Mapping single cysteine mutants of light chain 2 in chicken skeletal myosin.

The NH2- and COOH-terminal regions of the regulatory light chain (LC2) have been mapped to the head/rod junction by immunoelectron microscopy, using monoclonal and anti-fluorescyl antibodies as probes. In order to map the entire length of the light chain, we have engineered and purified mutants that contain a single cysteine residue at positions 2, 73, 94, 126, or 155. The single cysteine residues were labeled with either 5-iodoacetamido fluorescein or N-ethylmaleimide-biotin. We observed that the reactivity of Cys126 is far greater than that of Cys155, confirming that cysteine 126 is the fast-reacting thiol in wild-type light chain. The labeled light chains were exchanged into myosin stripped of its native LC2 by immunoaffinity chromatography, and the reconstituted myosin was reacted with anti-fluorescein antibody or avidin prior to rotary shadowing for electron microscopy. The position of the antibody or avidin was found to be near the head/rod junction for all mutants. These mapping studies, together with our finding that cysteines widely separated in the primary sequence can form multiple disulfide bonds (Saraswat, L.D., and Lowey, S. (1991) J. Biol. Chem. 226, 19777-19785), support a model for LC2 as a flexible, globular molecule localized mainly in the vicinity of the head/rod junction of myosin.     

Mapping myosin light chains by immunoelectron microscopy. Use of anti- fluorescyl antibodies as structural probes

The two classes of light chains in vertebrate fast muscle myosin have been selectively labeled with the thiol specific reagent 5- (iodoacetamido) fluorescein to determine their location in the myosin head. The alkali light chains (A1 and A2) were labeled at a single cysteine residue near the COOH terminus, whereas the regulatory light chain (LC2) was reacted at either cysteine 125 or 154. The two cysteines of LC2 appear to be near each other in the tertiary structure as evidenced by the ease of formation of an intramolecular disulfide bond. Besides having favorable spectral properties, fluorescein is a potent haptenic immunogen for raising high affinity antibodies. When anti-fluorescyl antibodies were added to the fluorescein-labeled light chains, the fluorescence was quenched by greater than 90%, thereby providing a simple method for determining an association constant. The interaction with antibody was the same for light chains exchanged into myosin as for free light chains. Complexes of antibody bound to light chain could be visualized in the electron microscope by rotary shadowing with platinum. By this approach we have shown that the COOH- terminal regions of the two classes of light chains are widely separated in myosin: the cysteine residues of LC2 lie close to the head/rod junction, whereas the single cysteine of A1 or A2 is located approximately 90 A distal to the junction. These sites correspond to the positions of the NH2 termini of the light chains mapped in earlier studies (Winkelmann, D. A., and S. Lowey. 1986. J. Mol. Biol. 188:595- 612; Tokunaga, M., M. Suzuki, K. Saeki, and T. Wakabayashi. 1987b. J. Mol. Biol. 194:245-255). We conclude that the two classes of light chains do not lie in a simple colinear arrangement, but instead have a more complex organization in distinct regions of the myosin head.     

Reaction of cardiac myosin with a purine disulfide analog of adenosine triphosphate. II. Stoichiometry and subunit location of labeling.

The reaction of bovine cardiac myosin with the site-specific purine disulfide analog of ATP, 6,6'-dithiobis (inosinyl imidodiphosphate), was studied to determine the stoichiometry of labeling and subunit location of the reactive cysteines. The analog inactivates myosin by forming a mixed disulfide between the thiopurine nucleotide and certain key cysteines. The thiopurine nucleotide was displaced quantitatively by 14CN to form the more stable thiocyanato-enzyme derivatives. In cardiac myosin, the reactive cysteines could be categorized into three classes, nonessential, critical, and noncritical. The modification of the critical cysteines (two per myosin) inactivated the EDTA and Ca2+ ATPase activities, the latter to a lesser extent. The nonessential cysteines (two to three per myosin) and the noncritical cysteines (two per myosin), differentiated by their rates of reaction, had no effect on the ATPase activities after modification. Thiocyanato-modified myosin was analyzed by sodium dodecyl sulfate gel electrophoresis to determine the distribution of 14CN in the subunits. The critical cysteines were found on the 21,000-dalton light chain (LC1) and the noncritical cysteines on the heavy chains. More specifically, the critical cysteine modified in cardiac LC1 (determined from the products after cyclization and chain cleavage at the thiocyanatoalanyl residues) was shown to be the thiol residue whose surrounding amino acid sequence is homologous to that of the single cysteine of the skeletal myosin alkali light chains, confirming the likely similar structure and function of these light chains in the two different muscle types.     

Subunit location of sulfhydryl groups of myosin labeled with a purine disulfide analog of adenosine triphosphate.

A purine disulfide analog of ATP, 6,6'-dithiobis(inosinyl imidodiphosphate), forms mixed disulfides with cysteine residues at what are believed to be ATP regulatory sites of myosin. Blocking these sites causes inactivation of the ATPase activity at the active sites. Two cysteine residues per head are specifically modifed by this disulfide analog. The thiopurine nucleotides can be stoichiometrically displaced from myosin by [14-C]cyanide to give a more stable thiocyanato derivative of the enzyme. [14-C]Thiocyanatomyosin (3.7 14-CN/myosin) was dissociated in 4 M urea and the individual subunits were isolated. The heavy chains each had 0.78 14-CN bound per 200,000 molecular weight unit. The light chain with molecular weight of 20,700 had 1.00 14-CN bound and the 16,500 molecular weight light chain had 0.65 14-CN bound. The two 19,000 molecular weight light chains were not labeled. The two labeled light chains have only a single cysteine which is stoichiometrically modified. These two light chains show a high degree of homology and presumably perform identical functions in myosin. Their specific modification by the purine disulfide analog and their other known properties suggest that they contribute directly to the ATP regulatory sites and may, in fact, function as regulatory subunits.     

Locations of disulfide bonds and free cysteines in the heavy and light chains of recombinant human factor VIII (antihemophilic factor A).

The locations of disulfide bonds and free cysteines in the heavy and light chains of recombinant human factor VIII were determined by sequence analysis of fragments produced by chemical and enzymatic digestions. The A1 and A2 domains of the heavy chain and the A3 domain of the light chain contain one free cysteine and two disulfide bonds, whereas the C1 and C2 domains of the light chain have one disulfide bond and no free cysteine. The positions of these disulfide bonds are conserved in factor V and ceruloplasmin except that the second disulfide bond in the A3 domain is missing in both factor V and ceruloplasmin. The positions of the three free cysteines of factor VIII are the same as three of the four cysteines present in ceruloplasmin. However, the positions of the free cysteines in factor VIII and ceruloplasmin are not conserved in factor V.     

Charge-based analysis of antibodies with engineered cysteines

THIOMABs are antibodies with an engineered unpaired cysteine residue on each heavy chain that can be used as intermediates to generate antibody-drug conjugates. Multiple charge variant peaks were observed during cation-exchange chromatography (CEX) and imaged capillary isoelectric focusing (cIEF) analysis of several different THIOMABs. This charge heterogeneity was due to cysteinylation and/or glutathionylation at the engineered and unpaired cysteines through disulfide bonds formed during the cell culture process. Cysteine treatment followed by analysis using CEX, LC/MS and electrophoresis demonstrates that cysteine is a mild reductant that can remove glutathione and cysteine bound to the engineered cysteines without disrupting the inter- or intra-chain disulfide bonds of antibodies. We further demonstrated that using a cysteine/cystine redox pair (rather than cysteine alone) can not only effectively remove glutathione at the engineered cysteines, but also generate homogeneously cysteinylated species, which resulted in one main peak in both CEX-HPLC and imaged cIEF assays for antibodies with engineered and unpaired cysteines.     

Role of disulfide bond formation in the folding of human chorionic gonadotropin beta subunit into an alpha beta dimer assembly-competent form.

The malignant trophoblastic cell line JAR was used as a model system to study protein folding in intact cells. We have used this model previously to identify conformational intermediates in the production of an assembly-competent form of the human chorionic gonadotropin beta subunit (Ruddon, R. W., Krzesicki, R. F., Norton, S. E., Beebe, J. S., Peters, B. P., and Perini, F. (1987) J. Biol. Chem. 262, 12533-12540). The earliest biosynthetic precursor of the human chorionic gonadotropin beta subunit detectable in JAR cells pulse labeled for 2 min is p beta 1, a form that lacks half of the six intrachain disulfide bonds observed in the fully processed dimer form of beta and that does not combine with the alpha subunit. p beta 1 is rapidly (t1/2 approximately 4 min) converted into p beta 2, which has a full complement of intrachain disulfide bonds and does combine with the alpha subunit. In this study, we have identified the three late forming disulfide bonds involved in the transition of p beta 1 into the assembly-compete form, p beta 2. The last three disulfide bonds to form are those between cysteines 9 and 90, 23 and 72, and 93 and 100. These were identified in JAR cell lysates that had been pulse labeled with [35S]cysteine for 2 or 5 min followed by trapping of the cysteine thiols with iodoacetic acid before immunopurification of the beta subunit forms. Immunopurified p beta 1 was treated with trypsin under nonreducing conditions to liberate [35S]cysteine-containing peptides from the disulfide-linked beta core polypeptide. These tryptic peptides were then separated by high performance liquid chromatography and sequenced to determine the location of the carboxymethyl-[35S]cysteine residues. The three late forming disulfide bonds are most likely the ones involved in stabilizing the conformation of the beta subunit that is required for combination with alpha to form the biologically functional alpha beta heterodimer.     

Free thiols of platelet thrombospondin. Evidence for disulfide isomerization

The free thiols of platelet thrombospondin (TSP) were derivatized with labeled N-ethylmaleimide (NEM) or iodoacetamide (IAM). When Ca2+ was chelated with EDTA, 2.9 mol of NEM or 2.6 mol of IAM reacted/mol of native TSP. No additional thiols were found after denaturation with urea. Since TSP has three apparently identical polypeptide chains, this suggests one free thiol/polypeptide chain. Ca2+ protected all of the thiols from reaction with IAM. In Ca2+ about half the thiols reacted normally with NEM and the others were unreactive, indicating that the thiols of TSP are not identical. The number of reactive thiols as a function of [Ca2+] revealed a sigmoidal curve with a transition midpoint of 207 microM. The ability of analogs of NEM to compete for derivatization of the thiols with labeled NEM was greater with larger, more hydrophobic agents. Gel electrophoretic separation of labeled TSP that had been partially digested with thrombin and trypsin indicated that some of the label was in the C-terminal tryptic fragment but that most was in the adjacent trypsin-sensitive region. After cyanogen bromide cleavage of the labeled and reduced protein, four labeled fractions were obtained from a gel filtration column. With subsequent combinations of tryptic digestion and reversed-phase high performance liquid chromatography, labeled peptides were purified from these four fractions, and the amino acid sequences were determined. Twelve labeled cysteines were identified, each with a specific radioactivity less than that of the thiol labeling reagent, indicating that only a fraction of that cysteine in a population of TSP molecules was a free thiol at the time of derivatization. While 2 labeled cysteines are in the non-repeating C-terminal portion of the molecule, the other 10 labeled cysteines are in the adjacent trypsin-sensitive type 3 repeats proposed (Lawler, J., and Hynes, R. O. (1986) J. Cell. Biol. 103, 1635-1648) as the calcium-binding region of the molecule. The disulfide bonds most sensitive to reduction by dithioerythritol were also stabilized by Ca2+, implying location in the Ca2(+)-sensitive part of the molecule. It is proposed that one equivalent of free thiol/polypeptide chain is distributed among 12 different cysteine residues through an intramolecular thioldisulfide isomerization.     

Co-translational modification of nascent immunoglobulin heavy and light chains

We have investigated the in vivo co-translational covalent modification of nascent immunoglobulin heavy and light chains. Nascent polypeptides were separated from completed polypeptides by ion-exchange chromatography of solubilized ribosomes on QAE-Sephadex. First, we have demonstrated that MPC 11 nascent heavy chains are quantitatively glycosylated very soon after the asparaginyl acceptor site passes through the membrane into the cisterna of the rough endoplasmic reticulum. Nonglycosylated completed heavy chains of various classes cannot be glycosylated after release from the ribosome, due either to rapid intramolecular folding and/or intermolecular assembly, which cause the acceptor site to become unavailable for the glycosylation enzyme. Second, we have shown that the formation of the correct intrachain disulfide loop within the first light chain domain occurs rapidly and quantitatively as soon as the appropriate cysteine residues of the nascent light chain pass through the membrane into the cisterna of the endoplasmic reticulum. The intrachain disulfide loop in the second or constant region domain of the light chain is not formed on nascent chains, because one of the cysteine residues involved in this disulfide bond does not pass through the endoplasmic reticulum membrane prior to chain completion and release from the ribosome. Third, we have demonstrated that some of the initial covalent assembly (formation of interchain disulfide bonds) occurs on nascent heavy chains prior to their release from the ribosome. The results are consistent with the pathway of covalent assembly of the cell line, in that completed light chains are assembled onto nascent heavy chains in MPC 11 cells (IgG₂b), where a heavy-light half molecule is the major initial covalent intermediate; and completed heavy chains are assembled onto nascent heavy chains in MOPC 21 cells (IgG₁), where a heavy chain dimer is the major initial disulfide linked intermediate.     

Functional regions in the essential light chain of smooth muscle myosin as revealed by the mutagenesis approach.

The endogenous essential light chain (LC17) of myosin from intestine smooth muscle was replaced with mutated essential light chains prepared using recombinant techniques. Complete exchange was observed with histidine-tagged derivatives of LC17a, LC17b and E122A-LC17a (LC17a and LC17b are the usual constituants of smooth muscle myosin), with small changes in the ATPase activity of reconstituted myosins. Much less exchange was observed with the light-chain derivative lacking the last 12 amino acid residues, demonstrating the importance of this segment, which may act as one arm of a pair of pincers to bind the myosin heavy chain.     

Studies on the subunits of myosin from muscle layer of Ascaris lumbricoides suum.

1. A purified preparation of Ascaris myosin was obtained from the muscle layer of Ascaris lumbricoides suum, using gel filtration and ion-exchange chromatography. 2. Ascaris myosin whether purified or unpurified, had almost the same ability for ATP-splitting and superprecipitation. 3. Ascaris myosin and rabbit skeletal myosin were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A significant difference in the number of light chains between both myosins was found. Ascaris myosin was found to have one heavy chain and two distinct light chain components (LC1-A and LC2-A), having molecular weights of 18000 and 16000, respectively. These light chains correspond in molecular weight to the light chain 2 (LC2-S) and light chain 3 (LC3-S) in rabbit skeletal myosin. 4. LC1-A could be liberated from the Ascaris myosin molecule reacted with 5,5'-dithio-bis(2-nirobenzoic acid( Nbs2) with recovery of ATPase activity by addition of dithiothreitol. These properties are equivalent to those of the LC2-S in rabbit skeletal myosin, although Ascaris myosin when treated with Nbs2-urea lost its ATPase activity.     

Detection and quantification of free sulfhydryls in monoclonal antibodies using maleimide labeling and mass spectrometry

The detection of free sulfhydryls in proteins can reveal incomplete disulfide bond formation, indicate cysteine residues available for conjugation, and offer insights into protein stability and structure. Traditional spectroscopic methods of free sulfhydryl detection, such as Ellman’s reagent, generally require a relatively large amount of sample, preventing their use for the analysis of biotherapeutics early in the development cycle. These spectroscopic methods also cannot accurately determine the location of the free sulfhydryl, further limiting their utility. Mass spectrometry was used to detect free sulfhydryl residues in intact proteins after labeling with Maleimide-PEG₂-Biotin. As little as 2% cysteine residues with free sulfhydryls (0.02 mol SH per mol protein) could be detected by this method. Following reduction, the free sulfhydryl abundance on antibody heavy and light chains could be measured. To determine free sulfhydryl location at peptide-level resolution, free sulfhydryls and cysteines involved in disulfide bonds were differentially labeled with N-ethylmaleimide and d₅-N-ethylmaleimide, respectively. Following enzymatic digestion and nanoLC-MS, the abundance of free sulfhydryls at individual cysteine residues was quantified down to 2%. The method was optimized to avoid non-specific labeling, disulfide bond scrambling, and maleimide exchange and hydrolysis. This new workflow for free sulfhydryl analysis was used to measure the abundance and location of free sulfhydryls in 3 commercially available monoclonal antibody standards (NIST Monoclonal Antibody Reference Material (NIST), SILu?Lite SigmaMAb Universal Antibody Standard (Sigma-Aldrich) and Intact mAb Mass Check Standard (Waters)) and 1 small protein standard (β-Lactoglobulin A).     

Polymorphism of myosin light chains. An electrophoretic and immunological study of rabbit skeletal-muscle myosins.

Antibodies specific for rabbit fast-twitch-muscle myosin LCIF light chain were purified by affinity chromatography and characterized by both non-competitive and competitive enzyme-linked immunosorbent assay (ELISA) and a gel-electrophoresis-derived assay (GEDELISA). The antibodies did not cross-react with myosin heavy chains, and were weakly cross-reactive with the LC2F [5,5'-dithio-(2-nitrobenzoic acid)-dissociated] light chain and with all classes of dissociated light chains (LC1Sa, LC1Sb and LC2S), as well as with the whole myosin, from hind-limb slow-twitch muscle. The immunoreactivity of myosins with a truly mixed light-chain pattern (e.g. vastus lateralis and gastrocnemius) correlated with percentage content of fast-twitch-muscle-type light chains. A more extensive immunoreactivity was observed with diaphragm and masseter myosins, which were also characterized, respectively, by a relative or absolute deficiency of LC1Sa light chain. Furthermore, it was found that the LC1Sb light chain of masseter myosin is antigenically different from its slow-twitch-muscle myosin analogue, and is immunologically related to the LC1F light chain. Rabbit masseter muscle from its metabolic and physiological properties and the content, activity and immunological properties of sarcoplasmic-reticulum adenosine triphosphatase, is classified as a red, predominantly fast-twitch, muscle. Therefore our results suggest that the two antigenically different iso-forms of LC1Sb light chain are associated with the myosins of fast-twitch red and slow-twitch red fibres respectively.     

Formation of intermolecular disulfide bonds on nascent immunoglobulin polypeptides.

The initial step of intermolecular covalent assembly of immunoglobulins molecules involves formation of heavy chain-light chain or heavy chain-heavy chain disulfide bonds. Using QAE-Sephadex chromatography to isolate microsomal nascent polypeptides, we have shown that this initial step of intermolecular covalent assembly occurs, to a substantial extent, on nascent heavy chains, as well as on completed heavy chains as previously demonstrated by others. In MPC 11 mouse myeloma cells, completed light chains are assembled covalently to nascent heavy chains, whereas in MOPC 21 mouse myeloma cells, completed heavy chains are assembled covalently to nascent heavy chains. These results are consisted with the heavy-light half-molecule being the major initial intermediate in the assembly of MPC 11 IgG2b and heavy-heavy dimer being the major initial intermediate formed in assembly of MOPC 21 IgG1. The nascent MPC 11 heavy chain must be at least 38,000 daltons in size before assembly with the light chain occurs, even though the heavy chain cysteine involved in this disulfide bond is 131 residues (approximately 15,000 daltons) from the NH2 terminus. In addition, pulse-chase labeling studies of MPC 11 cells have shown that the assembly of completed light chains with the nascent heavy chain must occur within a few minutes of the synthesis of the light chain even though a large excess of unassembled MPC 11 light chains remain inside the cell for an average time of 2 h before being secreted.     

Assignment of interchain disulfide bonds in platelet-derived growth factor (PDGF) and evidence for agonist activity of monomeric PDGF.

Platelet-derived growth factor (PDGF) is a dimeric factor stabilized by disulfide bonds. Using an approach involving partial reduction of PDGF, we have identified the 2nd and 4th cysteine residues in the PDGF chains as the cysteine residues forming interchain disulfide bonds. Analysis of PDGF mutants in which the 2nd and 4th cysteine residues were mutated to serine residues revealed that the disulfide bonds are arranged in a cross-wise manner, with the 2nd cysteine residue in one chain being linked to the 4th cysteine residue in the other. A PDGF B-chain mutant, in which both the 2nd and 4th cysteine residues were substituted with serine residues, migrated as a monomer in sodium dodecyl sulfate gel electrophoresis and retained receptor binding activity. When analyzed in receptor dimerization and autophosphorylation assays, this mutant showed agonistic activity. Thus, structural information has been obtained that will allow the large scale production of properly folded monomeric PDGF, as well as design of specific PDGF heterodimers.     

The susceptibility of disulfide bonds to modification in keratin fibers undergoing tensile stress

Cysteine residues perform a dual role in mammalian hairs. The majority help stabilize the overall assembly of keratins and their associated proteins, but a proportion of inter-molecular disulfide bonds are assumed to be associated with hair mechanical flexibility. Hair cortical microstructure is hierarchical, with a complex macro-molecular organization resulting in arrays of intermediate filaments at a scale of micrometres. Intermolecular disulfide bonds occur within filaments and between them and the surrounding matrix. Wool fibers provide a good model for studying various contributions of differently situated disulfide bonds to fiber mechanics. Within this context, it is not known if all intermolecular disulfide bonds contribute equally, and, if not, then do the disproportionally involved cysteine residues occur at common locations on proteins? In this study, fibers from Romney sheep were subjected to stretching or to their breaking point under wet or dry conditions to detect, through labeling, disulfide bonds that were broken more often than randomly. We found that some cysteines were labeled more often than randomly and that these vary with fiber water content (water disrupts protein-protein hydrogen bonds). Many of the identified cysteine residues were located close to the terminal ends of keratins (head or tail domains) and keratin-associated proteins. Some cysteines in the head and tail domains of type II keratin K85 were labeled in all experimental conditions. When inter-protein hydrogen bonds were disrupted under wet conditions, disulfide labeling occurred in the head domains of type II keratins, likely affecting keratin-keratin-associated protein interactions, and tail domains of the type I keratins, likely affecting keratin-keratin interactions. In contrast, in dry fibers (containing more protein-protein hydrogen bonding), disulfide labeling was also observed in the central domains of affected keratins. This central “rod” region is associated with keratin-keratin interactions between anti-parallel heterodimers in the tetramer of the intermediate filament.     

Identification of in vivo disulfide conformation of TRPA1 ion channel

TRPA1 (transient receptor potential ankyrin 1) is an ion channel expressed in the termini of sensory neurons and is activated in response to a broad array of noxious exogenous and endogenous thiol-reactive compounds, making it a crucial player in chemical nociception. A number of conserved cysteine residues on the N-terminal domain of the channel have been identified as critical for sensing these electrophilic pungent chemicals, and our recent EM structure with modeled domains predicts that these cysteines form a ligand-binding pocket, allowing for the possibility of disulfide bonding between the cysteine residues. Here, we present a comprehensive mass spectrometry investigation of the in vivo disulfide bonding conformation and in vitro reactivity of 30 of the 31 cysteine residues in the TRPA1 ion channel. Four disulfide bonds were detected in the in vivo TRPA1 structure: Cys-666-Cys-622, Cys-666-Cys-463, Cys-622-Cys-609, and Cys-666-Cys-193. All of the cysteines detected were reactive to N-methylmaleimide (NMM) in vitro, with varying degrees of labeling efficiency. Comparison of the ratio of the labeling efficiency at 300 μM versus 2 mM NMM identified a number of cysteine residues that were outliers from the mean labeling ratio, suggesting that protein conformation changes rendered these cysteines either more or less protected from labeling at the higher NMM concentrations. These results indicate that the activation mechanism of TRPA1 may involve N-terminal conformation changes and disulfide bonding between critical cysteine residues.     

Analysis of myosin light and heavy chain types in single human skeletal muscle fibers

In this study, myosin types in human skeletal muscle fibers were investigated with electrophoretic techniques. Single fibers were dissected out of lyophilized surgical biopsies and typed by staining for myofibrillar ATPase after preincubation in acid or alkaline buffers. After 14C-labelling of the fiber proteins in vitro by reductive methylation, the myosin light chain pattern was analysed on two-dimensional gels and the myosin heavy chains were investigated by one-dimensional peptide mapping. Surprisingly, human type I fibers, which contained only the slow heavy chain, were found to contain variable amounts of fast myosin light chains in addition to the two slow light chains LC1s and LC2s. The majority of the type I fibers in normal human muscle showed the pattern LC1s, LC2s and LC1f. Further evidence for the existence in human muscle of a hybrid myosin composed of a slow heavy chain with fast and slow light chains comes from the analysis of purified human myosin in the native state by pyrophosphate gel electrophoresis. With this method, a single band corresponding to slow myosin was obtained; this slow myosin had the light chain composition LC1s, LC2s and LC1f. Type IIA and IIB fibers, on the other hand, revealed identical light chain patterns consisting of only the fast light chains LC1f, LC2f and LC3f but were found to have different myosin havy chains. On the basis of the results presented, we suggest that the histochemical ATPase normally used for fibre typing is determined by the myosin heavy chain type (and not by the light chains). Thus, in normal human muscle a number of 'hybrid' myosins were found to occur, namely two extreme forms of fast myosins which have the same light chains but different heavy chains (IIA and IIB) and a continuum of slow forms consisting of the same heavy chain and slow light chains with a variable fast light chain composition. This is consistent with the different physiological roles these fibers are thought to have in muscle contraction.     

Recombinant production and solution structure of lipid transfer protein from lentil Lens culinaris

Lipid transfer protein, designated as Lc-LTP2, was isolated from seeds of the lentil Lens culinaris. The protein has molecular mass 9282.7 Da, consists of 93 amino acid residues including 8 cysteines forming 4 disulfide bonds. Lc-LTP2 and its stable isotope labeled analogues were overexpressed in Escherichia coli and purified. Antimicrobial activity of the recombinant protein was examined, and its spatial structure was studied by NMR spectroscopy. The polypeptide chain of Lc-LTP2 forms four α-helices (Cys4-Leu18, Pro26-Ala37, Thr42-Ala56, Thr64-Lys73) and a long C-terminal tail without regular secondary structure. Side chains of the hydrophobic residues form a relatively large internal tunnel-like lipid-binding cavity (van der Waals volume comes up to ∼600 Å³). The side-chains of Arg45, Pro79, and Tyr80 are located near an assumed mouth of the cavity. Titration with dimyristoyl phosphatidylglycerol (DMPG) revealed formation of the Lc-LTP2/lipid non-covalent complex accompanied by rearrangements in the protein spatial structure and expansion of the internal cavity. The resultant Lc-LTP2/DMPG complex demonstrates limited lifetime and dissociates within tens of hours.     

Disulfide bond assignment in human J chain and its covalent pairing with immunoglobulin M.

The assignment of disulfide bonds in human J chain and its covalent pairing with immunoglobulin M was determined under conditions which minimize disulfide bond interchange. We show that in J chain the three intradisulfide bridges are formed between Cys 12 and 100, Cys 71 and 91, and Cys 108 and 133. Previous reports [reviewed by Koshland, M. E. (1985) Annu. Rev. Immunol. 3, 425-453] have proposed that cysteines 12, 14, or 68 were linked to the penultimate cysteine 575 of two mu chain tails. In this work, we demonstrate that cysteines 14 and 68 are disulfide-bridged to mu chains. A revised, albeit putative, model of J chain folding is presented which takes into account the correct disulfide pairing and the predictive secondary structure assignment.