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 calcium binding proteins 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 α-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

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 β-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 char- acterize the progression toward type 2 diabetes and atherosclerosis. Here we report the discovery and initial charac- terization 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 exqui- sitely 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, inflamma- tion, 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, provid- ing 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

A comparative study of the conformational properties of Escherichia coli-derived rat intestinal and liver fatty acid binding proteins

Fourier transform infrared spectroscopy has been used to examine the conformation in aqueous solution of Escherichia coli-expressed rat intestinal and liver fatty-acid binding proteins (I-FABP and L-FABP, respectively). While I-FABP is known from X-ray analysis to have a predominantly \u3b2-structure with 10 antiparallel \u3b2-strands forming two orthogonal sheets that surround the ligand binding pocket, no structural data are available for L-FABP. As expected for homologous proteins with related functions, the secondary structures of I-FABP and I-FABP are very similar. In both proteins, the conformation-sensitive amide-I band shows the maximum absorption at around 1630 cm 121, proving that \u3b2-sheet is the major structural element. However, there are three critical differences between I-FABP and L-FABP; (i), a different solvent accessibility of the protein backbone; (ii), a different pH sensitivity and (iii), a different thermostability, with L-FABP being thermally more stable than I-FABP. These results suggest that, in spite of having a similar overall conformation, the architecture of these proteins is stabilized by slightly different interactions. Such dissimilarities, well-paralleled by fatty-acid binding studies, may provide a structural basis for their functional diversification.Peer reviewed: YesNRC publication: Ye

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 ¹H 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 ¹H spin–spin relaxation time constants (<i>T</i>₂) 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 <i>T</i>₂ components in biologically relevant fatty acids, assigned to nanometer-scale domains in different segments of the hydrocarbon chain. The <i>T</i>₂ 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 <i>cis</i>-double bond content. Moreover, the <i>T</i>₂ values for each domain exhibited positive linear correlations with fluidity. The TD-NMR <i>T</i>₂ 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 <i>T</i>₂ as a nanofluidity meter and demonstrate its potential for probing nanofluidity in other systems of biological interest

The RXR-alpha 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 (RXRα)-mediated gene expression. Here, we report a nuclear magnetic resonance (NMR) study of the RXRα ligand-binding domain complexed with 9-cis-retinoic acid and a glucocorticoid receptorinteracting 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 Kd derived from T462 intensity decay agreed with that derived from isothermal titration calorimetry. Furthermore, the exchange contribution to the 15N 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. Originally published Biochemical and Biophysical Research Communications, Vol. 366, No. 4, Feb 200

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 in- flammation, 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 magnet- ic fields. Current applications of compact NMR relaxometry include blood tests for candidiasis, hemostasis, malaria and insulin resistance