Sequence Lys Research Articles

A Peptide-Induced Self-Cleavage Reaction Initiates the Activation of Tyrosinase.

The conversion of inactive pro‐polyphenol oxidases (pro‐PPOs) into the active enzyme results from the proteolytic cleavage of its C‐terminal domain. Herein, a peptide‐mediated cleavage process that activates pro‐MdPPO1 (Malus domestica) is reported. Mass spectrometry, mutagenesis studies, and X‐ray crystal‐structure analysis of pro‐MdPPO1 (1.35 Å) and two separated C‐terminal domains, one obtained upon self‐cleavage of pro‐MdPPO1 and the other one produced independently, were applied to study the observed self‐cleavage. The sequence Lys 355–Val 370 located in the linker between the active and the C‐terminal domain is indispensable for the self‐cleavage. Partial introduction (Lys 352–Ala 360) of this peptide into the sequence of two other PPOs, MdPPO2 and aurone synthase (CgAUS1), triggered self‐cleavage in the resulting mutants. This is the first experimental proof of a self‐cleavage‐inducing peptide in PPOs, unveiling a new mode of activation for this enzyme class that is independent of any external protease.

Cyclic peptide models of the Ca 2+-binding loop of α-lactalbumin

A series of cyclic peptides with different linkers were designed and synthesized to model the elbow-type Ca 2+-binding loop of α-lactalbumin (LA). All amino acids of the Ca 2+-binding loop are strikingly well conserved among LAs of different species with the sequence Lys 79-Phe-Leu-Asp 82-Asp-Asp-Leu-Thr- Asp 87-Asp 88, where three carboxylates of Asp 82, Asp 87, and Asp 88 and the amide carbonyl oxygen atoms of Lys 79 and Asp 84 participate in Ca 2+ binding. Alanine-containing models were also prepared for monitoring the role of the binding (82, 87–88) and nonbinding Asp residues (83–84) in coordinating the cation. The structural features of synthetic peptides and their Ca 2+-binding properties were investigated in solution by circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy. In water, the CD curves show a strong negative band below 200 nm as a sign of the presence of unfolded conformers. In TFE, all cyclic peptides were found to have a CD spectrum, reflecting the presence of folded (turn) conformers. The effect of Ca 2+ was dependent on the structure and concentration of the model and the Ca 2+ to peptide ratio ( r cat). A surprising time dependence of the FTIR spectra of Ca 2+ complexes of the Ala-containing peptides was observed. The shape of the broad amide I band showed no more change after ∼60 min. Contrary to this, the deprotonation of the side chain COOH group(s) and formation of the final coordination sphere of Ca 2+ took more time. Infrared spectra showed that in the Ca 2+ complex of model comprising the binding Asp residues of LA, the cation is coordinated to the COO − groups of all three Asps, while in the complex of model comprising nonbinding Asp residues of LA, the two neighboring Asp side chains form a bridged Ca 2+-binding system.

Cysteine Labeling Studies Detect Conformational Changes in Region 106−132 of the Mitochondrial ADP/ATP Carrier of Saccharomyces cerevisiae

To know the structural and functional features of the cytosolic-facing first loop (LC1) including its surrounding region of the mitochondrial ADP/ATP carrier (AAC), we prepared 27 mutants, in which each amino acid residue between residues 106 and 132 of the yeast type 2 AAC (yAAC2) was replaced by a cysteine residue. For mutant preparation, we used a Cys-less AAC mutant, in which all four intrinsic cysteine residues were substituted with alanine residues, as a template [Hatanaka, T., Kihira, Y., Shinohara, Y., Majima, E., and Terada, H. (2001) Biochem. Biophys. Res. Commun. 286, 936-942]. From the labeling intensities of the membrane-impermeable SH-reagent eosin-5-maleimide (EMA), sequence Lys(108)-Phe(127) was suggested to constitute the LC1. The N-terminal half of this region (Lys(108)-Phe(115)) was suggested to change its location from the cytosol to a region close to the membrane on conversion from the c-state to the m-state in association with disruption or unwinding of its alpha-helical structure, whereas the C-terminal half region (Gly(116)-Phe(127)) was considered to extrude essentially into the cytosol, while keeping its alpha-helical structure. Hence, the conformation of m-state LC1 is greatly different from that of c-state LC1. Possibly the LC1 changes its location between the membranous region and the cytosol during ADP/ATP transport. Lys(108) in the LC1 of the yAAC2 was found to be associated with binding of the transport substrates, and its -NH(3)(+) moiety, to be of importance for the transport function. On the basis of these results, possible roles of the conformational changes of the LC1 in the transport activity are discussed.

Interaction of aluminium ions with some amino acids present in human blood.

The interaction of aluminium with some amino acids present in human blood was studied combining ion-chromatography (IC), atomic absorption spectrometry (AAS) and ultrafiltration (UF) techniques. An IC system for simultaneous determination of ornithine, lysine, glutamic acid, aspartic acid and tyrosine was developed. By adding aluminium to standard solutions of the amino acids and keeping the pH at 6 and 7 it was possible to verify that aluminium caused a reduction on the amino acid chromatographic signals. Similar experiment, carried out for copper showed the same behaviour (with different percentage of signal reductions) and validated the results for aluminium, considering that the interaction Cu-amino acid is well-established. The AAS analysis of sample fractions (500 microl) after the IC separation showed that aluminium (as copper as well) is not present in the fractions in which the amino acid peaks appear in the chromatogram. These approaches carried out with serum samples after UF showed that part of the "free" fraction of serum aluminium is distributed, besides other ligands, among these amino acids. It was found that in serum the affinity for aluminium followed the sequence Lys>Orn>Tyr>Glu approximately Asp.

Interaction of Inhibitor-2 with the Catalytic Subunit of Type 1 Protein Phosphatase: IDENTIFICATION OF A SEQUENCE ANALOGOUS TO THE CONSENSUS TYPE 1 PROTEIN PHOSPHATASE-BINDING MOTIF

Inhibitor-2 (I-2) is the regulatory subunit of a cytosolic type 1 Ser/Thr protein phosphatase (PP1) and potently inhibits the activity of the free catalytic subunit (CS1). Previous work from the laboratory had proposed that the interaction of I-2 with CS1 involved multiple sites (Park, I. K., and DePaoli-Roach, A. A. (1994) J. Biol. Chem. 269, 28919-28928). The present study refines the earlier analysis and arrives at a more detailed model for the interaction between I-2 and CS1. Although the NH(2)-terminal I-2 regions containing residues 1-35 and 1-64 have no inhibitory activity on their own, they increase the IC(50) for I-2 by approximately 30-fold, indicating the presence of a CS1-interacting site. Based on several experimental approaches, we have also identified the sequence Lys(144)-Leu-His-Tyr(147) as a second site of interaction that corresponds to the RVXF motif present in many CS1-binding proteins. The peptide I-2(135-151) significantly increases the IC(50) for I-2 and attenuates CS1 inhibition. Replacement of Leu and Tyr with Ala abolishes the ability to counteract inhibition by I-2. The I-2(135-151) peptide, but not I-2(1-35), also antagonizes inhibition of CS1 by DARPP-32 in a pattern similar to that of I-2. Furthermore, a peptide derived from the glycogen-binding subunit, R(GL)/G(M)(61-80), which contains a consensus CS1-binding motif, completely counteracts CS1 inhibition by I-2 and DARPP-32. The NH(2)-terminal 35 residues of I-2 bind to CS1 at a site that is specific for I-2, whereas the KLHY sequence interacts with CS1 at a site shared with other interacting proteins. Other results suggest the presence of yet more sites of interaction. A model is presented in which multiple "anchoring interactions" serve to position a segment of I-2 such that it sterically occludes the catalytic pocket but need not make high affinity contacts itself.

Biological activity of recombinant factor VIII variants lacking the central B-domain and the heavy-chain sequence Lys713-Arg740: discordant in vitro and in vivo activity.

Recombinant factor VIII variants with overlapping deletions spanning the region Lys713-Ile1668 have been expressed in mammalian cells, and analysed for biological activity both in vitro and in vivo. Two distinct assay systems were used to measure the activity in vitro. The one-stage coagulation assay served to assess factor VIII procoagulant activity while a spectrophotometric assay was used for the quantification of factor VIII cofactor activity in factor IXa-dependent factor X activation. Deletion of the entire B-domain (Ser741-Arg1648) resulted in a protein with similar procoagulant and cofactor activity. In contrast, factor VIII-del(713-1637), which has a deletion that also comprises the heavy-chain sequence Lys713-Arg740, had lost factor VIII procoagulant activity while factor VIII cofactor activity was retained. This functional inconsistency was further addressed by comparing purified factor VIII-del(713-1637) with factor VIII-del(868-1562), a mutant with normal in vitro activity. Kinetic studies of factor Xa formation revealed that higher concentrations of thrombin were required to develop the cofactor activity from factor VIII-del(713-1637) than needed for factor VIII-del(868-1562) or plasma factor VIII. The physiological significance of this finding was assessed in dogs with haemophilia A. Both deletion mutants were similar to plasma factor VIII with regard to binding to von Willebrand factor and half-life and recovery. Employing the cuticle bleeding time model, factor VIII-del(868-1562) was found to be indistinguishable from plasma factor VIII, whereas factor VIII-del(713-1637) was less effective. The increased thrombin-resistance of factor VIII-del(713-1637) thus limits both procoagulant activity and haemostatic efficacy in cuticle bleeding. These observations suggest that the heavy-chain sequence Lys713-Arg740, although dispensable for factor VIII cofactor function per se, is involved in the proteolytic activation of factor VIII both in vitro and in vivo.

Template assembled synthetic proteins: Condensation of a multifunctional peptide to a topological template via chemoselective ligation

A chemoselective ligation via oxime bond formation is used for the chemical synthesis of template assembled peptides according to the TASP (Template Assembled Synthetic Proteins) approach. Aminooxyacetylation of the multifunctional partial sequence Lys- Arg- Asp- Ser of lactoferrin and subsequent condensation in aqueous solution with a topological template containing four selectively addressable aldehyde functions as attachment sites gives readily access to the TASP molecule.