Structural comparison of recombinant human macrophage colony stimulating factor beta 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-CSFbeta) 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-CSFbeta and its derivative indicate that removing the disulfide bonds did not strongly alter the overall three-dimensional structure of rhm-CSFbeta. However, differences between deuterium exchange data of the intact proteins indicate that more NHs underwent fast deuterium exchange in the derivative than in rhm-CSFbeta. Regions located near the disulfide bond removal site were shown to exhibit faster deuterium exchange behavior in the derivative than in rhm-CSFbeta.

Reaction of neuronal nitric-oxide synthase with 2,6-dichloroindolphenol and cytochrome c3+: influence of the electron acceptor and binding of Ca2+-activated calmodulin on the kinetic mechanism.

Binding of Ca(2+)-activated calmodulin (Ca(2+)-CaM) to neuronal nitric-oxide synthase (nNOS) increases the rate of 2,6-dichloroindolphenol (DCIP) reduction 2-3-fold and that of cytochrome c(3+) 10-20-fold. Parallel initial velocity patterns indicated that both substrates were reduced via two-half reactions in a ping-pong mechanism. Product and dead-end inhibition data with DCIP were consistent with an iso ping-pong bi-bi mechanism; however, product and dead-end inhibition studies with cytochrome c(3+) were consistent with the (two-site) ping-pong mechanism previously described for the NADPH-cytochrome P450 reductase-catalyzed reduction of cytochrome c(3+) [Sem, D., and Kasper, C. (1994) Biochemistry 33, 12012--12021]. Dead-end inhibition by 2'-adenosine monophosphate (2'AMP) was competitive versus NADPH for both electron acceptors, although the value of the slope inhibition constant, K(is), was 25-30-fold greater with DCIP as the substrate than with cytochrome c(3+). The difference in the apparent affinity of 2'AMP is proposed to result from a rapidly equilibrating isomerization step that occurs in both mechanisms prior to the binding of NADPH. Thus, initial velocity, product, and dead-end inhibition data were consistent with a di-iso ping-pong bi-bi and an iso (two-site) ping-pong mechanism for the reduction of DCIP and cytochrome c(3+), respectively. The presence Ca(2+)-CaM did not alter the proposed kinetic mechanisms. The activated cofactor had a negligible effect on (k(cat)/K(m))(NADPH), while it increased (k(cat)/K(m))(DCIP) and (k(cat)/K(m))(cytc) 4.5- and 23-fold, respectively.

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.

Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometric analysis of the recombinant human macrophage colony stimulating factor beta 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 beta (rhM-CSF beta) 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 (approximately 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.

Kinetic and thermodynamic analysis of 9-cis-retinoic acid binding to retinoid X receptor alpha.

The interaction of retinoid X receptor alpha with 9-cis-retinoic acid was studied using stopped-flow fluorescence spectroscopy. Transient kinetic analyses of this interaction suggest a two-step binding mechanism involving a rapid, enthalpically driven pre-equilibrium followed by a slower, entropically driven reaction that may arise from a conformational change within the ligand binding domain of the receptor. The assignment of this kinetic mechanism was supported by agreement between the overall equilibrium constant, Kov, derived from kinetic studies with that determined by equilibrium fluorescence titrations. Although these analyses do not preclude ligand-induced alteration in the oligomerization state of the receptor in solution, the simplest model that can be applied to these data involves the stoichiometric interaction of 9-cis-retinoic acid with retinoid X receptor alpha monomers.

Thermal denaturation of Escherichia coli thioredoxin studied by hydrogen/deuterium exchange and electrospray ionization mass spectrometry: monitoring a two-state protein unfolding transition.

Thermally denatured oxidized Escherichia coli thioredoxin (TRX) in 2% acetic acid was examined by electrospray ionization mass spectrometry (ESI-MS) and circular dichroism. Conformational dynamics during thermal unfolding were probed by hydrogen/deuterium (H/D) exchange-in experiments. ESI-MS was used to determine the H/D ratios. TRX shows only a marginal change in negative ellipticity at 222 nm during thermal unfolding, but in the near-UV circular dichroism (240-350 nm) a clear transition is observed (Tm = 61 degrees C), and unfolding goes to completion. ESI mass spectra were recorded as a function of temperature, and the observed bimodal charge state distributions were analyzed assuming a two-state unfolding mechanism which allowed an estimation of the midpoint temperature, Tm = 64 degrees C. Under conditions at which the compact, folded conformational state is only marginally stable (80 degrees C, 2% acetic acid-d1), H/D exchange-in experiments in combination with ESI-MS resulted in mass spectra differing in the number of incorporated deuteriums which indicates the presence of two distinct populations of molecules after short incubation periods. As the exchange-in time increases, the population representing the unfolded state increases and the population which is protected against exchange decreases. The rate of conversion was used to estimate the rate constant of unfolding which was 2.1 +/- 0.2 min-1. The results presented here indicate that thermally denatured TRX under the conditions used may represent a collapsed unfolded state with properties often attributed to molten globule-like states, such as pronounced secondary structure but absence of rigid tertiary structure and, hence, lack of protection against H/D exchange.

Double mutant cycle analysis of aspartate 69, 97, and 103 to asparagine mutants in the m2 muscarinic acetylcholine receptor.

Double mutant cycles provide a method for analyzing the effects of a mutation at a defined position in the protein structure on the properties of an amino acid at a second site. This approach was used to map potential interactions between aspartates 69, 97, and 103 in the m2 muscarinic acetylcholine receptor transmembrane helices 2 and 3. Receptors containing single and double aspartate to asparagine mutants were expressed in Chinese hamster ovary cells and their effects on ligand binding, signal transduction, and thermal stability determined. Analysis of the double mutant cycles showed that the mutations had approximately additive effects on ligand binding, signal transduction, and thermal stability. Ligand binding and thermal inactivation results support the conclusion that aspartate-103 is the ligand amine counterion. Effector coupling properties of the mutant receptors showed that aspartate-103 was also required for signal transduction activity. The mutation of aspartate-69 to asparagine completely eliminated signal transduction by the agonists acetylcholine, carbachol, and pilocarpine but not oxotremorine M, which caused reduced but significant inhibition of adenylyl cyclase and stimulation of phospholipase C. In contrast, adenylyl cyclase stimulation by the asparagine-69 mutant was elicited only by acetylcholine and carbachol but not by oxotremorine M. The variation in agonist-dependent effector coupling properties provides evidence that the asparagine-69 mutant can exist in activated receptor states that are different from the wild-type m2 muscarinic receptor.

Site-directed mutagenesis on the m2 muscarinic acetylcholine receptor: the significance of Tyr403 in the binding of agonists and functional coupling.

The first step in the transmembrane signal mediated by G protein-coupled receptors is binding of agonist to receptors at the cell surface. The mechanism of the resulting receptor activation is not clear, but models based on the ternary complex model are capable of explaining most of the observations that have been reported in G protein-coupled receptors. This model suggests that a single agonist/receptor/G protein complex capable of activating G protein is formed as the result of agonist binding. Extensions of this basic model differ primarily in whether an equilibrium between active and inactive conformations is required to explain experimental results. We report results on ligand binding and coupling to physiological effector systems of the m2 muscarinic acetylcholine receptor site-directed mutant Y403F (residue 403 mutated from tyrosine to phenylalanine) expressed in Chinese hamster ovary cells and compare our results with results reported for the homologous Y506F mutation in the m3 muscarinic receptor [J. Biol. Chem. 267:19313-19319 (1992)]. The mutation in the m2 muscarinic receptor reduced absolute agonist affinities more dramatically than in the m3 muscarinic receptor. Unlike the results reported for the m3 subtype mutant, in which coupling to physiological effector systems was reduced, coupling to effector systems for the mutant in the m2 subtype was robust. In the Y403F m2 muscarinic receptor, the difference between the two agonist binding affinities was greater than in the wild-type receptor, whereas in the m3 subtype, the effect of the mutation was to decrease this difference. A prediction of the ternary complex model is that relative binding affinities will affect the steady state concentration of the agonist/receptor/G protein complex and, as the result, the extent of G protein coupling. These results can best be rationalized by this model, which suggests that the activation of G protein-coupled receptors is achieved by the relative affinity of agonist for two receptor states and does not require the existence of multiple states in conformational equilibrium.

Single amino acid substitutions in the pm2 muscarinic receptor alter receptor/G protein coupling without changing physiological responses.

The amino terminus of the third cytoplasmic loop of the porcine m2 muscarinic receptor plays an important role in receptor/effector coupling. Although large changes in coupling properties are easily detected, subtle changes are often overlooked. Three mutant receptors were characterized after expression in Chinese hamster ovary cells, and two of these exhibited subtle changes in coupling properties. Substitution of amino acids 219-223 (KDKKE) with those conserved in the m1/m3/m5 receptor subtype family (ELAAL) had little effect on coupling to effector systems, indicating that altering the charge distribution in this region did not affect receptor/G protein interactions. Substitution of alanine with glutamate at amino acid position 212 (A212E) or lysine with alanine in position 214 (K214A) resulted in receptors with IC50 values for inhibition of adenylyl cyclase that resembled those of wild-type, although maximal percent inhibition was reduced. All mutants moderately decreased coupling to phosphatidylinositol metabolism, but mutant A212E caused oxotremorine-M to become a weak partial agonist compared with carbachol, suggesting that receptor conformation is agonist dependent even for ligands normally thought of as full agonists. K214A coupled to PI metabolism through both PTX-sensitive and PTX-insensitive G proteins. The results indicated that these mutants superficially possessed effector coupling characteristics similar to those of wild-type, but on more detailed examination G protein/receptor interactions were altered.

Purification of recombinant porcine m2 muscarinic acetylcholine receptor from Chinese hamster ovary cells. Circular dichroism spectra and ligand binding properties.

The recombinant porcine m2 muscarinic acetylcholine receptor (rPm2R) from Chinese hamster ovary cells has been purified to homogeneity. Two mg of purified rPm2R, with a specific activity of 12 nmol of R-(-)-quinuclidinyl benzilate/mg of protein, were obtained from 30 ml of packed Chinese hamster ovary cells. The apparent molecular mass (78.5 kDa) and specific activity for the rPm2R preparation were the same as that for the Pm2R purified from atrial tissue, but the yield was 100 times greater. Purified rPm2R bound agonist and antagonist with the same affinities and coupled to the inhibitory guanine nucleotide-binding protein with the same efficiency as the purified native atrial Pm2R. Ligand binding studies were consistent with a single class of antagonist binding sites but two subclasses of agonist binding sites. The fraction of rPm2R having high affinity for agonists was increased by mM Mg2+, low detergent concentration, and low temperature. Circular dichroism spectra obtained for the purified rPm2R with and without agonists were indistinguishable, but spectra for the antagonist-occupied receptor showed reproducibly deeper characteristic negative deflections at 208 and 220 nm. Secondary structure analysis of the CD spectra predicted 53% alpha-helix for the free receptor and 49% alpha-helix for the R-(-)-quinuclidinyl benzilate-receptor complex.

Porcine m2 muscarinic acetylcholine receptor-effector coupling in Chinese hamster ovary cells.

The relationship between porcine m2 muscarinic receptor coupling to inhibition of cAMP formation and stimulation of phosphatidylinositol metabolism in Chinese hamster ovary cells was examined. Reduction of the number of receptors per cell with the slowly dissociating antagonist (-)-quinuclidinyl benzilate caused a decrease in maximal response with no effect on EC50 for coupling to phosphatidylinositol metabolism. Inhibition of cAMP formation showed the opposite dependence with no effect on maximal response but an increase in EC50 value as receptor density decreased. Pilocarpine appeared to be a partial agonist at low cell receptor density but displayed full agonism at higher receptor density. These results are compatible with a two-state model describing m2 muscarinic receptor acting via two different G proteins. This model is compatible with observations of negative antagonism where antagonists stimulated cAMP formation in adenylyl cyclase inhibition assays, and can also be used to estimate receptor affinities for G proteins in systems which display negative antagonism.

Kinetic and biophysical analysis of the m2 muscarinic receptor.

The recombinant Pm2 muscarinic receptor expressed in Chinese hamster ovary (CHO) cells was used as a model system to examine receptor-effector coupling and ligand binding. In CHO cells, equilibrium binding studies and the dependence on receptor number per cell of the maximum response and EC50 values for agonist stimulation of phosphatidylinositol metabolism and inhibition of cAMP formation were consistent with a modified ternary complex model of signal transduction that included a physiologically noncompetent receptor state. Detailed kinetic studies of oxotremorine M (Oxo-M) binding to CHO cell membranes suggested that agonist interactions at the high affinity class of binding sites are complicated and depend on receptor expression levels. At low levels of expression, kinetic data were consistent with a special case of a mechanism in which Oxo-M shifts the equilibrium between two receptor conformations while at high levels of expression, it was necessary to evoke receptor-receptor interactions to explain the kinetic data. Far ultraviolet circular dichroism studies of the purified recombinant receptor showed a high content of alpha-helical secondary structure and small changes in secondary structure upon antagonist, but not agonist, binding.

A kinetic model for oxotremorine M binding to recombinant porcine m2 muscarinic receptors expressed in Chinese hamster ovary cells.

The kinetic mechanism of super high affinity [3H]oxotremorine M binding to porcine m2 muscarinic receptors expressed in Chinese hamster ovary cells was examined. In cell lines expressing low receptor numbers (10(4) binding sites/cell) and in high expression (10(6) sites/cell) cell lines treated with cholate, [3H]oxotremorine M association and dissociation kinetics were monophasic. The reciprocal relaxation time for the association reaction was independent of [3H]oxotremorine M concentration and equaled the dissociation rate constant consistent with a special case for a mechanism involving a protein conformational change followed by ligand binding. Membranes from high expression cell lines and porcine atrial membranes showed complex kinetic behavior. Two kinetic phases were observed for [3H]oxotremorine M association, and both reciprocal relaxation times were independent of ligand concentration. The number of kinetic phases and their relative amplitudes seen in dissociation experiments were dependent on whether dissociation was initiated by dilution or by addition of unlabeled ligand(s) as well as on the fractional saturation of the receptor. Computer simulations of the data led to a model consistent with the existence of asymmetric receptor dimers as well as monomers and the ligand-dependent interconversion of fully occupied dimers and monomers.

Kinetics of the uracil-DNA glycosylase/inhibitor protein association. Ung interaction with Ugi, nucleic acids, and uracil compounds.

The bacteriophage PBS2 uracil-DNA glycosylase inhibitor (Ugi) inactivates Escherichia coli uracil-DNA glycosylase (Ung) by forming an Ung.Ugi protein complex with 1:1 stoichiometry. Stability of the Ung.Ugi complex was demonstrated by the inability of free Ugi to exchange with Ugi bound in preformed complex. Ung was reacted with fluorescein 5-isothiocyanate to produce fluorescent-Ung (F-Ung), which retained full uracil-DNA glycosylase activity and susceptibility to Ugi inactivation. Addition of Ugi to F-Ung under steady-state conditions resulted in saturable (15%) fluorescence quenching at a F-Ung.Ugi ratio of 1:1.4. Dissociation constants determined for the F-Ung interaction with M13 DNA, uracil-containing DNA, and poly(U) equaled 600, 220, and 190 microM, respectively. While F-Ung associated with nucleic acid polymers was able to bind Ugi efficiently, F-Ung bound in the F-Ung.Ugi complex could no longer effectively bind nucleic acid. Stopped-flow kinetic analysis suggested the F-Ung/Ugi association was described by a two-step mechanism. The first step entailed a rapid pre-equilibrium distinguished by the dissociation constant Kd = 1.3 microM. The second step led irreversibly to the formation of the final complex and was characterized by the rate constant k = 195 s-1. We infer Ugi inactivates Ung through the formation of an exceptionally stable protein-protein complex.

Partial agonist effects on the interaction between the atrial muscarinic receptor and the inhibitory guanine nucleotide-binding protein in a reconstituted system.

The mechanism of action of the partial muscarinic agonist pilocarpine was analyzed in a reconstituted system consisting of the purified porcine atrial muscarinic receptor and the purified porcine atrial inhibitory guanine nucleotide-binding protein, Gi. When GTPase activity was measured as a function of receptor.agonist complex concentration at saturating concentrations of either the full agonist carbachol or pilocarpine, both ligands gave similar values of kcat (4.3 +/- 0.2 min-1 for carbachol and 5.4 +/- 0.7 min-1 for pilocarpine); however, the observed dissociation constant for the ligand.receptor complex binding to Gi was about 4-fold lower for carbachol (0.81 +/- 0.19 nM) than for pilocarpine (3.02 +/- 0.83 nM). These results suggested that, in this system, the reduced activity of the partial agonist compared with the full agonist was the result of a decrease in affinity of the receptor.ligand complex for Gi, as opposed to differences in their relative abilities to activate the guanine nucleotide-binding protein. Several analogues of oxotremorine were also tested to determine their effects on the GTPase activity of Gi. Results from these studies indicate that the reconstituted system may be useful in determining structure-function relationships for muscarinic agonists with regard to receptor.Gi interactions.

Reconstitution of muscarinic receptor-mediated inhibition of adenylyl cyclase.

Inhibition of bovine brain calmodulin-sensitive adenylyl cyclase was examined in a system consisting of the reconstituted purified porcine atrial muscarinic acetylcholine receptor, the purified inhibitory guanine nucleotide-binding protein (Gi), and the partially purified stimulatory guanine nucleotide-binding protein.adenylyl cyclase complex. Under conditions where Gi existed mainly as the Gi.GDP complex, adenylyl cyclase was selectively preactivated with guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S). Addition of carbachol formed the receptor.carbachol complex, which catalyzed the exchange of GDP bound to Gi for GTP gamma S, initiating Gi-mediated inhibition of adenylyl cyclase. Adenylyl cyclase activated by calcium plus calmodulin was more sensitive to inhibition by carbachol than either unstimulated adenylyl cyclase or adenylyl cyclase activated by GTP gamma S or forskolin. Studies using the resolved subunits of Gi showed that the beta gamma subunit could inhibit adenylyl cyclase activated by GTP gamma S or calcium plus calmodulin, as well as the unactivated enzyme. The alpha subunit of Gi inhibited adenylyl cyclase only when adenylyl cyclase was activated by calcium plus calmodulin. Possible explanations for these results are discussed.

Agonist radioligand interactions with the solubilized porcine atrial A1 adenosine receptor.

Porcine atrial adenosine receptors have been solubilized using a detergent system consisting of digitonin and sodium cholate and characterized with the agonist radioligand N6[125I]hydroxyphenylisopropyl) adenosine [125I]HPIA. 125IHPIA labeled an apparently homogeneous population of solubilized recognition sites with a Bmax of 88 +/- 4 fmol/mg of protein and a KD of 1.4 +/- 0.1 nM. Solubilization resulted in a 2.5-fold enrichment of adenosine receptor specific activity and an enhanced signal to noise ratio over that observed for porcine atrial membrane preparations. Solubilized cardiac adenosine receptors were relatively stable and exhibited many of the properties of membrane-bound receptors. The rank order potency of adenosine receptor agonists inhibiting the binding of [125I]HPIA was consistent with the labeling of a solubilized A1 adenosine receptor. Association rate experiments suggested that the interaction of [125I]HPIA with solubilized cardiac adenosine receptors was consistent with that of a simple bimolecular reaction. The dissociation constant calculated from kinetic data (0.73 nM) was in good agreement with that determined by equilibrium binding measurements (1.4 nM). The interaction of cardiac A1 adenosine receptors and guanine nucleotide binding protein(s) G protein(s) was retained in this detergent system. Addition of guanosine-5'-O-(3-thio)triphosphate to an equilibrated mixture of solubilized cardiac adenosine receptors and [125I]HPIA resulted in a rapid and complete dissociation of [125I]HPIA. This dissociation was resolved into two kinetic phases, which appear to arise from two populations of independent, noninterconvertible receptor-G protein complexes that display differing sensitivities to guanine nucleotides. The A1 adenosine receptor-G protein complex solubilized in digitonin/cholate appears to provide an excellent system by which agonist radioligand-receptor-G protein interactions can be further studied.

Structure and regulation of muscarinic receptors.

Our knowledge of muscarinic receptor structure, mechanism, and regulation has increased enormously over the past five years. At this time, expression of receptor subtype and coupling via signal transducing elements to specific effector systems seems to be cell or tissue specific events. Determining the specific detailed molecular mechanisms of interaction for the various permutations of receptors and effector proteins and how the effector systems initiate physiological responses are challenging areas for future research.

Solubilization and hydrodynamic properties of pig atrial muscarinic acetylcholine receptor in dodecyl beta-D-maltoside.

The pig atrial muscarinic acetylcholine receptor (mAcChR) has been solubilized from the membrane-bound state in high yield and in stable conformation by the non-ionic detergent dodecyl beta-D-maltoside (DBM). The yield and selectivity for receptor solubilization is dependent on the detergent/protein ratio during extraction. Extraction at 2 mg of DBM/mg of protein gave a 75% yield of solubilized receptor with a 1.5-fold enrichment. A double-extraction procedure, in which non-receptor protein was first extracted at 0.4 mg of DBM/mg of protein and mAcChR was selectively solubilized by a second extraction at 0.35 mg of DBM/mg of protein, gave a 50% overall yield and a 2.8-fold enrichment. Both preparations had a half-life of about 20 days on ice without addition of muscarinic ligands. Receptor stability was decreased by the presence of cations, particularly bivalent cations, and enhanced by the agonist carbachol. Dissociation constants for the interaction of the DBM-solubilized receptor with the antagonist L-quinuclidinyl benzilate (Kd = 223 pM) and the agonist carbachol (Kd = 100 microM) were similar to those for the digitonin/cholate-solubilized receptor. Pig atrial mAcChR purified in digitonin/cholate and exchanged into DBM displayed reliable hydrodynamic behaviour during sucrose density sedimentation in gradients of 2H2O and H2O and during gel filtration in Sephacryl S-300. DBM is thus the first detergent which will solubilize a stable form of the ligand-free mAcChR in yields similar to those with digitonin, and is the only stabilizing detergent thus far suitable for hydrodynamic studies. DBM is also likely to be similarly useful in studying other membrane proteins for which digitonin has been the solubilizing detergent of choice.