Volumetric measurements are reported for fully hydrated lipid/cholesterol bilayer mixtures using the neutral flotation method. Apparent specific volume data were obtained with the lipids DOPC, POPC and DMPC at T = 30 °C, DPPC at 50 °C, and brain sphingomyelin (BSM) at 45 and 24 °C for mole fractions of cholesterol x from 0 to 0.5. Unlike previous cholesterol mixture studies, we converted our raw data to partial molecular volume V L of the lipid and V C of the cholesterol. The partial molecular volumes were constant for POPC and DOPC as x was varied, but had sharp breaks for the other lipids at values of x C near 0.25 ± 0.05. Results for x < x C clearly exhibit the condensation effect of cholesterol on DPPC, DMPC and BSM when measured at temperatures above their main transition temperatures T M . The break points at x C are compared to phase diagrams in the literature. For x > x C the values of the partial molecular volumes of cholesterol clustered near 630 ± 10 Å 3 in all the lipids when measured for T > T M ; we suggest that this is the most appropriate measure of the bare volume of cholesterol in lipid bilayers.

Piscidins are histidine-enriched antimicrobial peptides that interact with lipid bilayers as amphipathic α-helices. Their activity at acidic and basic pH in vivo makes them promising templates for biomedical applications. This study focuses on p1 and p3, both 22-residue-long piscidins with 68% sequence identity. They share three histidines (H3, H4 and H11) but p1, which is significantly more permeabilizing, has a fourth histidine (H17). This study investigates how variations in amphipathic character associated with histidines affect the permeabilization properties of p1 and p3. First, we show that the permeabilization ability of p3, but not p1, is strongly inhibited at pH 6.0 when the conserved histidines are partially charged and H17 is

The phosphorylation-specific peptidyl-prolyl isomerase Pin1 catalyzes the isomerization of the peptide bond preceding a proline residue between cis and trans isomers. To best understand the mechanisms of Pin1 regulation, rigorous enzymatic assays of isomerization are required. However, most measures of isomerase activity require significant constraints on substrate sequence and only yield rate constants for the cis isomer, kcatcis and apparent Michaelis constants, KMApp. By contrast, NMR lineshape analysis is a powerful tool for determining microscopic rates and populations of each state in a complex binding scheme. The isolated catalytic domain of Pin1 was employed as a first step towards elucidating the reaction scheme of the full-length enzyme. A 24-residue phosphopeptide derived from the amyloid precurser protein intracellular domain (AICD) phosphorylated at Thr668 served as a biologically-relevant Pin1 substrate. Specific 13C labeling at the Pin1-targeted proline residue provided multiple reporters sensitive to individual isomer binding and on-enzyme catalysis. We have performed titration experiments and employed lineshape analysis of phosphopeptide 13C-1H constant time HSQC spectra to determine Kcatcis, Kcattrans, KDcis, and KDtrans for the catalytic domain of Pin1 acting on this AICD substrate. The on-enzyme equilibrium value of [E·trans]/[E·cis] = 3.9 suggests that the catalytic domain of Pin1 is optimized to operate on this substrate near equilibrium in the cellular context. This highlights the power of lineshape analysis for determining the microscopic parameters of enzyme catalysis, and demonstrates the feasibility of future studies of Pin1-PPIase mutants to gain insights on the catalytic mechanism of this important enzyme.

Although conformational changes in TCRs and pMHC molecules often occur upon binding, their relationship to intrinsic flexibility and role in ligand selectivity are poorly understood. Here we used NMR to study TCR-pMHC binding, examining recognition of the QL9/H-2Ld complex by the 2C TCR. Although the majority of the CDR loops of the 2C TCR rigidify upon binding, the CDR3β loop remains mobile within the TCR-pMHC interface. Remarkably, the region of the QL9 peptide that interfaces with CDR3β is also mobile in the free pMHC and in the TCR-pMHC complex. Determination of conformational exchange kinetics revealed that the motions of CDR3β and QL9 are closely matched. The matching of conformational exchange in the free proteins and its persistence in the complex enhances the thermodynamic and kinetic stability of the TCR-pMHC complex and provides a mechanism for facile binding. We thus propose that matching of structural fluctuations is a component of how TCRs scan amongst potential ligands for those that can bind with sufficient stability to enable T cell signaling.