Dataset Information

HDX-ESI-MS reveals enhanced conformational dynamics of the amyloidogenic protein beta(2)-microglobulin upon release from the MHC-1.

ABSTRACT: The light chain of the major histocompatibility complex class 1 (MHC-1), the protein beta(2)-microglobulin (beta(2)m), has amyloidogenic properties that arise only upon its dissociation from the MHC-1. Here hydrogen/deuterium exchange electrospray ionization mass spectrometry (HDX-ESI-MS) has been used to compare the solution dynamics of beta(2)m in its MHC-1 bound state compared with those of beta(2)m as a free monomer. The capability of tandem mass spectrometry to dissociate the MHC-1 into its individual constituents in the gas phase following deuterium incorporation in solution has permitted the direct observation of the exchange properties of MHC-1 bound beta(2)m for the first time. The HDX-ESI-MS data show clearly that the H-->D exchange of MHC-1 bound beta(2)m follows EX2 kinetics and that about 20 protons remain protected from exchange after 17 days. Free from the MHC-1, monomeric beta(2)m exhibits significantly different HDX behavior, which encompasses both EX1 and EX2 kinetics. The EX2 kinetics indicate a tenfold increase in the rate of exchange compared with MHC-1 bound beta(2)m, with just 10 protons remaining protected from EX2 exchange and therefore exchanging only via the EX1 mechanism. The EX1 kinetics observed for unbound beta(2)m are consistent with unfolding of its exchange-protected core with a t(1/2) of 68 min (pH 7, 37 degrees C). Thus, upon dissociation from the stabilizing influence of the MHC-1, free beta(2)m becomes highly dynamic and undergoes unfolding transitions that result in an aggregation-competent protein.

SUBMITTER: Hodkinson JP

PROVIDER: S-EPMC2642988 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:Apolipoprotein-D is a glycosylated tetrameric lipocalin that binds and transports small hydrophobic molecules such as progesterone and arachidonic acid. Like other lipocalins, apolipoprotein-D adopts an eight-stranded ?-barrel fold stabilized by two intramolecular disulphide bonds, with an adjacent ?-helix. Crystallography studies of recombinant apolipoprotein-D demonstrated no major conformational changes upon progesterone binding. Amide hydrogen-deuterium exchange mass spectrometry (HDX-MS) reports structural changes of proteins in solution by monitoring exchange of amide hydrogens in the protein backbone with deuterium. HDX-MS detects changes in conformation and structural dynamics in response to protein function such as ligand binding that may go undetected in X-ray crystallography, making HDX-MS an invaluable orthogonal technique. Here, we report an HDX-MS protocol for apolipoprotein-D that solved challenges of high protein rigidity and low pepsin cleavage using rigorous quenching conditions and longer deuteration times, yielding 85% sequence coverage and 50% deuterium exchange. The relative fractional deuterium exchange of ligand-free apolipoprotein-D revealed apolipoprotein-D to be a highly structured protein. Progesterone binding was detected by significant reduction in deuterium exchange in eight peptides. Stabilization of apolipoprotein-D dynamics can be interpreted as a combined orthosteric effect in the ligand binding pocket and allosteric effect at the N-terminus and C-terminus. Together, our experiments provide insight into apolipoprotein-D structural dynamics and map the effects of progesterone binding that are relayed to distal parts of the protein. The observed stabilization of apolipoprotein-D dynamics upon progesterone binding demonstrates a common behaviour in the lipocalin family and may have implications for interactions of apolipoprotein-D with receptors or lipoprotein particles. Statement: We reveal for the first time how apolipoprotein-D, which is protective in Alzheimer's disease, becomes more ordered when bound to a molecule of steroid hormone. These results significantly extend the understanding of apolipoprotein-D structure from X-ray crystallography studies by incorporating information on how protein motion changes over time. To achieve these results an improved protocol was developed, suitable for proteins similar to apolipoprotein-D, to elucidate how proteins change flexibility when binding to small molecules.

Dataset Information

HDX-ESI-MS reveals enhanced conformational dynamics of the amyloidogenic protein beta(2)-microglobulin upon release from the MHC-1.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets