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Differential Scanning Fluorimetry and Hydrogen Deuterium Exchange Mass Spectrometry to Monitor the Conformational Dynamics of NBD1 in Cystic Fibrosis.

ABSTRACT: Cystic fibrosis (CF) is one of the most common, lethal autosomal recessive diseases in Caucasians with a life expectancy of 37-47 years. The CF transmembrane conductance regulator (CFTR) is a plasma membrane ion channel, confined to apical membrane of epithelia, and ensures transepithelial water and solute movement across secretory epithelia in several organs. Numerous CF mutations, including the most prevalent deletion of F508 (ΔF508) in the nucleotide binding domain 1 (NBD1) leads to CFTR global misfolding and premature intracellular degradation at the endoplasmic reticulum (ER). To better understand the misfolding mechanism caused by CF-causing point mutations in the NBD1, which is poorly understood, differential scanning fluorimetry (DSF) and hydrogen deuterium exchange coupled with mass spectrometry (HDX-MS) are the choice of techniques. These established methods can measure the conformational dynamics of the NBD1 globally and at peptide resolution level by monitoring backbone amide HDX, respectively, and will be instrumental to evaluate the mechanism of action of CF mutations and folding correctors that rescue CFTR folding defects via stabilizing the mutant NBD1.

SUBMITTER: Soya N

PROVIDER: S-EPMC8311558 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The use of Fourier transform mass spectrometry (FTMS) to monitor noncovalent complex formation in the gas phase under native conditions between the Link module from human tumor necrosis factor stimulated gene-6 (Link_TSG6) and hyaluronan (HA) oligosaccharides is reported. In particular, a titration experiment with increasing concentrations of octasaccharide (HA(8)) to protein produced a noncovalent complex with 1:1 stoichiometry when the oligosaccharide was in molar excess. However, in the presence of a molar excess of tetrasaccharide (HA(4)) nearly all proteins and oligosaccharides were observed in their unbound charge states. These results are consistent with solution-phase properties for this interaction in which HA(8), but not HA(4), supports high affinity Link_TSG6 binding. Hydrogen/deuterium amide exchange mass spectrometry (H/D-EX MS) was also utilized to investigate the level of global deuterium incorporation, over time, for Link_TSG6 in both the absence and presence of HA(8). After dilution into quenching conditions, deuterium incorporation reached limiting asymptotic values of 37 and 26 deuterons for the free and bound protein at 240 and 480 min, respectively, indicating that the oligosaccharide interferes with amide exchange on binding. To detect sequence-specific deuterium incorporation, pepsin digestion of Link_TSG6 in both the absence and presence of HA(8) was performed. A level of deuterium incorporation of 10-30% was observed for peptides analyzed in free Link_TSG6. Interestingly, HA(8) blocked some sites of proteolysis in Link_TSG6 compared to the free protein. Molecular modeling indicated that amino acids proximal to the ligand correlated with regions of the protein that were resistant to enzymatic digestion. Of the peptides that could be analyzed by H/D-EX MS in the presence of the ligand, a 30-60% reduction in deuterium incorporation, relative to the free protein, was observed, even for those sequences not directly involved in HA binding. These results support the utility of FTMS as a method for the characterization of protein-carbohydrate interactions.

Dataset Information

Differential Scanning Fluorimetry and Hydrogen Deuterium Exchange Mass Spectrometry to Monitor the Conformational Dynamics of NBD1 in Cystic Fibrosis.

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