No abstract

An uclear receptor, peroxisome proliferator-activated receptor g (PPARg ), is al igand-dependent transcription factor involved in glucose homeostasis anda dipocyted ifferentiation. PPARg is the molecular target of various natural ands ynthetic molecules, includinga nti-diabetic agents such as rosiglitazone. Amide hydrogen/deuterium-exchange (H/D-Ex), coupled with proteolysis and mass spectrometry,w as applied to study the dynamics of theP PAR g ligand binding domain (LBD) with or withoutmolecules that modulate PPARg activity.The H/D-Ex patterns of ligand-free PPARg LBD show that the ligand binding pocket of LBD is significantly more dynamic than ther est of the LBD. Presumably,t he bindingp ocketi si ntrinsically disordered in order to accommodated ifferentl igands. The presence of twof ull agonists (rosiglitazone and GW1929), ap artial agonist (nTZDpa), and ac ovalenta ntagonist (GW9662), changedt he dynamics/conformation of PPARg LBD ands lowedt he H/D exchanger ate in variousr egionso ft he protein.T he full agonists slowed theH /D exchange more globally andtoagreater extent than the partial agonist or the antagonist, indicating thatthe full agonist stabilizes the PPARg LBDmorethan thepartial agonist or the antagonist. Oneinteresting observation is that thetwo fullagonists significantly stabilized helix12whilethe partial agonist andthe antagonist did not perturb the H/D exchange of this region. Ther esults showedt hatt he change in proteind ynamics induced by ligandb inding may be an important factor fort he activation of genes and that H/D-Ex is au sefulm ethodf or analyzingt he biological activity of drug leads.

Statistical analysis of data from 39 proteins (13 766 amino acid residues) digested with immobilized porcine pepsin under conditions compatible with hydrogen/deuterium (H/D) exchange (<1 degrees C, <30 s) was performed to examine pepsin cleavage specificity. The cleavage of pepsin was most influenced by the amino acid residue at position P1. Phe and Leu are favored residues each with a cleavage probability greater than 40%. His, Lys, Arg, or Pro residues prohibit cleavage when found at the P1 position. Pro also cannot be at position P2 (cleavage probability <0.3%). Occupation of the P3 position by His, Lys, or Arg, or occupation of the P2' position by Pro, also leads to very little cleavage (cleavage probability <1.7%). The average cleavage probability over the entire data set was 13.6%, which is slightly lower than the value previously obtained by Powers et al. (14.8%). This is due, in part, to the larger protein sizes used in the current study. While the specificity of pepsin was similar to that previously observed, higher selectivity was observed in the present study due to less experimental variation in the conditions used to generate our database.

The epitope of horse cytochrome c against monoclonal antibody E8 was determined using amide hydrogen/deuterium (H/D) exchange combined with immobilized antibody, on-line pepsin proteolysis, liquid chromatography (LC), and mass spectrometry (MS). The results were generally in good agreement with contact residues identified by an X-ray co-crystal structure of the E8-cytochrome c complex and results obtained by H/D exchange with nuclear magnetic resonance (NMR) spectrometry. The H/D exchange reaction of cytochrome c was carried out in the presence or absence of immobilized E8 antibody. Regions that gained less deuterium in the presence of the antibody than in its absence are defined as the epitope by the H/D exchange MS method. Control experiments were carefully designed to help identify the epitope with high confidence.

No abstract

Structurally destabilizing mutations in acid β-glucosidase (GCase) can result in Gaucher disease (GD). The iminosugar isofagomine (IFG), a competitive inhibitor and a potential pharmacological chaperone of GCase, is currently undergoing clinical evaluation for the treatment of GD. An X-ray crystallographic study of the GCase-IFG complex revealed a hydrogen bonding network between IFG and certain active site residues. It was suggested that this network may translate into greater global stability. Here it is demonstrated that IFG does increase the global stability of wild-type GCase, shifting its melting curve by ~15 °C and that it enhances mutant GCase activity in pretreated N370S/N370S and F213I/L444P patient fibroblasts. Additionally, amide hydrogen/ deuterium exchange mass spectroscopy (H/D-Ex) was employed to identify regions within GCase that undergo stabilization upon IFG-binding. H/D-Ex data indicate that the binding of IFG not only restricts the local protein dynamics of the active site, but also propagates this effect into surrounding regions.

Backbone amide hydrogen exchange rates can be used to describe the dynamic properties of a protein. Amide hydrogen exchange rates in a native protein may vary from milliseconds (ms) to several years. Ideally, the rates of all amide hydrogens of the analyte protein can be determined individually. To achieve this goal, monitoring of a wider time window is critical, in addition to high sequence coverage and high sequence resolution. Significant improvements have been made to hydrogen/deuterium exchange mass spectrometry methods in the past decade for better sequence coverage and higher sequence resolution. On the other hand, little effort has been made to expand the experimental time window to accurately determine exchange rates of amide hydrogens. Many fast exchanging amide hydrogens are completely exchanged before completion of a typical short exchange time point (10-30 s) and many slow exchanging amide hydrogens do not start exchanging before a typical long exchanging time point (1-3 h). Here various experimental conditions, as well as a quenched-flow apparatus, are utilized to monitor cytochrome c amide hydrogen exchange behaviors over more than eight orders of magnitude (0.0044-1 000 000 s), when converted into the standard exchange condition (pH 7 and 23°C).

The practice of HDX-MS remains somewhat difficult, not only for newcomers but also for veterans, despite its increasing popularity. While a typical HDX-MS project starts with a feasibility stage where the experimental conditions are optimized and the peptide map is generated prior to the HDX study stage, the literature usually reports only the HDX study stage. In this protocol, we describe a few considerations for the initial feasibility stage, more specifically, how to optimize quench conditions, how to tackle the carryover issue, and how to apply the pepsin specificity rule. Two sets of quench conditions are described depending on the presence of disulfide bonds to facilitate the quench condition optimization process. Four protocols are outlined to minimize carryover during the feasibility stage: (1) addition of a detergent to the quench buffer, (2) injection of a detergent or chaotrope to the protease column after each sample injection, (3) back-flushing of the trap column and the analytical column with a new plumbing configuration, and (4) use of PEEK (or PEEK coated) frits instead of stainless steel frits for the columns. The application of the pepsin specificity rule after peptide map generation and not before peptide map generation is suggested. The rule can be used not only to remove falsely identified peptides, but also to check the sample purity. A well-optimized HDX-MS feasibility stage makes subsequent HDX study stage smoother and the resulting HDX data more reliable. Graphical Abstract ᅟ.