A Comparative Structural Analysis of the Flagellin Monomers of Caulobacter crescentus Indicates that These Proteins Are Encoded by Two Genes

The flagellum of Caulobacter crescentus is composed of two flagellin polypeptide monomers which are distinguished by molecular weight and are closely related by biochemical and immunological criteria (C. Lagenaur and N. Agabian, J. Bacteriol. 132:731-733, 1977). The synthesis and assembly of these two flagellin proteins are developmentally regulated, and the periodicity of expression for each is distinct (C. Lagenaur and N. Agabian, J. Bacteriol. 135:1062-1069, 1978; M. A. Osley, M. Sheffery, and A. Newton, Cell 12:393-400, 1977). To understand the genetic and functional relationship between the 25,000- and 27,500-molecular-weight flagellins of C. crescentus CB15, a detailed comparative analysis of their protein structure was made, using a number of techniques, including one- and two-dimensional peptide mapping, a novel procedure of peptide alignment, and amino terminal amino acid sequence analysis. The tryptic peptides generated by each of the flagellins were compared by two-dimensional thin-layer chromatography. This peptide map analysis indicated that approximately 36% of the peptides generated from these two proteins had similar migration properties. Together with biochemical and immunological criteria, the two-dimensional peptide map suggested some structural relatedness between the monomers. However, a comparison of peptide fragments generated during partial protease digestion of each protein by a method of one-dimensional mapping indicated that the two proteins are structurally unique. A peptide alignment technique was developed to directly compare the primary structure of these proteins. In the peptide alignment procedure the amino terminus of each protein is radioactively labeled. After partial enzymatic digestion, the peptides are fractionated by polyacrylamide gel electrophoresis: those labeled at the amino terminus are then resolved by subsequent autoradiography. Each digest contains a family of amino-terminal-labeled fragments, the sizes of which reflect the sequential alignment of cleavage sites in the protein. A comparison of the alignment of specific cleavage sites of the two flagellins by this technique further established that each flagellin is structurally unique, particularly in the carboxyl terminal region. Finally, comparison of the amino terminal amino acid sequences indicated that the amino terminal region of both flagellins is highly conserved, but that the two polypeptides are clearly not identical. These findings strongly indicate that the two flagellins are encoded by distinct genetic loci and are not the product of novel processing of a single larger precursor.     

Identification of residues important for cleavage of the extracellular signaling peptide CSF of Bacillus subtilis from its precursor protein

Extracellular Phr pentapeptides produced by gram-positive, spore-forming bacteria regulate processes during the transition from exponential- to stationary-phase growth. Phr pentapeptides are produced by cleavage of their precursor proteins. We determined the residues that direct this cleavage for the Bacillus subtilis Phr peptide, CSF, which is derived from the C terminus of PhrC. Strains expressing PhrC with substitutions in residues -1 to -5 relative to the cleavage site had a defect in CSF production. The mutant PhrC proteins retained a functional signal sequence for secretion, as assessed by secretion of PhrC-PhoA fusions. To determine whether the substitutions directly affected cleavage of PhrC to CSF, we tested cleavage of synthetic pro-CSF peptides that corresponded to the C terminus of PhrC and had an amino acid substitution at the -2, -3, or -4 position. The mutant pro-CSF peptides were cleaved less efficiently to CSF than the wild-type pro-CSF peptide whether they were incubated with whole cells, cell wall material, or the processing protease subtilisin or Vpr. To further define the range of amino acids that support CSF production, the amino acid at the -4 position of PhrC was replaced by the 19 canonical amino acids. Only four substitutions resulted in a >2-fold defect in CSF production, indicating that this position is relatively immune to mutational perturbations. These data revealed residues that direct cleavage of CSF and laid the groundwork for testing whether other Phr peptides are processed in a similar manner.     

Peptide mapping of bovine pancreatic ribonuclease A by reverse-phase high-performance liquid chromatography. I. Application to the reduced and S-carboxymethylated protein

Complete peptide maps of reduced and S-carboxymethylated ribonuclease A were obtained by reverse-phase high-performance liquid chromatography with the following peptide-chain cleavage techniques: cyanogen bromide cleavage, limited and extensive Staphylococcus aureus protease digestion, tryptic digestion, and tryptic followed by chymotryptic digestion. Commercial samples of S. aureus protease exhibited a broader specificity than had previously been reported, as demonstrated by its ability to cleave after glutamine residues. Cleavage after asparagine and serine residues was also strongly implicated. The procedures developed require roughly 0.1 to 1 mg of ribonuclease A for the peptide mapping of this protein. These procedures will be useful for the identification of the sites of a chemical modification and also for the isolation of a variety of peptides for further studies.     

Sites of methyl esterification and deamination on the aspartate receptor involved in chemotaxis

The receptors involved in bacterial chemotaxis are post-translationally modified by specific enzymes which catalyze the deamination of glutaminyl residues and the methyl esterification and demethylation of glutamyl residues. In this work we identify the sites of these covalent modifications on the aspartate receptor from Salmonella typhimurium. These were identified using the properties of the Staphylococcus aureus V8 protease which cleaves peptide bonds following glutamyl but not glutaminyl residues. We show here that bonds following methyl-esterified glutamyl residues are also resistant to the protease. A comparison of the fragments obtained after V8 protease cleavage of methyl-esterified (or deaminated) peptides with the fragments from the corresponding unmodified peptides immediately yields the sites of modification. Three of the four methyl-esterified glutamyl residues are located near the middle of the receptor amino acid sequence; one of these is synthesized as a glutaminyl residue and is deaminated by the esterase to form a glutamyl residue. The fourth site of methyl esterification is located near the carboxyl terminus. All four sites occupy analogous positions in a well-conserved arrangement of residues which may form a binding site for the esterase and the methyltransferase.     

Inhibition of protein carbamylation in urea solution using ammonium-containing buffers

Urea solution is one of the most commonly employed protein denaturants for protease digestion in proteomic studies. However, it has long been recognized that urea solution can cause carbamylation at the N termini of proteins/peptides and at the side chain amino groups of lysine and arginine residues. Protein/peptide carbamylation blocks protease digestion and affects protein identification and quantification in mass spectrometry analysis by blocking peptide amino groups from isotopic/isobaric labeling and changing peptide charge states, retention times, and masses. In addition, protein carbamylation during sample preparation makes it difficult to study in vivo protein carbamylation. In this study, we compared the peptide carbamylation in urea solutions of different buffers and found that ammonium-containing buffers were the most effective buffers to inhibit protein carbamylation in urea solution. The possible mechanism of carbamylation inhibition by ammonium-containing buffers is discussed, and a revised procedure for the protease digestion of proteins in urea and ammonium-containing buffers was developed to facilitate its application in proteomic research.     

The unique sites in SulA protein preferentially cleaved by ATP-dependent Lon protease from Escherichia coli.

SulA protein is known to be one of the physiological substrates of Lon protease, an ATP-dependent protease from Escherichia coli. In this study, we investigated the cleavage specificity of Lon protease toward SulA protein. The enzyme was shown to cleave approximately 27 peptide bonds in the presence of ATP. Among them, six peptide bonds were cleaved preferentially in the early stage of digestion, which represented an apparently unique cleavage sites with mainly Leu and Ser residues at the P1, and P1' positions, respectively, and one or two Gln residues in positions P2-P5. They were located in the central region and partly in the C-terminal region, both of which are known to be important for the function of SulA, such as inhibition of cell growth and interaction with Lon protease, respectively. The other cleavage sites did not represent such consensus sequences, though hydrophobic or noncharged residues appeared to be relatively preferred at the P1 sites. On the other hand, the cleavage in the absence of ATP was very much slower, especially in the central region, than in the presence of ATP. The central region was predicted to be rich in alpha helix and beta sheet structures, suggesting that the enzyme required ATP for disrupting such structures prior to cleavage. Taken together, SulA is thought to contain such unique cleavage sites in its functionally and structurally important regions whose preferential cleavage accelerates the ATP-dependent degradation of the protein by Lon protease.     

Impact of fluorination on proteolytic stability of peptides in human blood plasma

Several factors reduce the efficacy of natural peptides as drug candidates; chief among these is their rapid digestion by human proteases. Over the last few decades, a number of strategies have been employed to increase the enzymatic stability of peptides, including the introduction of non-natural amino acids. This study aims at the investigation of the effect of side chain fluorination on the stability of peptides in human blood plasma. Ten model peptides with different non-natural amino acids were designed, synthesized and subjected to enzymatic degradation in human blood plasma. The stability of the studied peptides was followed by HPLC analysis and compared to the control peptide built with only proteinogenic residues. Four main hydrolysis products were detected and identified by mass spectrometry, three of them being characteristic cleavage products of the serine protease Elastase. A final enzymatic study with isolated Elastase validated then the outcome of the plasma study. This case study contributes to the application of fluorinated amino acids in the design of proteolytically stable peptides and proteins with potential clinical relevance.     

Degradation of a signal peptide by protease IV and oligopeptidase A.

The degradation of the prolipoprotein signal peptide in vitro by membranes, cytoplasmic fraction, and two purified major signal peptide peptidases from Escherichia coli was followed by reverse-phase liquid chromatography (RPLC). The cytoplasmic fraction hydrolyzed the signal peptide completely into amino acids. In contrast, many peptide fragments accumulated as final products during the cleavage by a membrane fraction. Most of the peptides were similar to the peptides formed during the cleavage of the signal peptide by the purified membrane-bound signal peptide peptidase, protease IV. Peptide fragments generated during the cleavage of the signal peptide by protease IV and a cytoplasmic enzyme, oligopeptidase A, were identified from their amino acid compositions, their retention times during RPLC, and knowledge of the amino acid sequence of the signal peptide. Both enzymes were endopeptidases, as neither dipeptides nor free amino acids were formed during the cleavage reactions. Protease IV cleaved the signal peptide predominantly in the hydrophobic segment (residues 7 to 14). Protease IV required substrates with hydrophobic amino acids at the primary and the adjacent substrate-binding sites, with a minimum of three amino acids on either side of the scissile bond. Oligopeptidase A cleaved peptides (minimally five residues) that had either alanine or glycine at the P'1 (primary binding site) or at the P1 (preceding P'1) site of the substrate. These results support the hypothesis that protease IV is the major signal peptide peptidase in membranes that initiates the degradation of the signal peptide by making endoproteolytic cuts; oligopeptidase A and other cytoplasmic enzymes further degrade the partially degraded portions of the signal peptide that may be diffused or transported back into the cytoplasm from the membranes.     

Enzyme‐cleavable tandem peptides for quantitative studies in MS‐based proteomics

A novel type of peptide standard is introduced that consists of two peptides combined in one synthetic molecule and separated by a proteolytic cleavage site. Upon enzymatic digestion, the two peptides are released in a molar one‐to‐one ratio. This method enables the generation of exact equimolar mixtures of two peptides of any nature and origin, thereby providing a valuable tool for the investigation of fundamental phenomena in MS. The applicability of the method is exemplified by the analysis of the effect of peptide sequence variations on the relative ionization efficiency in ESI‐ and MALDI‐MS.     

Mapping protein surface accessibility via an electron transfer dissociation selectively cleavable hydrazone probe

A protein's surface influences its role in protein-protein interactions and protein-ligand binding. Mass spectrometry can be used to give low resolution structural information about protein surfaces and conformations when used in combination with derivatization methods that target surface accessible amino acid residues. However, pinpointing the resulting modified peptides upon enzymatic digestion of the surface-modified protein is challenging because of the complexity of the peptide mixture and low abundance of modified peptides. Here a novel hydrazone reagent (NN) is presented that allows facile identification of all modified surface residues through a preferential cleavage upon activation by electron transfer dissociation coupled with a collision activation scan to pinpoint the modified residue in the peptide sequence. Using this approach, the correlation between percent reactivity and surface accessibility is demonstrated for two biologically active proteins, wheat eIF4E and PARP-1 Domain C.     

Mechanism of inhibition of the retroviral protease by a Rous sarcoma virus peptide substrate representing the cleavage site between the gag p2 and p10 proteins.

The activity of the avian myeloblastosis virus (AMV) or the human immunodeficiency virus type 1 (HIV-1) protease on peptide substrates which represent cleavage sites found in the gag and gag-pol polyproteins of Rous sarcoma virus (RSV) and HIV-1 has been analyzed. Each protease efficiently processed cleavage site substrates found in their cognate polyprotein precursors. Additionally, in some instances heterologous activity was detected. The catalytic efficiency of the RSV protease on cognate substrates varied by as much as 30-fold. The least efficiently processed substrate, p2-p10, represents the cleavage site between the RSV p2 and p10 proteins. This peptide was inhibitory to the AMV as well as the HIV-1 and HIV-2 protease cleavage of other substrate peptides with Ki values in the 5-20 microM range. Molecular modeling of the RSV protease with the p2-p10 peptide docked in the substrate binding pocket and analysis of a series of single-amino acid-substituted p2-p10 peptide analogues suggested that this peptide is inhibitory because of the potential of a serine residue in the P1' position to interact with one of the catalytic aspartic acid residues. To open the binding pocket and allow rotational freedom for the serine in P1', there is a further requirement for either a glycine or a polar residue in P2' and/or a large amino acid residue in P3'. The amino acid residues in P1-P4 provide interactions for tight binding of the peptide in the substrate binding pocket.     

Structural characterization of the maytansinoid-monoclonal antibody immunoconjugate, huN901-DM1, by mass spectrometry

Immunoconjugates are being explored as novel cancer therapies with the promise of target-specific drug delivery. The immunoconjugate, huN901-DM1, composed of the humanized monoclonal IgG1 antibody, huN901, and the maytansinoid drug, DM1, is being tested in clinical trials to treat small cell lung carcinoma (SCLC). huN901-DM1 contains an average of three to four DM1 drug molecules per huN901 antibody molecule. The drug molecules are linked to huN901 through random modification of huN901 at epsilon-amino groups of lysine residues, thus yielding a heterogeneous population of conjugate species. We studied the drug distribution profile of huN901-DM1 by electrospray time-of-flight mass spectrometry(ESI-TOFMS), which showed that one to six DM1 drug molecules were attached to an antibody molecule. Both light and heavy chains contained linked drugs. The conjugation sites in both chains were determined by peptide mapping using trypsin and Asp-N protease digestion. Trypsin digestion identified modified lysine residues, since these residues were no longer susceptible to enzymatic cleavage after conjugation with the drug. With respect to Asp-N digestion, modified peptides were identified by observing a mass increase corresponding to the modification. The two digestion methods provided consistent results, leading to the identification of 20 modified lysine residues in both light and heavy chains. Each lysine residue was only partially modified. No conjugation sites were found in complementarity determining regions (CDRs). Using structural models of human IgG1, it was found that modified lysine residues were on the surface in areas of structural flexibility and had large solvent accessibility.     

A family of bacteriocin ABC transporters carry out proteolytic processing of their substrates concomitant with export

Lantibiotic and non-lantibiotic bacteriocins are synthesized as precursor peptides containing N-terminal extensions (leader peptides) which are cleaved off during maturation. Most non-lantibiotics and also some lantibiotics have leader peptides of the so- called double-glycine type. These leader peptides share consensus sequences and also a common processing site with two conserved glycine residues In positions -1 and 2. The double-glycine-type leader peptides are unrelated to the N-terminal signal sequences which direct proteins across the cytoplasmic membrane via the sec pathway. Their processing sites are also different from typical signal peptidase cleavage sites, suggesting that a different processing enzyme is involved. Peptide bacteriocins are exported across the cytoplasmic membrane by a dedicated ATP-binding cassette (ABC) transporter. Here we show that the ABC transporter is the maturation protease and that its proteolytic domain resides in the N-terminal part of the protein. This result demonstrates that the ABC transporter has a dual function: (i) removal of the leader peptide from its substrate, and (ii) translocation of its substrate across the cytoplasmic membrane. This represents a novel strategy for secretion of bacterial proteins.     

Complete amino acid sequence of the ubiquinone binding protein (QP-C), a protein similar to the 14,000-dalton subunit of the yeast ubiquinol-cytochrome c reductase complex

The amino acid sequence of the ubiquinone binding protein (QP-C) in the cytochrome bc1 region of the mitochondrial electron transfer chain was determined by analysis of peptides obtained by cyanogen bromide cleavage and staphylococcal protease digestion of succinylated derivatives. It was found to consist of 110 amino acid residues and its amino terminus to be blocked by an acetyl group, as determined by mass spectrometry of the amino-terminal peptide and a comparison with peptides chemically synthesized on high-performance liquid chromatography. The molecular weight of this ubiquinone binding protein including the acetyl group was calculated to be 13,389. The predicted secondary structure of QP-C has alpha-helical content of about 50% and QP-C was classified as an "all-alpha" or "alpha + beta" protein. This is the first report describing the amino acid sequence of the ubiquinone binding protein. A comparison of this sequence with that of the 14-kDa subunit of the yeast ubiquinol-cytochrome c reductase complex from the nucleotide sequence showed these two sequences to be quite similar.     

Amino acid sequence of bovine white matter proteolipid

The sequence of the bovine white matter proteolipid has been studied by a combination of proteolytic digestion and chemical cleavage at tryptophan residues. Alignment of peptides obtained by digestion with trypsin, chymotrypsin, clostripain, and Staphylococcus aureus protease gave the sequence of 52 residues at the amino terminus, 96 residues at the carboxyl terminus, and several additional segments. Peptides obtained by treatment of the protein with 2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine confirmed the alignment and extended the sequence. This information, combined with that of other investigators, permits us to propose the primary structure for the entire protein. On the basis of the sequence determination, the molecular weight of the proteolipid protein is 29,869.     

Structure of bovine milk lipoprotein lipase

The primary structure of bovine milk lipoprotein lipase (bLPL) was determined by alignment of peptides produced by tryptic digestion, Staphylococcus aureus V8 protease digestion, and cyanogen bromide cleavage. bLPL consists of 450 amino acid residues. Most tryptic peptides were isolated and analyzed, except for the dipeptide, Glu-Lys (position 423-424), and the 2 Lys at positions 416 and 488. Peptides resulting from digestion by S. aureus V8 protease and cyanogen bromide cleavage filled the missing part and completed the primary sequence of bLPL. The NH2 terminus of bLPL was determined to be Asp by sequencing the intact protein with a gas phase sequencer for up to 30 residues, whereas the COOH terminus was identified as Gly through, carboxyl peptidase Y cleavage. The enzyme contains 10 cysteine residues, all of which exist in disulfide linkages. They are formed between Cys29 and Cys42, Cys218 and Cys241, Cys266 and Cys285, Cys277, and Cys280, and Cys420 and Cys440. The sites of N-glycosylation were identified at Asn44 and Asn361. In accordance with a common structural homology of serine-type esterases, -G-X-S-X-G- (Yang, C. Y., Manoogian, D., Pao, Q., Lee, F., Knapp, R. D., Gotto, A. M., Jr., and Pownall, H. J. (1987) J. Biol. Chem., 262, 3086-3191), the active site serine of bLPL was assigned to the serine at position 134. The chymotrypsin nick of bLPL was determined to be between residues 390 and 391. A model of the enzyme is proposed on the basis of our data and available chemical data.     

Amino acid identity and/or position determines the proteasomal cleavage of the HLA-A*0201-restricted peptide tumor antigen MAGE-3271-279

The proteasome plays a crucial role in the proteolytic processing of antigens presented to T cells in the context of major histocompatibility complex class I molecules. However, the rules governing the specificity of cleavage sites are still largely unknown. We have previously shown that a cytolytic T lymphocyte-defined antigenic peptide derived from the MAGE-3 tumor-associated antigen (MAGE-3(271-279), FLWGPRALV in one-letter code) is not presented at the surface of melanoma cell lines expressing the MAGE-3 protein. By using purified proteasome and MAGE-3(271-279) peptides extended at the C terminus by 6 amino acids, we identified predominant cleavages after residues 278 and 280 but no detectable cleavage after residue Val(279), the C terminus of the antigenic peptide. In the present study, we have investigated the influence of Pro(275), Leu(278), and Glu(280) on the proteasomal digestion of MAGE-3(271-285) substituted at these positions. We show that positions 278 and 280 are major proteasomal cleavage sites because they tolerate most amino acid substitutions. In contrast, the peptide bond after Val(279) is a minor cleavage site, influenced by both distal and proximal amino acid residues.     

X-ray snapshots of peptide processing in mutants of tricorn-interacting factor F1 from Thermoplasma acidophilum

The tricorn-interacting factor F1 of the archaeon Thermoplasma acidophilum cleaves small hydrophobic peptide products of the proteasome and tricorn protease. F1 mutants of the active site residues that are involved in substrate recognition and catalysis displayed distinct activity patterns toward fluorogenic test substrates. Crystal structures of the mutant proteins complexed with peptides Phe-Leu, Pro-Pro, or Pro-Leu-Gly-Gly showed interaction of glutamates 213 and 245 with the N termini of the peptides and defined the S1 and S1' sites and the role of the catalytic residues. Evidence was found for processive peptide cleavage in the N-to-C direction, whereby the P1' product is translocated into the S1 site. A functional interaction of F1 with the tricorn protease was observed with the inactive F1 mutant G37A. Moreover, small angle x-ray scattering measurements for tricorn and inhibited F1 have been interpreted as formation of transient and substrate-induced complexes.     

Improvement of an in-gel tryptic digestion method for matrix-assisted laser desorption/ionization-time of flight mass spectrometry peptide mapping by use of volatile solubilizing agents

The combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), in-gel enzymatic digestion of proteins separated by two-dimensional gel electrophoresis and searches of molecular weight in peptide-mass databases is a powerful and well established method for protein identification in proteomics analysis. For successful protein identification by MALDI-TOF mass spectrometry of peptide mixtures, critical parameters include highly specific enzymatic cleavage, high mass accuracy and sufficient numbers and sequence coverage of the peptides which can be analyzed. For in-gel digestion with trypsin, the method employed should be compatible both with enzymatic cleavage and subsequent MALDI-TOF MS analysis. We report here an improved method for preparation of peptides for MALDI-TOF MS mass fingerprinting by using volatile solubilizing agents during the in-gel digestion procedure. Our study clearly demonstrates that modification of the in-gel digestion protocols by addition of dimethyl formamide (DMF) or a mixture of DMF/N,N-dimethyl acetamide at various concentrations can significantly increase the recovery of peptides. These higher yields of peptides resulted in more effective protein identification.