B133 (DSITKYFQMSLE), a laminin β1-derived peptide, contains distinct core sequences for both integrin α2β1-mediated cell adhesion and amyloid-like fibril formation

The B133 peptide (DSITKYFQMSLE, mouse laminin β1 chain 1319-1330) promotes cell attachment, and forms amyloid-like fibrils. Here, we evaluated the active core sequences using B133 deletion peptides. B133a, lacking the N-terminal Asp residue, promoted cell spreading via integrin α2β1, whereas B133g, lacking the C-terminal Glu residue, lost the activity. Congo red analysis using the truncated peptides determined that B133g forms amyloid-like fibrils but B133a did not. These results suggest that the N- and C-terminal amino acids contribute to integrin α2β1 binding and to fibril formation, respectively. Further analyses using the truncated peptides showed that the C-terminal eight residues (B133d: KYFQMSLE) are a minimum active sequence for integrin α2β1-mediated cell attachment and the N-terminal nine residues (B133i: DSITKYFQM) are critical for amyloid-like fibril formation. These results suggest that peptide B133 is multifunctional with two different active core sequences: integrin α2β1-mediated cell attachment and amyloid-like fibril formation. Moreover, alanine substitution analysis of B133a indicated that six amino acids, Ile, Thr, Tyr, Phe, Met, and Glu, are important for cell attachment activity. When the Ser residue at the 9th position of B133a was replaced with Ala, the cell attachment activity was enhanced. Further mutation analysis at the 9th position of B133a using various amino acids suggests that hydrophobic amino acids are effective for the integrin α2β1-mediated cell attachment activity. These findings define multifunctional and overlapping sites on the B133 peptide and are useful for designing multifunctional synthetic molecules.     

Cys-Cys cross-linking shows contact between the N-terminus of lethal factor and Phe427 of the anthrax toxin pore

Electrophysiological studies of wild-type and mutated forms of anthrax protective antigen (PA) suggest that the Phe clamp, a structure formed by the Phe427 residues within the lumen of the oligomeric PA pore, binds the unstructured N-terminus of the lethal factor and the edema factor during initiation of translocation. We now show by electrophysiological measurements and gel shift assays that a single Cys introduced into the Phe clamp can form a disulfide bond with a Cys placed at the N-terminus of the isolated N-terminal domain of LF. These results demonstrate direct contact of these Cys residues, supporting a model in which the interaction of the unstructured N-terminus of the translocated moieties with the Phe clamp initiates N- to C-terminal threading of these moieties through the pore.     

Comparison of the structure of two cardiac troponin T isoforms.

Two isoforms of troponin T have been isolated from bovine cardiac muscle. One isoform has an Mr of 31000 and a pI at about 7.1, the corresponding values for the second isoform being 33000 and 6.5. Both isoforms have identical C- and N-terminal sequences, and, according to the data from tryptic-peptide mapping, a similar structure of the central and C-terminal domains. The large N-terminal peptides of troponin T isoforms differ in the content of glutamine/glutamic acid and alanine. It is concluded that the isoform with Mr 33000 has an additional peptide enriched with glutamic acid and alanine that is inserted between the N-terminal pentapeptide and the cysteine located 40-60 residues from the N-terminus.     

Terminal truncations in amp C beta-lactamase from a clinical isolate of Pseudomonas aeruginosa.

AmpC beta-lactamases from strains of Pseudomonas aeruginosa have previously been shown to be heterogeneous with respect to their isoelectric point (pI). In order to elucidate the origin of this heterogeneity enzymes were isolated from a clinical isolate of a multiresistant P. aeruginosa strain and biochemically characterized. The purification was accomplished in four chromatographic steps comprising dye-affinity, size-exclusion, hydrophobic interaction chromatography, and chromatofocusing; this resulted in five forms with pI values of 9.1, 8.7, 8.3, 8.2, and 7.6. When analysed by SDS/PAGE and agarose IEF each separated beta-lactamase appeared to be both size- and charge-homogeneous. The specific activities of the variants were very similar. MS of each isolated beta-lactamase form showed minor differences in molecular mass (range 40.0-40.8 kDa). MS of the beta-lactamase with a pI of 8.2 demonstrated the presence of two subforms. The N-terminal sequences of three of the beta-lactamases were identical to the published sequence [Lodge, J.M. , Minchin, S.D., Piddock, L.J.V. & Busby, J.W. (1990) Biochem. J. 272, 627-631], while two variants were truncated by two amino-acid residues, one of which was acidic. The previously published sequence contains an alanine as the ultimate residue, but two of the beta-lactamases showed a substitution of Ala371 for arginine, whereas in the remaining forms C-terminal truncations by one and three residues were found. Our results indicate that the P. aeruginosa strain does not harbour multiple copies of the ampC gene, but rather that the five beta-lactamase isoforms are products of a single structural gene. The combinations of the identified N- and/or C-terminal truncations explained the multiple pI values of the beta-lactamase isoforms.     

Left handed �� helix models for mammalian prion fibrils

We propose models for in vitro grown mammalian prion protein fibrils based upon left handed beta helices formed both from the N-terminal and C-terminal regions of the proteinase resistant infectious prion core. The C-terminal threading onto a β-helical structure is almost uniquely determined by fixing the cysteine disulfide bond on a helix corner. In comparison to known left handed helical peptides, the resulting model structures have similar stability attributes including relatively low root mean square deviations in all atom molecular dynamics, substantial side-chain-to-side-chain hydrogen bonding, good volume packing fraction, and low hydrophilic/hydrophobic frustration. For the N-terminus, we propose a new threading of slightly more than two turns, which improves upon the above characteristics relative to existing three turn β-helical models. The N-terminal and C-terminal beta helices can be assembled into eight candidate models for the fibril repeat units, held together by large hinge (order 30 residues) domain swapping, with three amenable to fibril promoting domain swapping via a small (five residue) hinge on the N-terminal side. Small concentrations of the metastable C-terminal β helix in vivo might play a significant role in templating the infectious conformation and in enhancing conversion kinetics for inherited forms of the disease and explain resistance (for canines) involving hypothesized coupling to the methionine 129 sulfur known to play a role in human disease.Key words: prion, amyloid fibril, domain swap, beta helix, computational biology     

Terminal ion pairs stabilize the second beta-hairpin of the B1 domain of protein G

The effects of terminal ion pairs on the stability of a beta-hairpin peptide corresponding to the C-terminal residues of the B1 domain of protein G were determined using thermal unfolding as monitored by nuclear magnetic resonance and circular dichroism spectroscopy. Molecular dynamics (MD) simulations were also performed to examine the effect of ion pairs on the structures. Eight peptides were studied including the wild type (G41) and the N-terminal modified sequences that had the first residue deleted (E42), replaced with a Lys (K41), or extended by an additional Gly (G40). Acetylated variants were made to examine the effect of removing the positive N-terminal charge on beta-hairpin stability. The rank in stability determined experimentally is K41 > E42 approximately G41 approximately G40 > Ac-K41 > Ac-E42 approximately Ac-G41 > Ac-G40. The Tm of the K41 peptide is 12 degrees C higher than G41, while the Tm values for the acetylated peptides are less than their unacetylated forms by more than 15 degrees C. NOE cross-peaks between side-chain methylene groups at the N- and C-termini and larger CalphaH shifts compared to random values are seen for K41. The addition of 20% methanol increases the stability in K41 and G41. The MD studies complement these results by showing that the charged N-terminus is important to stability. The type of ion pair observed varies with peptide, and when formed the simulations show that the ion pair can prevent fraying of the beta-strands through electrostatic and hydrophobic contacts. Therefore, introducing favorable electrostatic interactions at the N- and C-termini can substantially enhance beta-hairpin stability and help define the structure.     

Cloning of the (Thr6)-phyllokinin precursor from Phyllomedusa sauvagei skin confirms a non-consensus tyrosine O-sulfation motif

Nine bradykinin-related peptides were identified in Phyllomedusa sauvagei skin secretion using QTOF MS/MS fragmentation sequencing. The major peptides were (Thr6)-bradykinin, (Hyp3, Thr6)-bradykinin, (Thr6)-phyllokinin and (Hyp3, Thr6)-phyllokinin. The phyllokinins occurred in both sulfated and non-sulfated forms. All (Thr6)-substituted bradykinins/phyllokinins could be generated from a common precursor by differential post-translational processing and modification. The open-reading frame of the cloned precursor cDNA consisted of 62 amino acid residues with a single bradykinin/phyllokinin coding sequence located at the C-terminus. Structural features included a Glu-Arg processing site at the N-terminus of the bradykinin/phyllokinin domain and the absence of an acidic amino acid residue adjacent to the C-terminal Tyr residue in the phyllokinins. However, the neutral amino acid residue (Ile) at position -1 and the basic amino acid residue (Arg) at position -2 from the Tyr residue, constitute a sulfation motif previously identified only in a protochordean.     

Association of the N- and C-terminal domains of phospholipase D. Contribution of the conserved HKD motifs to the interaction and the requirement of the association for Ser/Thr phosphorylation of the enzyme

Rat brain phospholipase D1 (rPLD1) belongs to a superfamily defined by the highly conserved catalytic motif (H(X)K(X)(4)D, denoted HKD. rPLD1 contains two HKD domains, located in the N- and C-terminal regions. The integrity of the two HKD domains is essential for enzymatic activity. Our previous studies showed that the N-terminal half of rPLD1 containing one HKD motif can associate with the C-terminal half containing the other HKD domain to reconstruct wild type PLD activity (Xie, Z., Ho, W.-T. and Exton, J. H. (1998) J. Biol. Chem. 273, 34679-34682). In the present study, we have shown by mutagenesis that conserved amino acids in the HKD domains are important for both the catalytic activity and the association between the two halves of rPLD1. Furthermore, we found that rPLD1 could be modified by Ser/Thr phosphorylation. The modification occurred at the N-terminal half of the enzyme, however, the association of the N-terminal domain with the C-terminal domain was required for the modification. The phosphorylation of the enzyme was not required for its catalytic activity or response to PKCalpha and small G proteins in vitro, although the phosphorylated form of rPLD1 was localized exclusively in the crude membrane fraction. In addition, we found that the individually expressed N- and C-terminal fragments did not interact when mixed in vitro and were unable to reconstruct PLD activity under these conditions. It is concluded that the association of the N- and C-terminal halves of rPLD1 requires their co-expression in vivo and depends on conserved residues in the HKD domains. The association is also required for Ser/Thr phosphorylation of the enzyme.     

Conformational analysis of N-terminal O-glycopeptide sequences of interleukin-2]

The preferred conformations of eight O-glycopeptide sequences from the N-terminus of interleukin-2 containing two to ten amino acids, monoglycosylated at Thr3 with a 2-acetamido-2-deoxy-alpha-D-galactopyranosyl group, were determined by means of n.m.r. spectroscopic methods. The preferred conformation of the N-terminal sequence, L-Ala-L-Pro-[alpha-D-GalpNAc-(1----3)]-L-Thr-L-Ser, including the O-glycosidically linked 2-acetamido-2-deoxy-alpha-D-galactopyranosyl group is not substantially influenced by the linkage of additional amino acids at the C-terminal end. Extended conformations were observed for all peptide units. Measurements of the relaxation times of the 13C atoms showed that the 2-acetamido-2-deoxy-D-galactose bound to the central amino acids has the lowest mobility, whereas the terminal amino acid residues and peptide side-chains are flexible. Calculations with the force-field program AMBER yielded conformations of minimized energies that were in good agreement with the n.m.r. spectroscopic data. This was only true when n.m.r. parameters that can be used as starting values for the calculations were available. Comparison with a nonglycosylated, N-terminal tetrapeptide sequence analog did not suggest changes in the peptide conformation when Thr3 is glycosylated with a 2-acetamido-2-deoxy-alpha-D-galactopyranosyl group.     

The N- and C-termini of the human Nogo molecules are intrinsically unstructured: bioinformatics, CD, NMR characterization, and functional implications

RTN4 or Nogo proteins are composed of three alternative splice forms, namely 1192-residue Nogo-A, 373-residue Nogo-B, and 199-residue Nogo-C. Nogo proteins have received intense attentions because they have been implicated in a variety of critical cellular processes including CNS neuronal regeneration, vascular remodeling, apoptosis, interaction with beta-amyloid protein converting enzyme, and generation/maintenance of the tubular network of the endoplasmic reticulum (ER). Despite their significantly-different N-terminal lengths, they share a conserved C-terminal reticulon-homology domain consisting of two transmembrane fragments, a 66-residue extracellular loop Nogo-66 and a 38-residue C-tail carrying ER retention motif. Nogo-A owns the largest N-terminus with 1016 residues while the Nogo-B has an N-terminus almost identical to the first 200 residues of Nogo-A. So far, except for our previous determination of the Nogo-66 solution structure, no structural characterization of the other Nogo regions has been reported. In the present study, we initiated a systematically investigation of structural properties of Nogo molecules by a combined use of bioinformatics, CD, and NMR spectroscopy. The results led to two striking findings: (1) in agreement with bioinformatics prediction, the N- and C-termini of Nogo-B were experimentally demonstrated to be intrinsically unstructured by CD, two-dimensional 1H 15N NMR HSQC, hydrogen exchange, and 15N heteronuclear NOE characterization. (2) Further studies showed that the 1016-residue N-terminus of Nogo-A was again highly disordered. Therefore, it appears that being intrinsically-unstructured allows Nogo molecules to serve as double-faceted functional players, with one set of functions involved in cellular signaling processes essential for CNS neuronal regeneration, vascular remodeling, apoptosis and so forth and with another in generating/maintaining membrane-related structures. We propose that this mechanism may represent a general strategy to place the formation/maintenance of membrane-related structures under the direct regulation of the cellular signaling.     

Localization of the N-terminal methionine of rat liver cytochrome P-450 in the lumen of the endoplasmic reticulum

Recent cumulative evidence suggests that liver microsomal cytochrome P-450 (P-450) is exposed to the cytosol with the exception of the N-terminal peptide (amino acid residues 1 to 21), or two peptides (residues 1 to 60). We tested the localization of the N-terminal methionine residue of P-450IIB1 of rat liver microsomes in the natural membrane with the site-specific reagent fluorescein isothiocyanate. The N-terminus of isolated P-450 was stoichiometrically modified in solution with fluorescein isothiocyanate. In intact microsomes, the N-terminus was not modified but became accessible to the reagent when the membrane was dissolved with Triton X-100. Our results indicate that the N-terminus faces the lumen of the endoplasmic reticulum, and we propose that P-450 spans the membrane only once with amino acid residues 1 to 21.     

Characterization of a self-splicing mini-intein and its conversion into autocatalytic N- and C-terminal cleavage elements: facile production of protein building blocks for protein ligation

The determinants governing the self-catalyzed splicing and cleavage events by a mini-intein of 154 amino acids, derived from the dnaB gene of Synechocystis sp. were investigated. The residues at the splice junctions have a profound effect on splicing and peptide bond cleavage at either the N- or C-terminus of the intein. Mutation of the native Gly residue preceding the intein blocked splicing and cleavage at the N-terminal splice junction, while substitution of the intein C-terminal Asn154 resulted in the modulation of N-terminal cleavage activity. Controlled cleavage at the C-terminal splice junction involving cyclization of Asn154 was achieved by substitution of the intein N-terminal cysteine residue with alanine and mutation of the native C-extein residues. The C-terminal cleavage reaction was found to be pH-dependent, with an optimum between pH6.0 and 7.5. These findings allowed the development of single junction cleavage vectors for the facile production of proteins as well as protein building blocks with complementary reactive groups. A protein sequence was fused to either the N-terminus or C-terminus of the intein, which was fused to a chitin binding domain. The N-terminal cleavage reaction was induced by 2-mercaptoethanesulfonic acid and released the 43kDa maltose binding protein with an active C-terminal thioester. The 58kDa T4 DNA ligase possessing an N-terminal cysteine was generated by a C-terminal cleavage reaction induced by pH and temperature shifts. The intein-generated proteins were joined together through a native peptide bond. This intein-mediated protein ligation approach opens up novel routes in protein engineering.     

Impact of N-terminal acetylation of α-synuclein on its random coil and lipid binding properties

N-Terminal acetylation of α-synuclein (aS), a protein implicated in the etiology of Parkinson's disease, is common in mammals. The impact of this modification on the protein's structure and dynamics in free solution and on its membrane binding properties has been evaluated by high-resolution nuclear magnetic resonance and circular dichroism (CD) spectroscopy. While no tetrameric form of acetylated aS could be isolated, N-terminal acetylation resulted in chemical shift perturbations of the first 12 residues of the protein that progressively decreased with the distance from the N-terminus. The directions of the chemical shift changes and small changes in backbone (3)J(HH) couplings are consistent with an increase in the α-helicity of the first six residues of aS, although a high degree of dynamic conformational disorder remains and the helical structure is sampled <20% of the time. Chemical shift and (3)J(HH) data for the intact protein are virtually indistinguishable from those recorded for the corresponding N-terminally acetylated and nonacetylated 15-residue synthetic peptides. An increase in α-helicity at the N-terminus of aS is supported by CD data on the acetylated peptide and by weak medium-range nuclear Overhauser effect contacts indicative of α-helical character. The remainder of the protein has chemical shift values that are very close to random coil values and indistinguishable between the two forms of the protein. No significant differences in the fibrillation kinetics were observed between acetylated and nonacetylated aS. However, the lipid binding properties of aS are strongly impacted by acetylation and exhibit distinct behavior for the first 12 residues, indicative of an initiation role for the N-terminal residues in an "initiation-elongation" process of binding to the membrane.     

Amino acid sequence of the N-terminal non-collagenous segment of dermatosparactic sheep procollagen type I.

The non-collagenous N-terminal segment of type I procollagen from dermatosparactic sheep skin was isolated in the form of the peptide Col 1 from a collagenase digest of the protein. The peptide has a blocked N-terminus, which was identified as pyrrolid-2-one-5-carboxylic acid. Appropriate overlapping fragments were prepared from reduced and alkylated peptide Col 1 by cleavage with trypsin at lysine, arginine and S-aminoethyl-cysteine residues and by cleavage with staphylococcal proteinase at glutamate residues. Amino acid sequence analysis of these fragments by Edman degradation and mass spectrometry established the whole sequence of peptide Col 1 except for a peptide junction (7--8) and a single Asx residue (44), and demonstrated that peptide Col 1 consists of 98 amino acid residues. The N-terminal portion of peptide Col 1 (86 residues) shows an irregular distribution of glycine, whereas the C-terminal portion (12 residues) possesses the triplet structure Gly-Xy and is apparently derived from the precursor-specific collagenous domain of procollagen. The central region of the peptide contains ten cysteine residues located between positions 18 and 73 and shows alternating polar and hydrophobic sequence elements. The regions adjacent to the cysteine-rich portion have a hydrophilic nature and are abundant in glutamic acid. The data are consistent with previous physicochemical and immunological evidence that distinct regions at the N- and C-termini of the non-collagenous domain possess a less rigid conformation than does the central portion of the molecule.     

Molecular dissection of Salmonella FliH,a regulator of the ATPase FliI and the type III flagellar protein export pathway

FliH is a soluble component of the flagellar export apparatus that binds to the ATPase FliI, and negatively regulates its activity. The 235-amino-acid FliH dimerizes and interacts with FliI to form a hetero-trimeric (FliH)2FliI complex. In the present work, the importance of different regions of FliH was examined. A set of 24 scanning deletions of 10 amino acids was constructed over the entire FliH sequence, along with several combined deletions of 40 amino acids and truncations of both N- and C-termini. The mutant proteins were examined with respect to (i) complementation; (ii) dominance and multicopy effects; (iii) interaction with wild-type FliH; (iv) interaction with FliI; (v) inhibition of the ATPase activity of FliI; and (vi) interaction with the putative general chaperone FliJ. Analysis of the deletion mutants revealed a clear functional demarcation between the FliH N- and C-terminal regions. The 10-amino-acid deletions throughout most of the N-terminal half of the sequence complemented and were not dominant, whereas those throughout most of the C-terminal half did not complement and were dominant. FliI binding was disrupted by C-terminal deletions from residue 101 onwards, indicating that the C-terminal domain of FliH is essential for interaction with FliI. FliH dimerization was abolished by deletion of residues 101-140 in the centre of the sequence, as were complementation, dominance and interaction with FliI and FliJ. The importance of this region was confirmed by the fact that fragment FliHC2 (residues 99-235) interacted with FliH and FliI, whereas fragment FliHC1 (residues 119-235) did not. FliHC2 formed a relatively unstable complex with FliI and showed biphasic regulation of ATPase activity, suggesting that the FliH N-terminus stabilizes the (FliH)2FliI complex. Several of the N-terminal deletions tested permitted close to normal ATPase activity of FliI. Deletion of the last five residues of FliH caused a fivefold activation of ATPase activity, suggesting that this region of FliH governs a switch between repression and activation of FliI. Deletion of the first 10 residues of FliH abolished complementation, severely reduced its interaction with FliJ and uncoupled its role as a FliI repressor from its other export functions. Based on these data, a model is presented for the domain construction and function of FliH in complex with FliI and FliJ.     

The expression of multiple forms of troponin T in chicken-fast-skeletal muscle may result from differential splicing of a single gene

Troponin T isolated from chicken fast skeletal muscle has been shown to be present in three different molecular forms, one in breast and two in leg muscle. The three forms differ in both size and charge. Troponin T from breast muscle has a molecular mass of 33.5 kDa and a pI of about 7. Of the two leg muscle forms the larger has a molecular mass of 30.5 kDa and a pI of about 8.5 and the smaller a molecular mass of 29.8 kDa and a pI of about 10. Considerably more heterogeneity has been found in the leg than in the breast muscle proteins although this is not reflected in their N-terminal sequences. The reason for this is not clear. Troponin T from breast or leg muscle can be phosphorylated with troponin T kinase at the single serine residue at the N-terminus. No difference in the rate or extent of phosphorylation could be found between proteins from breast or leg muscle. The three proteins have been shown to differ only in the amino acid sequence of their N-terminal tryptic peptides. These peptides are of different length, that from breast troponin T being 58 residues and those from leg troponin T being 36 and 42 residues, these differences account for the difference in molecular mass of the parent proteins. Despite this difference the sequence of the first 12 and last 14 residues is identical in all three N-terminal peptides. The remainder of the sequence of the smallest peptide is also repeated in the other two but they each contain an extra piece of unique sequence. On the basis of these sequences it is proposed that chicken troponin T is coded for by a single gene containing, at the 5' end, a number of small exons and that three different mRNA molecules may be produced by alternative pathways of RNA splicing. The possible significance of these N-terminal sequence variations is discussed.     

Conformational changes in phospholipase A2 upon binding to micellar interfaces in the absence and presence of competitive inhibitors. A 1H and 15N NMR study.

An NMR study has been made of porcine pancreatic phospholipase A2 (PLA) in three environments: free in solution, in a binary complex with dodecylphosphocholine micelles, and in a ternary complex with a micelle and the substrate-like inhibitor (R)-1-octyl-2-(N-dodecanoylamino)-2-deoxyglycero-3-phosph oglycol. 1H and 15N chemical shifts, amide exchange rates, and NOE intensities are compared for the enzyme in different environments. From these data, structural differences are found for the N-terminal part, the end of the surface loop at residues Tyr69 and Thr70, and the active site residue His48, and also for the Ca-binding loop (residues 28-32). Specifically, when binding to a micelle, the side chains of residues Ala1, Trp3, and Tyr69, as well as all protons of Thr70, are found to be closer together. After subsequent introduction of the competitive inhibitor, further changes are found for these residues. The N-terminus is flexible in PLA free in solution, in contrast with the crystal structures where it adopts an alpha-helical conformation. According to the NMR data, this helix is rigidly formed only in the ternary complex. Furthermore, in the ternary complex, the N-terminal amino group and the exchangeable hydrogen at N3 of the ring of His48 are observed. We propose that PLA is activated in two steps. An initial conformational change occurs upon binding to a micellar interface. The catalytically active conformation of the enzyme, which has an extensive network of hydrogen bonds, is formed only when binding a substrate or competitive inhibitor at a lipid-water interface.     

Serratia protease. Amino acid sequences of both termini, the 53 residues in the middle region containing the sole methionine residue, and a probable zinc-binding region

Reexamination of the molecular mass and the amino acid composition of Serratia protease revealed the presence of 1 mol of methionine per mol of protein (about 46K daltons), and this was confirmed by BrCN cleavage followed by separation of the two fragments. The sole methionine residue was located near the middle region of the molecule. The amino(N)-terminal sequence was determined by Edman degradation of the protein and studies of several proteolytic peptides, establishing a sequence of 18 residues with a heterogeneous N-terminus. The carboxyl(C)-terminal sequence was determined by carboxypeptidase A digestion and tritium-labeling of the citraconylated C-terminal half segment to be -Phe-Ile-Val. The sequences of a total of 53 residues containing the methionine residue and a total of 38 residues containing two histidine residues were established by the application of various conventional methods to a BrCN peptide and several proteolytic peptides. The segment containing the histidine residues was homologous with that containing the two histidine residues chelating the zinc atom of thermolysin. The 38-residue segment may be directly connected to the 53-residue segment.     

The presence of N-terminal methionine on nascent protein of rat liver and rabbit reticulocytes and its cleavage during polypeptide-chain elongation

1. The size of nascent globin peptides from which the N-terminal methionine residue is cleaved has been investigated by comparing the proportion of N-terminal methionine and valine in short and long chains. Nascent chains were labelled in rabbit reticulocyte lysates, fractionated according to length by chromatography on Sephadex G-50, and analysed by the Edman degradation of selected pooled fractions. It was found that different peptide fractions contained either methionine or valine, but not both, as the N-terminal residue. Methionine was present at the N-terminus of globin chains containing up to approx. 50 amino acids whereas valine was found to be the N-terminal amino acid of longer peptides. 2. In similar experiments with nascent proteins of rat liver, labelled either in vivo or in a cell-free system containing microsomal material and cell sap, evidence was obtained for the presence of methionine at the N-terminus of nascent chains up to approx. 65 amino acid residues long. Thus protein synthesis in liver appears to be initiated also by methionine, but in this case cleavage takes place somewhat later during peptide elongation than in globin synthesis.