Computational and biochemical docking of the irreversible cocaine analog RTI 82 directly demonstrates ligand positioning in the dopamine transporter central substrate-binding site.

The dopamine transporter (DAT) functions as a key regulator of dopaminergic neurotransmission via re-uptake of synaptic dopamine (DA). Cocaine binding to DAT blocks this activity and elevates extracellular DA, leading to psychomotor stimulation and addiction, but the mechanisms by which cocaine interacts with DAT and inhibits transport remain incompletely understood. Here, we addressed these questions using computational and biochemical methodologies to localize the binding and adduction sites of the photoactivatable irreversible cocaine analog 3ß-(p-chlorophenyl)tropane-2ß-carboxylic acid, 4'-azido-3'-iodophenylethyl ester ([(125)I]RTI 82). Comparative modeling and small molecule docking indicated that the tropane pharmacophore of RTI 82 was positioned in the central DA active site with an orientation that juxtaposed the aryliodoazide group for cross-linking to rat DAT Phe-319. This prediction was verified by focused methionine substitution of residues flanking this site followed by cyanogen bromide mapping of the [(125)I]RTI 82-labeled mutants and by the substituted cysteine accessibility method protection analyses. These findings provide positive functional evidence linking tropane pharmacophore interaction with the core substrate-binding site and support a competitive mechanism for transport inhibition. This synergistic application of computational and biochemical methodologies overcomes many uncertainties inherent in other approaches and furnishes a schematic framework for elucidating the ligand-protein interactions of other classes of DA transport inhibitors.

Hypothiocyanous acid oxidation of tubulin cysteines inhibits microtubule polymerization.

Thiol oxidation is a probable outcome of cellular oxidative stress and is linked to degenerative disease progression. In addition, protein thiol redox reactions are increasingly identified as a mechanism to regulate protein structure and function. We assessed the effect of hypothiocyanous acid on the cytoskeletal protein tubulin. Total cysteine oxidation by hypothiocyanous and hypochlorous acids was monitored by labeling tubulin with 5-iodoacetamidofluorescein and by detecting higher molecular weight inter-chain tubulin disulfides by Western blot under nonreducing conditions. Hypothiocyanous acid induced nearly stoichiometric oxidation of tubulin cysteines (1.9 mol cysteine/mol oxidant) and no methionine oxidation was observed. Because disulfide reducing agents restored all the polymerization activity that was lost due to oxidant treatment, we conclude that cysteine oxidation of tubulin inhibits microtubule polymerization. Hypothiocyanous acid oxidation of tubulin cysteines was markedly decreased in the presence of 4% glycerol, a component of the tubulin purification buffer. Due to its instability and buffer- and pH-dependent reactivity, hypothiocyanous acid studies require careful consideration of reaction conditions.

Mapping the eosinophil cationic protein antimicrobial activity by chemical and enzymatic cleavage.

The eosinophil cationic protein (ECP) is a human antimicrobial protein involved in the host immune defense that belongs to the pancreatic RNase A family. ECP displays a wide range of antipathogen activities. The protein is highly cationic and its bactericidal activity is dependant on both cationic and hydrophobic surface exposed residues. Previous studies on ECP by site-directed mutagenesis indicated that the RNase activity is not essential for its bactericidal activity. To further understand the ECP bactericidal mechanism, we have applied enzymatic and chemical limited cleavage to search for active sequence determinants. Following a search for potential peptidases we selected the Lys-endoproteinase, which cleaves the ECP polypeptide at the carboxyl side of its unique Lys residue, releasing the N-terminal fragment (0-38). Chemical digestion using cyanogen bromide released several complementary peptides at the protein N-terminus. Interestingly, ECP treatment with cyanogen bromide represents a new example of selective chemical cleavage at the carboxyl side of not only Met but also Trp residues. Recombinant ECP was denatured and carboxyamidomethylated prior to enzymatic and chemical cleavage. Irreversible denaturation abolishes the protein bactericidal activity. The characterization of the digestion products by both enzymatic and chemical approaches identifies a region at the protein N-terminus, from residues 11 to 35, that retains the bactericidal activity. The most active fragment, ECP(0-38), is further compared to ECP derived synthetic peptides. The region includes previously identified stretches related to lipopolysaccharide binding and bacteria agglutination. The results contribute to define the shortest ECP minimized version that would retain its antimicrobial properties. The data suggest that the antimicrobial RNase can provide a scaffold for the selective release of cytotoxic peptides.

Recombinant self-assembling peptides as biomaterials for tissue engineering.

Synthetic nanostructures based on self-assembling systems that aim to mimic natural extracellular matrix are now being used as substrates in tissue engineering applications. Peptides are excellent starting materials for the self-assembly process as they can be readily synthesised both chemically and biologically. P11-4 is an 11 amino acid peptide that undergoes triggered self-assembly to form a self-supporting hydrogel. It exists as unimers of random coil conformations in water above pH 7.5 but at low pH adopts an antiparallel ß-sheet conformation. It also self-assembles under physiological conditions in a concentration-dependent manner. Here we describe an unimer P11-4 production system and the use of a simple site-directed mutagenesis approach to generate a series of other P11-family peptide expression vectors. We have developed an efficient purification strategy for these peptide biomaterials using a simple procedure involving chemical cleavage with cyanogen bromide then repeated filtration, lyophilisation and wash steps. We report peptide-fusion protein yields of ca. 4.64 g/L and we believe the highest reported recovery of a recombinant self-assembling peptide at 203 mg/L of pure recombinant P11-4. This peptide forms a self-supporting hydrogel under physiological conditions with essentially identical physico-chemical properties to the chemically synthesised peptide. Critically it also displays excellent cytocompatibility when tested with primary human dermal fibroblasts. This study demonstrates that high levels of a series of recombinant self-assembling peptides can be purified using a simple process for applications as scaffolds in tissue engineering.

Eukaryote-specific motif of ribosomal protein S15 neighbors A site codon during elongation and termination of translation.

The eukaryotic ribosomal protein S15 is a key component of the decoding site in contrast to its prokaryotic counterpart, S19p, which is located away from the mRNA binding track on the ribosome. Here, we determined the oligopeptide of S15 neighboring the A site mRNA codon on the human 80S ribosome with the use of mRNA analogues bearing perfluorophenyl azide-modified nucleotides in the sense or stop codon targeted to the 80S ribosomal A site. The protein was cross-linked to mRNA analogues in specific ribosomal complexes that were obtained in the presence of eRF1 in the experiments with mRNAs bearing stop codon. Digestion of modified S15 with various specific proteolytic agents followed by identification of the resulting modified oligopeptides showed that cross-link was in C-terminal fragment in positions 131-145, most probably, in decapeptide 131-PGIGATHSSR-140. The position of cross-linking site on the S15 protein did not depend on the nature of the A site-bound codon (sense or stop codon) and on the presence of polypeptide chain release factor eRF1 in the ribosomal complexes with mRNA analogues bearing a stop codon. The results indicate an involvement of the mentioned decapeptide in the formation of the ribosomal decoding site during elongation and termination of translation. Alignment of amino acid sequences of eukaryotic S15 and its prokaryotic counterpart, S19p from eubacteria and archaea, revealed that decapeptide PGIGATHSSR in positions 131-140 is strongly conserved in eukaryotes and has minor variations in archaea but has no homology with any sequence in C-terminal part of eubacterial S19p, which suggests involvement of the decapeptide in the translation process in a eukaryote-specific manner.

Probing the steroid binding domain-like I (SBDLI) of the sigma-1 receptor binding site using N-substituted photoaffinity labels.

Radioiodinated photoactivatable photoprobes can provide valuable insights regarding protein structure. Previous work in our laboratory showed that the cocaine derivative and photoprobe 3-[ (125)I]iodo-4-azidococaine ([ (125)I]IACoc) binds to the sigma-1 receptor with 2-3 orders of magnitude higher affinity than cocaine [Kahoun, J. R. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 1393-1397]. Using this photoprobe, we demonstrated the insertion site for [ (125)I]IACoc to be Asp188 [Chen, Y. (2007) Biochemistry 46, 3532-3542], which resides in the proposed steroid binding domain-like II (SBDLII) region (amino acids 176-194) [Pal, A. (2007) Mol. Pharmacol. 72, 921-933]. An additional photoprobe based on the sigma-1 receptor ligand fenpropimorph, 1- N-(2-3-[ (125)I]iodophenyl)propane ([ (125)I]IAF), was found to label a peptide in both the SBDLII and steroid binding domain-like I (SBDLI) (amino acids 91-109) [Pal, A. (2007) Mol. Pharmacol. 72, 921-933]. In this report, we describe two novel strategically positioned carrier-free, radioiodinated photoaffinity labels specifically designed to probe the putative "nitrogen interacting region" of sigma-1 receptor ligands. These two novel photoprobes are (-)-methyl 3-(benzoyloxy)-8-2-(4-azido-3-[ (125)I]iodobenzene)-1-ethyl-8-azabicyclo[3.2.1]octane-2-carboxylate ([ (125)I]-N-IACoc) and N-propyl- N-(4-azido-3-iodophenylethyl)-3-(4-fluorophenyl)propylamine ([ (125)I]IAC44). In addition to reporting their binding affinities to the sigma-1 and sigma-2 receptors, we show that both photoaffinity labels specifically and covalently derivatized the pure guinea pig sigma-1 receptor (26.1 kDa) [Ramachandran, S. (2007) Protein Expression Purif. 51, 283-292]. Cleavage of the photolabeled sigma-1 receptor using Endo Lys C and cyanogen bromide (CNBr) revealed that the [ (125)I]-N-IACoc label was located primarily in the N-terminus and SBDLI-containing peptides of the sigma-1 receptor, while [ (125)I]IAC44 was found in peptide fragments consistent with labeling of both SBDLI and SBDLII.

The role of the S-S bridge in retroviral protease function and virion maturation.

Retroviral proteases are translated as a part of Gag-related polyproteins, and are released and activated during particle release. Mason-Pfizer monkey virus (M-PMV) Gag polyproteins assemble into immature capsids within the cytoplasm of the host cells; however, their processing occurs only after transport to the plasma membrane and subsequent release. Thus, the activity of M-PMV protease is expected to be highly regulated during the replication cycle. It has been proposed that reversible oxidation of protease cysteine residues might be responsible for such regulation. We show that cysteine residues in M-PMV protease can form an intramolecular S-S bridge. The disulfide bridge shifts the monomer/dimer equilibrium in favor of the dimer, and increases the proteolytic activity significantly. To investigate the role of this disulfide bridge in virus maturation and replication, we engineered an M-PMV clone in which both protease cysteine residues were replaced by alanine (M-PMV(PRC7A/C106A)). Surprisingly, the cysteine residues were dispensable for Gag polyprotein processing within the virus, indicating that even low levels of protease activity are sufficient for polyprotein processing during maturation. However, the long-term infectivity of M-PMV(PRC7A/C106A) was noticeably compromised. These results show clearly that the proposed redox mechanism does not rely solely on the formation of the stabilizing S-S bridge in the protease. Thus, in addition to the protease disulfide bridge, reversible oxidation of cysteine and/or methionine residues in other domains of the Gag polyprotein or in related cellular proteins must be involved in the regulation of maturation.

Nucleotide-binding sites in the voltage-dependent anion channel: characterization and localization.

In this study, we addressed the presence and location of nucleotide-binding sites in the voltage-dependent anion channel (VDAC). VDAC bound to reactive red 120-agarose, from which it was eluted by ATP, less effectively by ADP and AMP, but not by NADH. The photoreactive ATP analog, benzoyl-benzoyl-ATP (BzATP), was used to identify and characterize the ATP-binding sites in VDAC. [alpha-(32)P]BzATP bound to purified VDAC at two or more binding sites with apparent high and low binding affinities. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) analysis of BzATP-labeled VDAC confirmed the binding of at least two BzATP molecules to VDAC. The VDAC BzATP-binding sites showed higher specificity for purine than for pyrimidine nucleotides and higher affinity for negatively charged nucleotide species. VDAC treatment with the lysyl residue modifying reagent, fluorescein 5'-isothiocyanate, markedly inhibited VDAC labeling with BzATP. The VDAC nucleotide-binding sites were localized using chemical and enzymatic cleavage. Digestion of [alpha-(32)P]BzATP-labeled VDAC with CNBr or V8 protease resulted in the appearance of approximately 17- and approximately 14-kDa labeled fragments. Further digestion, high performance liquid chromatography separation, and sequencing of the selected V8 peptides suggested that the labeled fragments originated from two different regions of the VDAC molecule. MALDI-TOF analysis of BzATP-labeled, tryptic VDAC fragments indicated and localized three nucleotide binding sites, two of which were at the N and C termini of VDAC. Thus, the presence of two or more nucleotide-binding sites in VDAC is suggested, and their possible function in the control of VDAC activity, and, thereby, of outer mitochondrial membrane permeability is discussed.

Antibodies against the CB10 fragment of type II collagen in rheumatoid arthritis.

Antibodies against intact type II collagen (CII) are a feature of rheumatoid arthritis (RA) but have limited diagnostic value. Here we assess whether either of the two major cyanogen bromide fragments of CII, namely CB10 or CB11, are more sensitive substrates for the detection of antibodies in RA. Cleavage of bovine CII with cyanogen bromide yielded CB10 and CB11; these were purified by column chromatography for use in an enzyme-linked immunosorbent assay. Serum antibodies were measured in patients with RA, psoriatic arthritis (PsA), osteoarthritis (OA) and blood donors. Results were compared with those using intact CII. Antibodies against CB10 were found in as many as 88% of 96 patients with long-standing RA, but only 12% of 33 patients with PsA, 6% of 34 patients with OA and 3% of 93 control sera. Lower frequencies for these diseases were obtained on testing for antibodies against CB11: 50%, 6%, 21% and 2%, respectively. The sensitivity of detection in RA of antibodies against CB10 compared with antibodies against intact CII (88% versus 24%) was not at the expense of specificity, which remained high at 94%. The much higher frequency of antibodies against CB10 in RA than in other rheumatic diseases or control sera indicates that CB10 is clearly a more sensitive substrate than the intact collagen molecule and, combined with other assays (rheumatoid factor, anti-cyclic citrullinated peptide [anti-CCP]), might comprise a panel with a highly reliable predictive value. Moreover, our findings should encourage renewed interest in the role of collagen autoimmunity in the pathogenesis of RA.

Isolation of N-terminal protein sequence tags from cyanogen bromide cleaved proteins as a novel approach to investigate hydrophobic proteins.

A novel method for the isolation of protein sequence tags to identify proteins in a complex mixture of hydrophobic proteins is described. The PST (Protein Sequence Tag) technology deals with the isolation and MS/MS based identification of one N-terminal peptide from each polypeptide fragment generated by cyanogen bromide cleavage of a mixture of proteins. PST sampling takes place after sub-cellular fractionation of a complex protein mixture to give enrichment of mitochondrial proteins. The method presented here combines effective sample preparation with a novel peptide isolation protocol involving chemical and enzymatic cleavage of proteins coupled to chemical labeling and selective capture procedures. The overall process has been very successful for the analysis of complex mixtures of hydrophobic proteins, particularly membrane proteins. This method substantially reduces the complexity of a protein digest by "sampling" the peptides present in the digest. The sampled digest is amenable to analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS). Methods of "sampling" protein digests have great value' if they can provide sufficient information to identify substantially all of the proteins in the sample while reducing the complexity of the sample to maximize the efficient usage of LC-MS/MS capacity. The validity of the process is demonstrated for mitochondrial samples from S. cerevisiae. The proteins identified by the PST technology are compared to the proteins identified by the conventional technology 2-D gel electrophoresis as a control.

Transition-metal complexes as enzyme-like reagents for protein cleavage: complex cis-[Pt(en)(H2O)2]2+ as a new methionine-specific protease.

Complex cis-[Pt(en)(H(2)O)(2)](2+) promotes selective hydrolytic cleavage of two proteins, horse cytochrome c and bovine beta-casein. The cleavage is completed in 24 h under relatively mild conditions, at about pH 2.5, and a temperature as low as 40 degrees C. The results of HPLC and TSDS PAGE separations, MALDI mass spectrometry, and Edman sequencing showed that cleavage occurred exclusively at the peptide bond involving the C-terminus of each methionine residue, both such residues in cytochrome c and all six such residues in beta-casein. While having the same selectivity as cyanogen bromide (CNBr), the most common chemical protease, cis-[Pt(en)(H(2)O)(2)](2+) has several advantages. It is nonvolatile, easy to handle, and recyclable. Its cleavage is residue-selective, the rest of the polypeptide backbone remains intact, and the other side chains remain unmodified. It is applied in approximately equimolar amounts with respect to methionine residues, creates free amino and carboxylic groups, and cleaves even the Met-Pro bond, which is resistant to CNBr and most proteolytic enzymes. Finally the complex also works in the presence of the denaturing reagent sodium dodecyl sulfate. Experiments with the synthetic peptides, AcAla-Lys-Tyr-Gly-Gly-Met-Ala-Ala-Arg-Ala (termed Met-peptide) and AcVal-Lys-Gly-Gly-His-Ala-Lys-Tyr-Gly-Gly-Met-Ala-Ala-Arg-Ala (termed HisMet-peptide) as substrates, revealed structural and mechanistic features of the proteolytic reactions. We explain why two similar complexes with similar metal ions, cis-[Pt(en)(H(2)O)(2)](2+) and cis-[Pd(en)(H(2)O)(2)](2+), differ in selectivity as proteolytic reagents. The selectivity of cleavage is governed by the selectivity of the cis-[Pt(en)(H(2)O)(2)](2+) binding to the methionine side chain. The proteolytic activity is governed by the modes of coordination, which control the approach of the anchored Pt(II) ion to the scissile peptide bond. The cleavage occurs with a small, but significant, catalytic turnover of more than 18 after 7 days. The ability of cis-[Pt(en)(H(2)O)(2)](2+) to cleave proteins at relatively few sites, with explicable selectivity and catalytic turnover, bodes well for its use in biochemical practice.

Evidence of oxidative stress in familial amyloidotic polyneuropathy type 1.

OBJECTIVE: To evaluate the oxidative state in patients with familial amyloidotic polyneuropathy type 1 (FAP1). DESIGN: From 3 unrelated families, patients with FAP1 carrying a transthyretin Met-30 mutation were studied. The diagnosis was confirmed by genetic analysis. Eleven of 21 patients carried the mutation; all were symptomatic and were clinically assessed using a clinical score. All of the patients were evaluated for copper-zinc superoxide dismutase type 1 activity in red blood cells using spectrophotometry. Plasma total reactive antioxidant potential was studied using a chemiluminescent method. The results were compared with those obtained from an age-matched control group. SETTING: A public and academic multidisciplinary research clinic. RESULTS: Six of the 11 FAP1-positive patients disclosed superoxide dismutase type 1 activity values greater than 55 U/mg of protein (upper control limit), whereas 9 of 10 patients in whom total reactive antioxidant potential was measured had values below the lower limit of the control group. No relationship was found between the levels of superoxide dismutase type 1 activity and the severity of the clinical involvement. CONCLUSIONS: Oxidative stress may be part of the mechanisms leading to tissue damage in patients with FAP1. The lack of correlation between the laboratory findings and the severity of clinical involvement may signal that oxidative processes are at work throughout the natural history of the disease.

DNA duplexes containing photoactive derivatives of 2'-deoxyuridine as photocrosslinking probes for EcoRII DNA methyltransferase-substrate interaction.

EcoRII DNA methyltransferase (M.EcoRII) recognizes the DNA sequence 5'.CC*T/AGG.3' and catalyzes the transfer of the methyl group from S-adenosyl-L-methionine to the C5 position of the inner cytosine residue (C*). We obtained several DNA duplexes containing photoactive 5-iodo-2'-deoxyuridine (i(5)dU) or 5-[4-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenyl]-2'-deoxyuridine (Tfmdp-dU) to characterize regions of M.EcoRII involved in DNA binding and to investigate the DNA double helix conformational changes that take place during methylation. The efficiencies of methylation, DNA binding affinities and M.EcoRII-DNA photocrosslinking yields strongly depend on the type of modification and its location within the EcoRII recognition site. The data obtained agree with the flipping of the target cytosine out of the DNA double helix for catalysis. To probe regions of M.EcoRII involved in DNA binding, covalent conjugates M.EcoRII-DNA were cleaved by cyanogen bromide followed by analysis of the oligonucleotide-peptides obtained. DNA duplexes containing i(5)dU or Tfmdp-dU at the central position of the recognition site, or instead of the target cytosine were crosslinked to the Gly(268)-Met(391) region of the EcoRII methylase. Amino acid residues from this region may take part both in substrate recognition and stabilization of the extrahelical target cytosine residue.

A fusion protein system for the recombinant production of short disulfide-containing peptides.

A recombinant fusion protein system for the production, oxidation, and purification of short peptides containing a single disulfide bond is described. The peptides are initially expressed in Escherichia coli as a fusion to an engineered mutant of the N-terminal SH2 domain of the intracellular phosphatase, SHP-2. This small protein domain confers several important properties which facilitate the production of disulfide-containing peptides: (i) it is expressed at high levels in E. coli; (ii) it can be purified via a hexahistidine tag and reverse-phase HPLC; (iii) it contains no endogenous cysteine residues, allowing the formation of an intrapeptide disulfide bond while still attached to the fusion partner; (iv) it is highly soluble in native buffers, facilitating the production of very hydrophobic peptides and the direct use of fusion products in biochemical assays; (v) it contains a unique methionine residue at the junction of the peptide and fusion partner to facilitate peptide cleavage by treatment with cyanogen bromide (CNBr). This method is useful for producing peptides, which are otherwise difficult to prepare through traditional chemical synthesis approaches, and this has been demonstrated by preparing a number of hydrophobic disulfide-containing peptides derived from phage-display libraries.

The catalytic center of glucose-6-phosphatase. HIS176 is the nucleophile forming the phosphohistidine-enzyme intermediate during catalysis.

Glucose-6-phosphatase (G6Pase), a key enzyme in glucose homeostasis, is anchored to the endoplasmic reticulum by nine transmembrane helices. The amino acids comprising the catalytic center of G6Pase include Lys(76), Arg(83), His(119), Arg(170), and His(176). During catalysis, a His residue in G6Pase becomes phosphorylated generating an enzyme-phosphate intermediate. It was predicted that His(176) would be the amino acid that acts as a nucleophile forming a phosphohistidine-enzyme intermediate, and His(119) would be the amino acid that provides the proton needed to liberate the glucose moiety. However, the phosphate acceptor in G6Pase has eluded molecular characterization. To identify the His residue that covalently bound the phosphate moiety, we generated recombinant adenoviruses carrying G6Pase wild type and active site mutants. A 40-kDa [(32)P]phosphate-G6Pase intermediate was identified after incubating [(32)P]glucose 6-phosphate with microsomes expressing wild type but not with microsomes expressing either H119A or H176A mutant G6Pase. Human G6Pase contains five methionine residues at positions 1, 5, 121, 130, and 279. After cyanogen bromide cleavage, His(119) is predicted to be within a 116-amino acid peptide of 13.5 kDa with an isoelectric point of 5.3 (residues 6-121), and His(176) is predicted to be within a 149-amino acid peptide of 16.8 kDa with an isoelectric point of 9.3 (residues 131-279). We show that after digestion of a non-glycosylated [(32)P]phosphate-G6Pase intermediate by cyanogen bromide, the [(32)P]phosphate remains bound to a peptide of 17 kDa with an isoelectric point above 9, demonstrating that His(176) is the phosphate acceptor in G6Pase.

The unusual cartilaginous tissues of jawless craniates, cephalochordates and invertebrates.

A collagenous extracellular matrix was previously considered to be a requirement for classification of true cartilage. Data from the lamprey and hagfish now clearly indicate that both of these jawless craniates have extensive non-collagenous, yet cartilaginous endoskeletons. Non-collagenous cartilages are present in the cephalochordates (amphioxus) and in the invertebrates, although collagen-containing cartilages also are found in the invertebrates. This review summarizes current knowledge of the morphological, biochemical and molecular characteristics of the unusual non-collagenous cartilages in jawless craniates and the cartilaginous tissues in amphioxus and invertebrates. A least two types of non-collagenous cartilage matrix proteins are found in both the hagfishes and the lampreys, all of which are resistant to digestion by cyanogen bromide (CNBr). Although all four of these matrices show some similarities with each other, suggesting a family of non-collagenous, elastin-like proteins, it is clear that the major matrix proteins of each are different. New morphological and biochemical information on the cartilaginous tissues in squid, horseshoe crab and amphioxus reveals the presence of CNBr-insoluble, non-collagenous matrix proteins, potentially extending the jawless craniate family of cartilaginous proteins into the invertebrates. Details of the evolutionary relationships between these non-collagenous matrix proteins and the significance of the occurrence of these proteins as the major components of the cartilaginous tissues of jawless craniates, amphioxus, horseshoe crab and squid, all of which are capable of producing a variety of collagens in other tissues, remain to be investigated.

The ricinosomes of senescing plant tissue bud from the endoplasmic reticulum.

The ricinosome (synonym, precursor protease vesicle) is a novel organelle, found so far exclusively in plant cells. Electron microscopic studies suggest that it buds off from the endoplasmic reticulum in senescing tissues. Biochemical support for this unusual origin now comes from the composition of the purified organelle, which contains large amounts of a 45-kDa cysteine endoprotease precursor with a C-terminal KDEL motif and the endoplasmic reticulum lumen residents BiP (binding protein) and protein disulfide isomerase. Western blot analysis, peptide sequencing, and mass spectrometry demonstrate retention of KDEL in the protease proform. Acidification of isolated ricinosomes causes castor bean cysteine endopeptidase activation, with cleavage of the N-terminal propeptide and the C-terminal KDEL motif. We propose that ricinosomes accumulate during senescence by programmed cell death and are activated by release of protons from acidic vacuoles.

Chemical cleavage of proteins on membranes.

Described in this unit are five basic protocols that are widely used for specific and efficient chemical cleavage of proteins bound to membranes. Cyanogen bromide (CNBr) cleaves at methionine (Met) residues; BNPS-skatole cleaves at tryptophan (Trp) residues; formic acid cleaves at aspartic acid-proline (Asp-Pro) peptide bonds; hydroxylamine cleaves at asparagine-glycine (Asn-Gly) peptide bonds, and 2-nitro-5-thiocyanobenzoic acid (NTCB) cleaves at cysteine (Cys) residues. Because the above loci are at relatively low abundance in most proteins, digestion with these agents will yield relatively long peptides. In addition, Alternate Protocol an describes CNBr cleavage of PVDF-bound protein previously analyzed by Edman degradation. Finally, a Support Protocol discusses preferred methods of separating and analyzing peptide fragments generated by the chemical cleavage reactions described in the basic protocols.

Characterization of an anti-Borrelia burgdorferi OspA conformational epitope by limited proteolysis of monoclonal antibody-bound antigen and mass spectrometric peptide mapping.

Lyme borreliosis is a multisystem disorder caused by the spirochete Borrelia burgdorferi that is transmitted to humans by the tick Ixodes dammini. The immune response against the 31 kDa OspA, which is one of the most abundant B. burgdorferi proteins, appears to be critical in preventing infection and tissue inflammation. Detailed knowledge of the immunological and molecular characteristics of the OspA protein is important for the development of reliable diagnostic assays. In this study, we characterized a new conformational epitope present within the middle part of B. burgdorferi OspA. Our approach used enzymatic proteolyses of the immune complex followed by mass spectrometric identification of the peptides bound to the antibody. It appears to be one of the first reports on the characterization of a discontinuous epitope using mass spectrometry.

Intrahelical arrangement in the integral membrane protein rhodopsin investigated by site-specific chemical cleavage and mass spectrometry.

Site-specific cleavage on the interhelical loop I on the cytoplasmic face of rhodopsin has been observed after activation of a Cu-phenanthroline tethered cleavage reagent attached on the cytoplasmic loop IV. The characterization of the reaction products by mass spectrometry, both of the membrane-bound protein and of the CNBr-cleaved peptides, allows the site of cleavage to be determined precisely. The specific cleavage of the peptide bond between Q64 and H65 on loop I leaves the N-terminal peptide (M1-Q64) intact, confirmed by MALDI-MS detection of the two N-linked glycosyl groups near the N-terminus of rhodopsin. The limited extension of the tether side chain requires a interresidue distance between the cleavage site, Q64, and the site of ligand attachment, C316, of less than 12 A. Upon photoactivation of the receptor, no change in the cleavage pattern is observed; however, a simulated Meta II intermediate activation state indicates a much more complex cleavage pattern. The development of this cleavage method, previously used primarily as a "chemical nuclease", in combination with mass spectrometry, may provide a powerful method on membrane protein conformation studies that can be used to complement other biophysical characterizations.