Michael I. Schimerlik

Large Scale Preparation and Characterization of Membrane-Bound and Detergent-Solubilized Muscarinic Acetylcholine Receptor from Pig Atria

The muscarinic acetylcholine receptor (mAcChR) has been prepared from pig atrial membranes by new large scale procedures which result in 30-40 fold enrichment of the receptor in the membrane-bound state and a further three fold enrichment during solubilization. The membrane-bound receptor was prepared by differential and sucrose density gradient centrifugation in 25 mM imidazole, 1 mM EDTA, pH 7.4. A double extraction procedure using a mixed digitonin/cholate detergent was used to solubilize the receptor at a 60-70% yield. The membrane and solubilized preparations had specific activities of 3.5-5 and 8-12 pmol [3H]L-quinuclidinyl benzilate (QNB) binding sites per mg of protein, respectively. The presence of imidazole, which behaved as a weak muscarinic ligand, stabilized the receptor during solubilization and storage. Both the membrane-bound and detergent-solubilized mAcChR bound antagonists at a single class of sites and agonists at two subclasses of QNB sites. The proportion of high affinity agonist sites in the solubilized receptor was about 1/3 that in the membrane receptor. [3H]Propylbenzilylcholine mustard covalently labeled a single prominent atropine-sensitive component with an apparent molecular weight of 70-74,000 on SDS-polyacrylamide gels for both the membrane and solubilized receptor.

Glycoprotein Properties of the Solubilized Atrial Muscarinic Acetylcholine Receptor

Abstract: The muscarinic acetylcholine receptor from porcine atria exhibits sialoglycoprotein characteristics based on its sensitivity to neuraminidase digestion and its ability to interact specifically with lectin affinity resins when solubilized with a digitonin/cholate mixed detergent system. Differential lectin binding properties of the neuraminidase‐treated and untreated receptor suggest that high‐affinity binding to immobilized wheat germ agglutinin is accomplished through the presence of both terminal sialic acid and internal N‐acetylglucosamine or its β(1 → 4)‐linked oligomers.

Solubilization of the atrial muscarinic acetylcholine receptor: A new detergent system and rapid assays☆

Abstract The muscarinic acetylcholine receptor, identified by tritiated l -quinuclidinyl benzilate ( l -[ 3 H]QNB) binding, was solubilized from porcine atrial membranes using a 5:1 ( w w ) ratio of digitonin and cholate. Specific binding activities of the solubilized receptor solutions usually exceeded 1.0 nmol l -[ 3 H]QNB sites per gram of protein, representing 75–98% total site recovery and a two- to threefold enrichment over untreated atrial membranes. Two rapid assays for measuring the binding activities of detergent extracts were devised and compared with equilibrium dialysis. All three methods gave similar results. The equilibrium dissociation constant of the solubilized receptor for l -[ 3 H]QNB as determined by the three methods varied from 230 to 450 p m depending on the method and temperature.

Hydrogen/deuterium exchange and mass spectrometric analysis of a protein containing multiple disulfide bonds: Solution structure of recombinant macrophage colony stimulating factor-beta (rhM-CSFβ)

Studies with the homodimeric recombinant human macrophage colony‐stimulating factor beta (rhM‐CSFβ), show for the first time that a large number (9) of disulfide linkages can be reduced after amide hydrogen/deuterium (H/D) exchange, and the protein digested and analyzed successfully for the isotopic composition by electrospray mass spectrometry. Analysis of amide H/D after exchange‐in shows that in solution the conserved four‐helix bundle of (rhM‐CSFβ) has fast and moderately fast exchangeable sections of amide hydrogens in the αA helix, and mostly slow exchanging sections of amide hydrogens in the αB, αC, and αD helices. Most of the amide hydrogens in the loop between the β1 and β4 sheets exhibited fast or moderately fast exchange, whereas in the amino acid 63–67 loop, located at the interface of the two subunits, the exchange was slow. Solvent accessibility as measured by H/D exchange showed a better correlation with the average depth of amide residues calculated from reported X‐ray crystallographic data for rhM‐CSFα than with the average B‐factor. The rates of H/D exchange in rhM‐CSFβ appear to correlate well with the exposed surface calculated for each amino acid residue in the crystal structure except for the αD helix. Fast hydrogen isotope exchange throughout the segment amino acids 150–221 present in rhM‐CSFβ, but not rhM‐CSFα, provides evidence that the carboxy‐terminal region is unstructured. It is, therefore, proposed that the anomalous behavior of the αD helix is due to interaction of the carboxy‐terminal tail with this helical segment.

Rapid kinetic studies on calcium interactions with native and fluorescently labeled calmodulin

Abstract Stopped-flow studies on calcium binding to calmodulin showed that under pseudo first order conditions the reaction was complete within 2.5 milliseconds. The time course for calcium dissociation from the native protein showed a single kinetic phase (τ1 −1 = 10S −1 ) while that from the dansylated derivative revealed a second slower kinetic phase (τ1 −1 = 10S −1 , τ2 −1 = 0.31S −1 ) that accounted for about one-half of the total fluorescence decrease. Therefore the dansyl derivative of calmodulin may provide a useful tool for studying conformational changes in the protein not reflected by the active site tyrosines.

Deuterium exchange and mass spectrometry reveal the interaction differences of two synthetic modulators of RXRα LBD

Protein amide hydrogen/deuterium (H/D) exchange was used to compare the interactions of two antagonists, UVI 2112 and UVI 3003, with that of the agonist, 9‐cis‐retinoic acid, upon binding to the human retinoid X receptor alpha ligand‐binding domain (hRXRα LBD) homodimer. Analysis of the H/D content by mass spectrometry showed that in comparison to 9‐cis‐retinoic acid, the antagonists provide much greater protection toward deuterium exchange‐in throughout the protein, suggesting that the protein–antagonist complex adopts a more restricted conformation or ensemble of conformations in which solvent accesses to amide protons are reduced. A comparison between the two antagonists shows that UVI 3003 is more protective in the C‐terminal region due to the extra hydrophobic interactions derived from the atoms in the benzene ring of the carboxylic acid chain. It was less protective within regions comprising peptides 271–278 and 326–330 due to differences in conformational orientation, and/or shorter carboxylic acid chain length. Decreased deuterium exchange‐in in the segment 234–239 where the residues do not involve interactions with the ligand was observed with the two antagonists, but not with 9‐cis‐RA. The amide protons of helix 12 of the agonist‐ or antagonist‐occupied protein in solution have the same deuterium exchange rates as the unliganded protein, supporting a suggestion made previously that helix 12 can cover the occupied binding cavity only with the cofactor present to adjust its location.

N-Methyl-D-aspartate Receptor Subunits Are Non-myosin Targets of Myosin Regulatory Light Chain

Excitatory synapses contain multiple members of the myosin superfamily of molecular motors for which functions have not been assigned. In this study we characterized the molecular determinants of myosin regulatory light chain (RLC) binding to two major subunits of the N-methyl-d-aspartate receptor (NR). Myosin RLC bound to NR subunits in a manner that could be distinguished from the interaction of RLC with the neck region of non-muscle myosin II-B (NMII-B) heavy chain; NR-RLC interactions did not require the addition of magnesium, were maintained in the absence of the fourth EF-hand domain of the light chain, and were sensitive to RLC phosphorylation. Equilibrium fluorescence spectroscopy experiments indicate that the affinity of myosin RLC for NR1 is high (30 nm) in the context of the isolated light chain. Binding was not favored in the context of a recombinant NMII-B subfragment one, indicating that if the RLC is already bound to NMII-B it is unlikely to form a bridge between two binding partners. We report that sequence similarity in the “GXXXR” portion of the incomplete IQ2 motif found in NMII heavy chain isoforms likely contributes to recognition of NR2A as a non-myosin target of the RLC. Using site-directed mutagenesis to disrupt NR2A-RLC binding in intact cells, we find that RLC interactions facilitate trafficking of NR1/NR2A receptors to the cell membrane. We suggest that myosin RLC can adopt target-dependent conformations and that a role for this light chain in protein trafficking may be independent of the myosin II complex.

Structural comparison of recombinant human macrophage colony stimulating factor β and a partially reduced derivative using hydrogen deuterium exchange and electrospray ionization mass spectrometry

Hydrogen deuterium exchange, monitored by electrospray ionization mass spectrometry, has been employed to characterize structural features of a derivative of recombinant human macrophage colony stimulating factor beta (rhm‐CSFβ) in which two of the nine disulfide bridges (Cys157/Cys159–Cys′157/Cys′159) were selectively reduced and alkylated. Removal of these two disulfide bridges did not affect the biological activity of the protein. Similarities between CD and fluorescence spectra for rhm‐CSFβ and its derivative indicate that removing the disulfide bonds did not strongly alter the overall three‐dimensional structure of rhm‐CSFβ. However, differences between deuterium exchange data of the intact proteins indicate that more NHs underwent fast deuterium exchange in the derivative than in rhm‐CSFβ. Regions located near the disulfide bond removal site were shown to exhibit faster deuterium exchange behavior in the derivative than in rhm‐CSFβ.

Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometric analysis of the recombinant human macrophage colony stimulating factor β and derivatives

The potential of electrospray ionization (ESI) Fourier transform ion cyclotron mass spectrometry (FTICR-MS) to assist in the structural characterization of monomeric and dimeric derivatives of the macrophage colony stimulating factor β (rhM-CSFβ) was assessed. Mass spectrometric analysis of the 49 kDa protein required the use of sustained off-resonance irradiation (SORI) in-trap cleanup to reduce adduction. High resolution mass spectra were acquired for a fully reduced and a fully S-cyanylated monomeric derivative (∼25 kDa). Mass accuracy for monomeric derivatives was better than 5 ppm, after applying a new calibration method (i.e., DeCAL) which eliminates space charge effects upon high accuracy mass measurements. This high mass accuracy allowed the direct determination of the exact number of incorporated cyanyl groups. Collisionally induced dissociation using SORI yielded b- and y-fragment ions within the N- and C-terminal regions for the monomeric derivatives, but obtaining information on other regions required proteolytic digestion, or potentially the use of alternative dissociation methods.

Overview of Membrane Protein Solubilization

The solubilization of membrane proteins (i.e., extraction of the protein from the membrane in a disperse state in aqueous solution) in a stable state that retains the native activity of the membrane‐bound form is more an empirical art than a protocol‐driven science. Although a large number of molecules have been invented that display detergent properties, selection of the proper solubilizing agent is largely a matter of trial and error that depends on the protein of interest and the particular goals of the investigator. The purpose of this overview is to describe the methodology for finding the optimal conditions for detergent solubilization and to relate the selection of a specific detergent to explicit experimental goals.