCM patients with Hispanic CCM1 mutation.

Introduction: Cerebral cavernous malformations (CCMs) are abnormal clusters of capillaries in the nervous system. This pilot study analyzed the cardiometabolic health status of individuals with familial CCMs caused by a rare mutation in the CCM1 gene (fCCM1). The aim was to compare plasma water T2 values from individuals with fCCM1 with values from metabolically unhealthy and healthy individuals with no known CCM mutations. Design: This observational, cross-sectional study included 75 participants: 11 fCCM1 patients, 24 metabolically unhealthy and 40 metabolically healthy individuals. Plasma water T2, an early, global and practical marker of cardiometabolic health, was measured in the time domain using benchtop magnetic resonance relaxometry. The results were stratified by age (≤ 45 vs. >45 years). Group means were compared using Welch's one-way ANOVA and post hoc Tukey-Kramer tests. Multivariable linear regression, with T2 as the outcome variable, was used to explore associations with age, gender, Hispanic ethnicity and fCCM1 status. Results: In the younger age stratum, the fCCM1 group had a mean plasma water T2 value comparable to the metabolically healthy group (p=0.6388), but higher than the unhealthy group (p<0.0001). By contrast, in the older stratum, the mean plasma water T2 value for the fCCM1 group was comparable to the metabolically unhealthy group (p=0.7819) and lower than the healthy group (p=0.0005). Multivariable linear regression revealed that age and the interaction between age and fCCM1 status were significant predictors of T2, even after adjusting for gender and Hispanic ethnicity. Conclusion: Plasma water T2 shows potential as a biomarker for assessing the health status of individuals with fCCM1. Further research is needed to validate these preliminary observations and elucidate the association between CCMs and cardiometabolic health.

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.

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.

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.

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.

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.

Novel functions of CCM1 delimit the relationship of PTB/PH domains.

BACKGROUND: Three NPXY motifs and one FERM domain in CCM1 makes it a versatile scaffold protein for tethering the signaling components together within the CCM signaling complex (CSC). The cellular role of CCM1 protein remains inadequately expounded. Both phosphotyrosine binding (PTB) and pleckstrin homology (PH) domains were recognized as structurally related but functionally distinct domains. METHODS: By utilizing molecular cloning, protein binding assays and RT-qPCR to identify novel cellular partners of CCM1 and its cellular expression patterns; by screening candidate PTB/PH proteins and subsequently structurally simulation in combining with current X-ray crystallography and NMR data to defined the essential structure of PTB/PH domain for NPXY-binding and the relationship among PTB, PH and FERM domain(s). RESULTS: We identified a group of 28 novel cellular partners of CCM1, all of which contain either PTB or PH domain(s), and developed a novel classification system for these PTB/PH proteins based on their relationship with different NPXY motifs of CCM1. Our results demonstrated that CCM1 has a wide spectrum of binding to different PTB/PH proteins and perpetuates their specificity to interact with certain PTB/PH domains through selective combination of three NPXY motifs. We also demonstrated that CCM1 can be assembled into oligomers through intermolecular interaction between its F3 lobe in FERM domain and one of the three NPXY motifs. Despite being embedded in FERM domain as F3 lobe, F3 module acts as a fully functional PH domain to interact with NPXY motif. The most salient feature of the study was that both PTB and PH domains are structurally and functionally comparable, suggesting that PTB domain is likely evolved from PH domain with polymorphic structural additions at its N-terminus. CONCLUSIONS: A new ß1A-strand of the PTB domain was discovered and new minimum structural requirement of PTB/PH domain for NPXY motif-binding was determined. Based on our data, a novel theory of structure, function and relationship of PTB, PH and FERM domains has been proposed, which extends the importance of the NPXY-PTB/PH interaction on the CSC signaling and/or other cell receptors with great potential pointing to new therapeutic strategies. GENERAL SIGNIFICANCE: The study provides new insight into the structural characteristics of PTB/PH domains, essential structural elements of PTB/PH domain required for NPXY motif-binding, and function and relationship among PTB, PH and FERM domains.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.