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Structural studies of the N-terminus of Connexin 32 using 1H NMR spectroscopy.

ABSTRACT: The amino terminus of gap junction proteins, connexins, plays a fundamental role in voltage gating and ion permeation. We have previously shown with (1)H NMR that the structure of the N-terminus of a representative connexin molecule contains a flexible turn around glycine 12 [P.E. Purnick, D.C. Benjamin, V.K. Verselis, T.A. Bargiello, T.L. Dowd, Arch. Biochem. Biophys. 381 (2000) 181-190] allowing the N-terminus to reside at the cytoplasmic entry of the channel forming a voltage-sensor. Previous functional studies or neuropathies have shown that the mutation G12Y and G12S form non-functional channels while functional channels are formed from G12P. Using 2D (1)H NMR we show that similar to G12, the structure of the G12P mutant contains a more flexible turn around residue 12, whereas the G12S and G12Y mutants contain tighter, helical turns in this region. These results suggest an unconstrained turn is required around residue 12 to position the N-terminus within the pore allowing the formation of the cytoplasmic channel vestibule, which appears to be critical for proper channel function.

SUBMITTER: Kalmatsky BD

PROVIDER: S-EPMC4510928 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature

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Structural studies of the N-terminus of Connexin 32 using 1H NMR spectroscopy.

Kalmatsky B D BD Bhagan S S Tang Q Q Bargiello T A TA Dowd T L TL

Archives of biochemistry and biophysics 20090726 1

The amino terminus of gap junction proteins, connexins, plays a fundamental role in voltage gating and ion permeation. We have previously shown with (1)H NMR that the structure of the N-terminus of a representative connexin molecule contains a flexible turn around glycine 12 [P.E. Purnick, D.C. Benjamin, V.K. Verselis, T.A. Bargiello, T.L. Dowd, Arch. Biochem. Biophys. 381 (2000) 181-190] allowing the N-terminus to reside at the cytoplasmic entry of the channel forming a voltage-sensor. Previous ...[more]

PMID: 19638273

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Project description:BackgroundAmbiguity in malignant transformation of glioma has made prognostic diagnosis very challenging. Tumor malignant transformation is closely correlated with specific alterations of the metabolic profile. Exploration of the underlying metabolic alterations in glioma cells of different malignant degree is therefore vital to develop metabolic biomarkers for prognosis monitoring.MethodsWe conducted (1)H nuclear magnetic resonance (NMR)-based metabolic analysis on cell lines (CHG5, SHG44, U87, U118, U251) developed from gliomas of different malignant grades (WHO II and WHO IV). Several methods were applied to analyze the (1)H-NMR spectral data of polar extracts of cell lines and to identify characteristic metabolites, including principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), fuzzy c-means clustering (FCM) analysis and orthogonal projection to latent structure with discriminant analysis (OPLS-DA). The expression analyses of glial fibrillary acidic protein (GFAP) and matrix metal proteinases (MMP-9) were used to assess malignant behaviors of cell lines. GeneGo pathway analysis was used to associate characteristic metabolites with malignant behavior protein markers GFAP and MMP-9.ResultsStable and distinct metabolic profiles of the five cell lines were obtained. The metabolic profiles of the low malignancy grade group (CHG5, SHG44) were clearly distinguished from those of the high malignancy grade group (U87, U118, U251). Seventeen characteristic metabolites were identified that could distinguish the metabolic profiles of the two groups, nine of which were mapped to processes related to GFAP and MMP-9. Furthermore, the results from both quantitative comparison and metabolic correlation analysis indicated that the significantly altered metabolites were primarily involved in perturbation of metabolic pathways of tricarboxylic acid (TCA) cycle anaplerotic flux, amino acid metabolism, anti-oxidant mechanism and choline metabolism, which could be correlated with the changes in the glioma cells' malignant behaviors.ConclusionsOur results reveal the metabolic heterogeneity of glioma cell lines with different degrees of malignancy. The obtained metabolic profiles and characteristic metabolites are closely associated with the malignant features of glioma cells, which may lay the basis for both determining the molecular mechanisms underlying glioma malignant transformation and exploiting non-invasive biomarkers for prognosis monitoring.

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Structural studies of the N-terminus of Connexin 32 using 1H NMR spectroscopy.

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Structural studies of the N-terminus of Connexin 32 using 1H NMR spectroscopy.

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