Specificity of zinc binding to myelin basic protein

Z²⁺ appears to stabilize the myelin sheath but the mechanism of this effect is unknown. In a previous report we have shown that zinc binds to CNS myelin basic protein (MBP) in the presence of phosphate and this results in MBP aggregation. For this paper we used a solid phase zinc blotting assay to identify which myelin proteins bind zinc. MBP and a 58 kDa band were found to be the major targets of⁶⁵Zn binding. Moreover, using fluorescence, light scattering and electron microscopy we investigated the binding of zinc and other cations to purified MBP in solution. Among the cations tested for their ability to interfere with the binding of zinc, the most effective were cadmium, mercury and copper, but only cadmium and mercury increased the scattering intensity, whereas MBP aggregation was not inhibited by copper ions. Thus, the effect of zinc on the formation of MBP clusters seems to be specific.     

Interactions of the 18.5-kDa isoform of myelin basic protein with Ca(2+)-calmodulin: in vitro studies using fluorescence microscopy and spectroscopy.

The interactions of the 18.5-kDa isoform of myelin basic protein (MBP) with calmodulin (CaM) in vitro have been investigated using fluorescence microscopy and spectroscopy. Two forms of MBP were used: the natural bovine C1 charge isomer (bMBP/C1) and a hexahistidine-tagged recombinant murine product (rmMBP), with only minor differences in behaviour being observed. Fragments of each protein generated by digestion with cathepsin D (EC 3.4.23.5) were also evaluated. Using fluorescence microscopy, it was shown that MBP and CaM interacted in the presence of Ca2+ under a variety of conditions, including high urea and salt concentrations, indicating that the interaction was specific and not merely electrostatic in nature. Using cathepsin D digestion fragments of MBP, it was further shown that the carboxyl-terminal domain of MBP interacted with Ca(2+)-CaM, consistent with our theoretical prediction. Spectroscopy of the intrinsic fluorescence of the sole Trp residue of MBP showed that binding was cooperative in nature. The dissociation constants for formation of a 1:1 MBP-Ca(2+)-CaM complex were determined to be 2.1 +/- 0.1 and 2.0 +/- 0.2 microM for bMBP/C1 and rmMBP, respectively. Fluorescence spectroscopy using cathepsin D digestion fragments indicated also that the carboxyl-terminal region of each protein interacted with Ca(2+)-CaM, with dissociation constants of 1.8 +/- 0.2 and 2.8 +/- 0.9 microM for the bMBP/C1 and rmMBP fragments, respectively. These values show a roughly 1000-fold lower affinity of MBP for CaM than other CaM-binding peptides, such as myristoylated alanine-rich C-kinase substrate, that are involved in signal transduction.     

Mechanism of interaction of myelin basic protein and S-100 protein: metal binding and fluorescence studies.

Abstract— It has been reported that myelin basic protein (MBP) forms a specific complex with S-100 protein in the presence of either Ca²⁺ or Mn²⁺, as detected by Immunoelectrophoresis. We have now studied the binding of Ca²⁺ and Mn²⁺ to these two proteins. We find that MBP binds 1 mol of Mn²⁺/mol of protein, and this binding produces an increment in its fluorescence, indicating a conformational change. Ca²⁺ does not bind to MBP nor does it affect the fluorescence of MBP. S-100 protein, as has been reported, binds about 10 mol of Ca²⁺/mol and this binding produces a conformational change. S-100 protein also has 25 binding sites for Mn²⁺, but this binding does not alter fluorescence and does not appear to affect conformation. Competitive binding experiments demonstrate that the binding sites of S-100 protein for Ca²⁺ and Mn²⁺ are independent. The alteration of electrophoretic migration in gels of S-100 protein produced by Ca²⁺ and of MBP produced by Mn²⁺ are in accord with the observations based on fluorescence. Mn²⁺ does not affect the electrophoretic mobility of S-100. These results indicate that the formation of the complex between MBP and S-100 protein in the presence of either Ca²⁺ or Mn²⁺ is due to the conformational change induced by these ions in S-100 protein, MBP, or both.     

Phosphorylation of myelin basic protein and peptides by ganglioside-stimulated protein kinase

Rabbit myelin basic protein (MBP) was phosphorylated by a ganglioside-stimulated protein kinase to a stoichiometry of 1.4 and 2.1 mol phosphate/mol MBP in the presence and absence of GTlb, respectively. Two-dimensional peptide mapping analyses revealed that two of the sites of phosphorylation were distinct from those catalyzed by cAMP-dependent protein kinase or protein kinase C. Phosphorylation of one of these sites by ganglioside-stimulated protein kinase was inhibited by GTlb, suggesting that the inhibitory effect of gangliosides on MBP phosphorylation may be substrate-directed. Although ganglioside-stimulated protein kinase did not phosphorylate MBP at a domain containing residues 82-117, a synthetic peptide Arg-Phe-Ser-Trp-Gly-Ala-Glu-Gly-Gln-Lys corresponding to residues 111-120 was phosphorylated by the kinase in a ganglioside-stimulated manner. These findings suggest that the conformation of MBP may be important in determining its phosphorylatability.     

Interaction of eosinophil granule major basic protein with synthetic lipid bilayers: A mechanism for toxicity

Summary Eosinophil granule major basic protein (MBP) is a potent toxin for mammalian cells and helminths, but the mechaism of its toxicity is not known. Here we tested whether MBP toxicity is exerted through its effect on the lipid bilayer of its targets. Liposomes prepared from synthetic phospholipids were used as targets for MBP and their properties examined by fluorescence and circular dichroism (CD) spectroscopy. MBP caused a change in the temperature transition profiles of acidic liposomes (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidyl serine or an equimolar mixture of 1,2-dimyristoyl-sn-glycero-3-phosphocholine and 1,2-dimyristoyl-sn-glycero-3-phosphatidic acid) and induced their aggregation as shown by fluorescence resonance energy transfer experiments. The CD spectra and fluorescence characteristics of MBP itself were altered by its interaction with acidic lipids. Blue shifts in the emission maxima of the Trp, and of the dimethylaminonaphthyl moiety in acrylodan-labeled MBP, and a reduction in the effectiveness of quenching of Trp fluorescence by acrylamide were observed in the presence of acidic lipids. None of these effects were noted with zwitterionic lipids. This MBP : lipid bilayer interaction resulted in fusion and lysis of liposomes as indicated by the fluorescent indicator calcein. The results demonstrate that MBP associates with acidic lipids and that it disrupts, aggregates, fuses, and lyses liposomes prepared from such lipids. Such interaction might account for its wide range of toxicity.Abbreviations used Acrylodan 6-acryloyl-2-dimethylam-inonaphthalene - CD circular dichroism - DMPA 1,2-dimyrist-oyl-sn-glycero-3-phosphatidic acid - DMPC 1,2-dimyristoyl-sn-glycero-3-phosphocholine - DPH 1,6-diphenyl-1,3,5-hexatriene - DTT dithiothreitol - FRET fluorescence resonance energy transfer - HEPES N-2-hydroxyethyl piperazine-N-2-ethane sulfonic acid - K _sv Stern-Volmer constant - K _q bimolecular quenching coefficient - _em emission wavelength - _ex excitation wavelength - MBP major basic protein - MOPC 1-oleoyl-2-hydroxy-sn-glycero-3-phosphocholine - NBD-PE N-(7-nitro-2,1,3-benzo-xadiazol-4-yl)-phosphatidylethanolamine - nMBP native major basic protein - PBS phosphate-buffered saline - POPC 1-palmit-oyl-2-oleoyl-sn-glycero-3-phosphocholine - POPS 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidyl serine - raMBP reduced and alkylated major basic protein - RHO-PE rhodamine-phosphatidylethanolamine - Tes N-tris[hydroxymethyl]-methyl-2-amino-ethane-sulfonic acid - Tris tris[hydroxymethyl]-amino-methane We would like to thank Dr. Predrag J.K. Ilich for assistance with initial data analysis, Dr. Salah S. Sedarous for the lifetime data and for helpful discussions, Dr. S. Yu. Venyaminov for helpful discussions, Mr. Kenneth D. Peters and Mr. Peter J. Callahan for assistance with some of the illustrations, and Ms. Jill Wagner for performing the radioimmunoassays. We would also like to thank Ms. Jill Kappers for excellent secretarial work. This work was supported in part by a Fellowship grant from the American Heart Association, Minnesota Affiliate, and by grants from the National Institutes of Health AI 09728 and from the Mayo Foundation. RIA-G is a Fellow of the American Heart Association.     

Fluorescence spectral resolution of myelin basic protein conformers in complexes with lysophosphatidylcholine

The structure of (Deibler) myelin basic protein in solution and in a lysolecithin lipid complex has been studied by using the emission properties of the single tryptophan residue of the protein (Trp-115). The studies have been carried out using both static and time-resolved fluorescence techniques. Relative to the free protein, the lipid bound myelin basic protein showed a, twofold increase in fluorescence intensity and a marked blue-shift in the emission maximum wavelength. The multiexponential fluorescence decays and the decay associated spectra indicated that the protein exists in at least three different conformations both in buffer and in lipids. Fluorescence polarization and acrylamide quenching experiments showed that the tryptophan containing region of the protein is embedded in the lipid matrix. The binding of the protein to the lipid appears to be comparable with that predicted for the interaction of amphipathic helices with nonpolar lipids.     

Thermodynamics of maltose binding protein unfolding.

The maltose binding protein (MBP or MalE) of Escherichia coli is the periplasmic component of the transport system for malto-oligosaccharides. It is used widely as a carrier protein for the production of recombinant fusion proteins. The melting of recombinant MBP was studied by differential scanning and titration calorimetry and fluorescence spectroscopy under different solvent conditions. MBP exhibits a single peak of heat absorption with a delta(Hcal)/delta(HvH) ratio in the range of 1.3-1.5, suggesting that the protein comprises two strongly interacting thermodynamic domains. Binding of maltose resulted in elevation of the Tm by 8-15 degrees C, depending of pH. The presence of ligand at neutral pH, in addition to shifting the melting process to higher temperature, caused it to become more cooperative. The delta(Hcal)/delta(HvH) ratio decreased to unity, indicating that the two domains melt together in a single two-state transition. This ligand-induced merging of the two domains appears to occur only at neutral pH, because at low pH maltose simply stabilized MBP and did not cause a decrease of the delta(Hcal)/delta(HvH) ratio. Binding of maltose to MBP is characterized by very low enthalpy changes, approximately -1 kcal/mol. The melting of MBP is accompanied by an exceptionally large change in heat capacity. 0.16 cal/K-g, which is consistent with the high amount of nonpolar surface--0.72 A2/g--that becomes accessible to solvent in the unfolded state. The high value of delta Cp determines a very steep delta G versus T profile for this protein and predicts that cold denaturation should occur above freezing temperatures. Evidence for this was provided by changes in fluorescence intensity upon cooling the protein. A sigmoidal cooperative transition with a midpoint near 5 degrees C was observed when MBP was cooled at low pH. Analysis of the melting of several fusion proteins containing MBP illustrated the feasibility of assessing the folding integrity of recombinant products prior to separating them from the MBP carrier protein.     

Fluorescence spectral resolution of myelin basic protein conformers in complexes with lysophosphatidylcholine

The structure of (Deibler) myelin basic protein in solution and in a lysolecithin++ lipid complex has been studied by using the emission properties of the single tryptophan residue of the protein (Trp-115). The studies have been carried out using both static and time-resolved fluorescence techniques. Relative to the free protein, the lipid bound myelin basic protein showed a twofold increase in fluorescence intensity and a marked blue-shift in the emission maximum wavelength. The multiexponential fluorescence decays and the decay associated spectra indicated that the protein exists in at least three different conformations both in buffer and in lipids. Fluorescence polarization and acrylamide quenching experiments showed that the tryptophan containing region of the protein is embedded in the lipid matrix. The binding of the protein to the lipid appears to be comparable with that predicted for the interaction of amphipathic helices with nonpolar lipids.     

Real-time fluorogenic kinase assay using protein as substrate

A real-time fluorogenic kinase assay using myelin basic protein (MBP) as a substrate is reported. MBP is part of a noncovalent complex with a negatively charged, dye-labeled lipopeptide, (N-heptadecanoyl)-K(dye2)-linker-EEIYGEF-amide. The complex is approximately 20 times less fluorescent than the free lipopeptide. The MBP-lipopeptide complex serves as a protein substrate for several Ser/Thr kinases. We infer that the observed fluorescence increase on the addition of kinase and ATP is due to the phosphorylation of MBP, which decreases the affinity of MBP with the negatively charged, dye-labeled lipopeptide. Several protein kinases (protein kinase C βII, mitogen-activated protein kinase [MAPK] Erk1, and MAPK Erk2) were tested with the assay. The assay exhibited a fivefold fluorescence increase over background, provided kinetic values comparable to literature values (apparent K_m^ATP), and produced inhibitor constants comparable to literature values for a typical inhibitor, namely staurosporine.     

Mannan-binding protein blocks the activation of metalloproteases meprin alpha and beta

Mannan-binding protein (MBP) is a C-type serum lectin that is known to be a host defense factor involved in innate immunity, and recognizes mannose, fucose, and N-acetylglucosamine residues. Although some exogenous MBP ligands have been reported, little is known about its endogenous ligands. In the present study, we found that endogenous MBP ligands are highly expressed in the brush border epithelial cells of kidney-proximal tubules by immunohistochemistry, and both meprin alpha and beta (meprins), as novel endogenous MBP ligands, have been identified through affinity chromatography and mass spectrometry. Meprins are membrane-bound and secreted zinc metalloproteases extensively glycosylated and highly expressed in kidney and small intestinal epithelial cells, leukocytes, and certain cancer cells. Meprins are capable of cleaving growth factors, extracellular matrix proteins, and biologically active peptides. Deglycosylation experiments indicated that the MBP ligands on meprins are high mannose- or complex-type N-glycans. The interaction of MBP with meprins resulted in significant decreases in the proteolytic activity and matrix-degrading ability of meprins. Our results suggest that core N-linked oligosaccharides on meprins are associated with the optimal enzymatic activity and that MBP is an important regulator for modulation of the localized meprin proteolytic activity via N-glycan binding. Because meprins are known to be some of the major matrix-degrading metalloproteases in the kidney and intestine, MBP, which functions as a natural and effective inhibitor of meprins, may contribute, as a potential therapeutic target, to tumor progression by facilitating the migration, intravasation, and metastasis of carcinoma cells, and to acute renal failure and inflammatory bowel diseases.     

Purification and in vitro characterization of the maltose-binding protein of the plant pathogen Xanthomonas citri

The uptake of maltose and maltodextrins in gram-negative bacteria is mediated by an ATP-dependent transport complex composed of a periplasmic maltose-binding protein (MBP) and membrane-associated proteins responsible for the formation of a membrane pore and generation of energy to drive the translocation process. In this work, we report the purification and in vitro functional analysis of MBP, encoded by the malE gene, of the plant pathogen Xanthomonas citri, responsible for the canker disease affecting citrus plants throughout the world. The X. citri MBP is composed of 456 amino acids, displaying a low amino acid identity (16% throughout the sequence) compared to the Escherichia coli K12 ortholog. The X. citri malE gene was cloned into a pET28a vector, and the encoded protein was expressed and purified by affinity chromatography as a His-tag N-terminal fusion peptide produced by the E. coli BL21 strain. Enhanced levels of soluble protein were achieved with static cultures kept overnight at 23 degrees C. Ability to bind immobilized amylose, the emission of intrinsic fluorescence and circular dichroism spectra indicated that the purified recombinant protein preserved both conformation and biological activity of the native protein. The availability of the recombinant MBP will contribute to the functional and structural analysis of the maltose and maltodextrin uptake system of the plant pathogen X. citri.     

Detection of conformationally changed MBP using intramolecular FRET

The principal objective of this study was to explore protein conformational changes using fluorescence resonance energy transfer (FRET) technology. Maltose binding protein (MBP) was adopted as a target model, due to its well-characterized structure and ligand specificity. To the best of our knowledge, this is the first report to provide information regarding the biological distance between the two lobes of MBP upon maltose binding. For the FRET pair, ECFP and EYFP were used as the donor and the acceptor, and were linked genetically to the C-terminal and N-terminal regions of MBP (ECFP:MBP:EYFP), respectively. After the FRET reaction, maltose-treated MBP was shown to exhibit a considerable energy transfer (FRET efficiency (E) = ∼0.11, Distance (D) = ∼6.93 nm) at the ensemble level, which was regarded as reflective of the increase in donor quenching and the upshift in acceptor emission intensity, thereby suggesting that the donor and the acceptor had been brought close together as the result of structural alterations in MBP. However, upon glucose treatment, no FRET phenomenon was detected, thereby implying the specificity of interaction between MBP and maltose. The in vitro FRET results were also confirmed via the acceptor photobleaching method. Therefore, our data showed that maltose-stimulated conformational changes of MBP could be measured by FRET, thereby providing biological information, including the FRET efficiency and the intramolecular distance.     

Monitoring of conformational change in maltose binding protein using split green fluorescent protein

In this study, we describe a novel method for the detection of conformational changes in proteins, which is predicated on the reconstitution of split green fluorescent protein (GFP). We employed fluorescence complementation assays for the monitoring of the conformationally altered proteins. In particular, we used maltose binding protein (MBP) as a model protein, as MBP undergoes a characteristic hinge-twist movement upon substrate binding. The common feature of this approach is that GFP, as a reporter protein, splits into two non-fluorescent fragments, which are genetically fused to the N- and C-termini of MBP. Upon binding to maltose, the chromophores move closer together, resulting in the generation of fluorescence. This split GFP method also involves the reconstitution of GFP, which is determined via observations of the degree to which fluorescence intensity is restored. As a result, reconstituted GFP has been observed to generate fluorescence upon maltose binding in vitro, thereby allowing for the direct detection of changes in fluorescence intensity in response to maltose, in a concentration- and time-dependent fashion. Our findings showed that the fluorescence complementation assay can be used to monitor the conformational alterations of a target protein, and this ability may prove useful in a number of scientific and medical applications.     

Abeta produced as a fusion to maltose binding protein can be readily purified and stably associates with copper and zinc

The 42 amino acid Alzheimer's Abeta peptide has been produced in E. coli as a soluble fusion to maltose binding protein (MBP). Affinity purification on amylose columns of MBP-Abeta and MBP led to the recovery of proteins at purities that were suited for physicochemical analyses. MBP-Abeta was able to bind approximately 2 mole equivalents of copper or 4 mole equivalents of zinc, while MBP alone bound negligible amounts of zinc or copper. We conclude that Abeta can bind 2 copper or 4 zinc ions in its fusion format. Because MBP-Abeta is a convenient protein to work with, this system is well suited for further studies on the structure of Abeta and its interactions with metals.     

Phosphorylation of myelin basic protein by glycogen phosphorylase kinase

The ability of homogeneous glycogen phosphorylase kinase (Phk) from rabbit skeletal muscle to phosphorylate bovine brain myelin basic protein (MBP) was investigated. Phk could incorporate a maximum of 1.9 mol phosphate/mol MBP. The apparent Km and Vmax for Phk phosphorylation of MBP were 27 microM and 90 nmol/min per mg enzyme, respectively. Properties of MBP phosphorylation by Phk are similar to those of phosphorylase as a substrate. Only serine residues of MBP are phosphorylated by Phk. Phosphorylation sites of MBP by Phk are not identical to those by cAMP-dependent protein kinases.     

Effects of the osmolyte trimethylamine-N-oxide on conformation,self-association,and two-dimensional crystallization of myelin basic protein

The osmolyte trimethylamine-N-oxide (TMAO) is a naturally in vivo occurring "chemical chaperone" that has been shown to stabilise the folding of numerous proteins. Myelin basic protein (MBP) is a molecule that has not yet been suitably crystallized either in three dimensions for X-ray crystallography or in two dimensions for electron crystallography. Here, we describe lipid monolayer crystallization experiments of two species of recombinant murine MBP in the presence of TMAO. One protein was unmodified, whereas the other contained six Arg/Lys-->Gln substitutions to mimic the effects of deimination (i.e., the enzymatic modification of Arg to citrulline), which reduces the net positive charge. Planar arrays of both proteins were formed on binary lipid monolayers containing a nickel-chelating lipid and a phosphoinositide. In the presence of TMAO, the diffraction spots of these arrays became sharper and more distinct than in its absence, indicating some improvement of crystallinity. The osmolyte also induced the formation of epitaxial growth of protein arrays, especially with the mutant protein. However, none of these assemblies was sufficiently ordered to extract high-resolution structural information. Circular dichroic spectroscopy showed that MBP gained no increase in ordered secondary structure in the presence of TMAO in bulk solution, whereas it did in the presence of lipids. Dynamic light-scattering experiments confirmed that the MBP preparations were monomodal under the optimal crystallization conditions determined by electron microscopy trials. The salt and osmolyte concentrations used were shown to result in a largely unassociated population of MBP. The amino acid composition of MBP overwhelmingly favours a disordered state, and a neural-network-based scheme predicted large segments that would be unlikely to adopt a regular conformation. Thus, this protein has an inherently disordered nature, which mitigates strongly against its crystallization for high-resolution structure determination.     

Interaction of the 18.5-kD isoform of myelin basic protein with Ca2+ -calmodulin: effects of deimination assessed by intrinsic Trp fluorescence spectroscopy,dynamic light scattering,and circular dichroism

The effects of deimination (conversion of arginyl to citrullinyl residues) of myelin basic protein (MBP) on its binding to calmodulin (CaM) have been examined. Four species of MBP were investigated: unmodified recombinant murine MBP (rmMBP-Cit(0)), an engineered protein with six quasi-citrullinyl (i.e., glutaminyl) residues per molecule (rmMBP-qCit(6)), human component C1 (hMBP-Cit(0)), and human component C8 (hMBP-Cit(6)), both obtained from a patient with multiple sclerosis (MS). Both rmMBP-Cit(0) and hMBP-Cit(0) bound CaM in a Ca(2+)-dependent manner and primarily in a 1:1 stoichiometry, which was verified by dynamic light scattering. Circular dichroic spectroscopy was unable to detect any changes in secondary structure in MBP upon CaM-binding. Inherent Trp fluorescence spectroscopy and a single-site binding model were used to determine the dissociation constants: K(d) = 144 +/- 76 nM for rmMBP-Cit(0), and K(d) = 42 +/- 15 nM for hMBP-Cit(0). For rmMBP-qCit(6) and hMBP-Cit(6), the changes in fluorescence were suggestive of a two-site interaction, although the dissociation constants could not be accurately determined. These results can be explained by a local conformational change induced in MBP by deimination, exposing a second binding site with a weaker association with CaM, or by the existence of several conformers of deiminated MBP. Titration with the collisional quencher acrylamide, and steady-state and lifetime measurements of the fluorescence at 340 nm, showed both dynamic and static components to the quenching, and differences between the unmodified and deiminated proteins that were also consistent with a local conformational change due to deimination.     

Zinc Ions Stabilise the Association of Basic Protein with Brain Myelin Membranes

Myelin basic protein (MBP) dissociated from brain myelin membranes when they were incubated (37 degrees C; pH 7.4) at physiological ionic strength. Zinc ions inhibited, and calcium promoted, this process. Protease activity in the membrane preparations cleaved the dissociated MBP into both small (less than 4 kilodaltons) and large (greater than 8 kilodaltons) fragments. The latter were detected, together with intact MBP, by gel electrophoresis of incubation media. Zinc ions appeared to act in two distinct processes. In the presence or absence of added CaCl2, zinc ions in the range 0.1-1 mM inhibited MBP-membrane dissociation. This process was relatively insensitive to heat and Zn2+ could be substituted by either copper (II) or cobalt (II) ions. A second effect was evident only in the presence of added calcium ions, when lower concentrations of Zn2+ (less than 0.1 mM) inhibited MBP-membrane dissociation and the accumulation of intact MBP in incubation media. This process was heat sensitive and only copper (II), but not cobalt (II), ions could replace Zn2+. To determine whether endogenous zinc in myelin membranes is bound to MBP, preparations were solubilised in buffers containing Triton X-100/2 mM CaCl2 and subjected to gel filtration. Endogenous zinc, as indicated by a dithizone-binding method, eluted with fractions containing both MBP and proteolipid protein (PLP). Thus, one means whereby zinc stabilises association of MBP with brain myelin membranes may be by promoting its binding to PLP.     

Fluorescence labeling, purification, and immobilization of a double cysteine mutant calmodulin fusion protein for single-molecule experiments

We present a method of labeling and immobilizing a low-molecular-weight protein, calmodulin (CaM), by fusion to a larger protein, maltose binding protein (MBP), for single-molecule fluorescence experiments. Immobilization in an agarose gel matrix eliminates potential interactions of the protein and the fluorophore(s) with a glass surface and allows prolonged monitoring of protein dynamics. The small size of CaM hinders its immobilization in low-weight-percentage agarose gels; however, fusion of CaM to MBP via a flexible linker provides sufficient restriction of translational mobility in 1% agarose gels. Cysteine residues were engineered into MBP.CaM (MBP-T34C,T110C-CaM) and labeled with donor and acceptor fluorescent probes yielding a construct (MBP.CaM-DA) which can be used for single-molecule single-pair fluorescence resonance energy transfer (spFRET) experiments. Mass spectrometry was used to verify the mass of MBP.CaM-DA. Assays measuring the activity of CaM reveal minimal activity differences between wild-type CaM and MBP.CaM-DA. Single-molecule fluorescence images of the donor and acceptor dyes were fit to a two-dimensional Gaussian function to demonstrate colocalization of donor and acceptor dyes. FRET is demonstrated both in bulk fluorescence spectra and in fluorescence trajectories of single MBP.CaM-DA molecules. The extension of this method to other biomolecules is also proposed.     

Membrane proteins in reverse micelles: myelin basic protein in a membrane-mimetic environment

The solubility, reactivity, and conformational dynamics of myelin basic protein (MBP) from bovine brain were studied in reverse micelles of sodium bis(2-ethylhexyl) sulfosuccinate (AOT)-isooctane and water. Such a membrane-mimetic system resembles the aqueous spaces of native myelin sheath in terms of physicochemical properties as reflected in the high affinity of MBP for interfacial bound water. This is marked by the unusual profile of the solubility curve of the protein in reverse micelles, which shows optimal solubility at a much lower molar ratio of water to surfactant ([ H2O]/[AOT] = w0) than that reported for other water-soluble proteins. The role of counterions and/or charged polar head groups in the solubilization process is revealed by comparison of the solubility of MBP in nonionic surfactant micellar solutions. Whereas MBP is unfolded in aqueous solutions, insertion into reverse micelles generates a more folded structure, characterized by the presence of 20% alpha-helix. This conformation is unaffected by variations in the water content of the system (in the 2.0-22.4 w0 range). The reactivity of epsilon-amino groups of lysine residues with aqueous solutions of o-phthalaldehyde demonstrates that segments of the peptide chain are accessible to water. Similar results were obtained with the sequence involved in heme binding. In contrast, the sole tryptophan residue, Trp-117, is shielded from the aqueous solvent, as indicated by lack of reaction with N-bromosuccinimide. The invariance of the wavelength maximum emission in the fluorescence spectra as a function of w0 is consistent with this result.(ABSTRACT TRUNCATED AT 250 WORDS)