William R. Kearney

Vps27-Hse1 and ESCRT-I complexes cooperate to increase efficiency of sorting ubiquitinated proteins at the endosome

Ubiquitin (Ub) attachment to cell surface proteins causes their lysosomal degradation by incorporating them into lumenal membranes of multivesicular bodies (MVBs). Two yeast endosomal protein complexes have been proposed as Ub-sorting “receptors,” the Vps27-Hse1 complex and the ESCRT-I complex. We used NMR spectroscopy and mutagenesis studies to map the Ub-binding surface for Vps27 and Vps23. Mutations in Ub that ablate only Vps27 binding or Vps23 binding blocked the ability of Ub to serve as an MVB sorting signal, supporting the idea that both the Vps27-Hse1 and ESCRT-I complexes interact with ubiquitinated cargo. Vps27 also bound Vps23 directly via two PSDP motifs present within the Vps27 COOH terminus. Loss of Vps27-Vps23 association led to less efficient sorting into the endosomal lumen. However, sorting of vacuolar proteases or the overall biogenesis of the MVB were not grossly affected. In contrast, disrupting interaction between Vps27 and Hse1 caused severe defects in carboxy peptidase Y sorting and MVB formation. These results indicate that both Ub-sorting complexes are coupled for efficient recognition of ubiquitinated cargo.

Orientation and Dynamics of an Antimicrobial Peptide in the Lipid Bilayer by Solid-State NMR Spectroscopy

The orientation and dynamics of an 18-residue antimicrobial peptide, ovispirin, has been investigated using solid-state NMR spectroscopy. Ovispirin is a cathelicidin-like model peptide (NH(2)-KNLRRIIRKIIHIIKKYG-COOH) with potent, broad-spectrum bactericidal activity. (15)N NMR spectra of oriented ovispirin reconstituted into synthetic phospholipids show that the helical peptide is predominantly oriented in the plane of the lipid bilayer, except for a small portion of the helix, possibly at the C-terminus, which deviates from the surface orientation. This suggests differential insertion of the peptide backbone into the lipid bilayer. (15)N spectra of both oriented and unoriented peptides show a reduced (15)N chemical shift anisotropy at room temperature compared with that of rigid proteins, indicating that the peptide undergoes uniaxial rotational diffusion around the bilayer normal with correlation times shorter than 10(-4) s. This motion is frozen below the gel-to-liquid crystalline transition temperature of the lipids. Ovispirin interacts strongly with the lipid bilayer, as manifested by the significantly reduced (2)H quadrupolar splittings of perdeuterated palmitoyloleoylphosphatidylcholine acyl chains upon peptide binding. Therefore, ovispirin is a curved helix residing in the membrane-water interface that executes rapid uniaxial rotation. These structural and dynamic features are important for understanding the antimicrobial function of this peptide.

Lanthanide-binding helix-turn-helix peptides: Solution structure of a designed metallonuclease

A designed lanthanide-binding chimeric peptide based on the strikingly similar geometries of the EF-hand and helix-turn-helix (HTH) motifs was investigated by NMR and CD spectroscopy and found to retain the same overall solution structure of the parental motifs. CD spectroscopy showed that the 33-mer peptide P3W folds on binding lanthanides, with an increase in α-helicity from 20% in the absence of metal to 38% and 35% in the presence of excess Eu(III) and La(III) ions, respectively. The conditional binding affinities of P3W for La(III) (5.9 ± 0.3 μM) and for Eu(III) (6.2 ± 0.3 μM) (pH 7.8, 5 mM Tris) were determined by tryptophan fluorescence titration. The La(III) complex of peptide P3, which differs from P3W by only one Trp-to-His substitution, has much less signal dispersion in the proton NMR spectra than LaP3W, indicating that the Trp residue is a critical hydrophobic anchor for maintaining a well-folded helix-turn-helix structure. A chemical-shift index analysis indicates the metallopeptide has a helix-loop-helix secondary structure. A structure calculated by using nuclear Overhauser effect and other NMR constraints reveals that P3W not only has a tightly folded metal-binding loop but also retains the α−α corner supersecondary structure of the parental motifs. Although the solution structure is undefined at both the N and C termini, the NMR structure confirms the successful incorporation of a metal-binding loop into a HTH sequence.

Bronchoscopic assessment of airway retention time of aerosolized xylitol

BackgroundHuman airway surface liquid (ASL) has abundant antimicrobial peptides whose potency increases as the salt concentration decreases. Xylitol is a 5-carbon sugar that has the ability to lower ASL salt concentration, potentially enhancing innate immunity. Xylitol was detected for 8 hours in the ASL after application in airway epithelium in vitro. We tested the airway retention time of aerosolized iso-osmotic xylitol in healthy volunteers.MethodsAfter a screening spirometry, volunteers received 10 ml of nebulized 5% xylitol. Bronchoscopy was done at 20 minutes (n = 6), 90 minutes (n = 6), and 3 hours (n = 5) after nebulization and ASL was collected using microsampling probes, followed by bronchoalveolar lavage (BAL). Xylitol concentration was measured by nuclear magnetic resonance spectroscopy and corrected for dilution using urea concentration.ResultsAll subjects tolerated nebulization and bronchoscopy well. Mean ASL volume recovered from the probes was 49 ± 23 μl. The mean ASL xylitol concentration at 20, 90, and 180 minutes was 1.6 ± 1.9 μg/μl, 0.6 ± 0.6 μg/μl, and 0.1 ± 0.1 μg/μl, respectively. Corresponding BAL concentration corrected for dilution was consistently lower at all time points. The terminal half-life of aerosolized xylitol obtained by the probes was 45 minutes with a mean residence time of 65 minutes in ASL. Corresponding BAL values were 36 and 50 minutes, respectively.ConclusionAfter a single dose nebulization, xylitol was detected in ASL for 3 hours, which was shorter than our in vitro measurement. The microsampling probe performed superior to BAL when sampling bronchial ASL.

Glycosylation of Aromatic Amines I: Characterization of Reaction Products and Kinetic Scheme

The reactions of aliphatic and aromatic amines with reducing sugars are important in both drug stability and synthesis. The formation of glycosylamines in solution, the first step in the Maillard reaction, does not typically cause browning but results in decreased potency and is hence significant from the aspect of drug instability. The purpose of this research was to present (1) unreported ionic equilibria of model reactant (kynurenine), (2) the analytical methods used to characterize and measure reaction products, (3) the kinetic scheme used to measure reaction rates and (4) relevant properties of various reducing sugars that impact the reaction rate in solution. The methods used to identify the reversible formation of two products from the reaction of kynurenine and monosaccharides included LC mass spectrometry, UV spectroscopy, and 1-D and 2-D 1H–1H COSY NMR spectroscopy. Kinetics was studied using a stability-indicating HPLC method. The results indicated the formation of α and β glycosylamines by a pseudo first-order reversible reaction scheme in the pH range of 1–6. The forward reaction was a function of initial glucose concentration but not the reverse reaction. It was concluded that the reaction kinetics and equilibrium concentrations of the glycosylamines were pH-dependent and also a function of the acyclic content of the reacting glucose isomer.

Low-Barrier Hydrogen Bond in the Catalytic Triad of Serine Enzymes

Acetylcholinesterase (AChE) and chymotrypsin are serine enzymes whose catalytic mechanism involves a nucleophilic attack (serine) and a general acid-base moiety (histidine). The incipient imidiazolium which is formed as a result of the nucleophilic attack by serine is stabilized by the negatively charged carboxylate (Glu or Asp). This transition state stabilization is thought to be gained by a low-barrier hydrogen bond (LBHB) in which the proton is centrally located between N5 and the carboxylate oxygen.