Interactions of BPN' and Carlsberg subtilisins with peptides containing aromatic amino acids at the C-terminus. Specific rate enhancement due to the secondary enzyme-substrate interaction.

The interaction between BPN' or Carlsberg subtilisins and peptides of the type Ac-Glyn-X-OMe (n = 0, 1, 2, 3), where X denotes one of five different aromatic amino acids, was investigated to elucidate the effect of the secondary interaction on catalysis in relation to the nature of the X residue. The increase in interaction upon elongation of the chain was accompanied by a large increase in kcat but with no marked change in Km in all the series of sensitive substrates. The peptides containing 2-(2-nitro-4-carboxyphenylsulfenyl)-tryptophan, however, acted as competitive inhibitors and exhibited an invariant dissociation constant in spite of the different chain lengths. These observations suggest that the secondary enzyme-substrate interaction induces a conformational change in the active site of the enzyme or in the substrate in such a way as to lower the activation energy and to form a stabilized transient complex. In this respect, BPN' and Carlsberg subtilisins are similar to porcine pepsin and Streptomyces griseus protease 1 rather than to alpha-chymotrypsin.     

Interactions of tryptophan, tryptophan peptides, and tryptophan alkyl esters at curved membrane interfaces

Motivated by ongoing efforts to understand the mechanism of membrane protein crystallogenesis and transport in the lipidic cubic phase, the nature of the interaction between tryptophan and the bilayer/aqueous interface of the cubic phase has been investigated. The association was quantified by partitioning measurements that enabled the free energy of interaction to be determined. Temperature-dependent partitioning was used to parse the association free energy change into its enthalpic and entropic components. As has been observed with tryptophan derivatives interacting with glycerophospholipid bilayers in vesicles, tryptophan partitioning in the cubic phase is enthalpy driven. This is in contrast to partitioning into apolar solvents, which exhibits the classic hydrophobic effect whose hallmark is a favorable entropy change. These results with tryptophan are somewhat surprising given the simplicity, homogeneity, and curvature of the interface that prevails in the case of the cubic phase. Nevertheless, the interaction between tryptophan and the mesophase is very slight as revealed by its low partition coefficient. Additional evidence in support of the interaction was obtained by electronic absorption and fluorescence spectroscopy and fluorescence quenching. Partitioning proved insensitive to the lipid composition of the membrane, examined by doping with glycerophospholipids. However, the interaction could be manipulated in meaningful ways by the inclusion in the aqueous medium of salt, glycerol, or urea. The effects seen with tryptophan were amplified rationally when measurements were repeated using tryptophan alkyl esters and with tryptophan peptides of increasing length. These findings are interpreted in the context of the insertion, folding, and function of proteins in membranes.     

Cellular uptake and biophysical properties of galactose and/or tryptophan containing cell-penetrating peptides

Glycosylated cell penetrating peptides (CPPs) have been conjugated to a peptide cargo and the efficiency of cargo delivery into wild type Chinese hamster ovary (CHO) and proteoglycan deficient CHO cells has been quantified by MALDI-TOF mass spectrometry and compared to tryptophan- or alanine containing CPPs. In parallel, the behavior of these CPPs in contact with model membranes has been characterized by different biophysical techniques: Differential Scanning and Isothermal Titration Calorimetries, Imaging Ellipsometry and Attenuated Total Reflectance IR spectroscopy. With these CPPs we have demonstrated that tryptophan residues play a key role in the insertion of a CPP and its conjugate into the membrane: galactosyl residues hampered the internalization when introduced in the middle of the amphipathic secondary structure of a CPP but not when added to the N-terminus, as long as the tryptophan residues were still present in the sequence. The insertion of these CPPs into membrane models was enthalpy driven and was related to the number of tryptophans in the sequence of these secondary amphipathic CPPs. Additionally, we have observed a certain propensity of the investigated CPP analogs to aggregate in contact with the lipid surface.     

An autoradiographic technique for the detection of cystine and peptides containing cystine or its derivatives in keratin digests

Summary A technique is described for the detection of cystine or peptides containing cystine or its derivatives in digests of keratin. The technique involves the preparation of radioactive wool by the intravenous injection of experimental animals with (³⁵S) cystine followed by the growth of keratinolytic fungi upon the labelled wool and the detection of soluble compounds in the wool digests which contain cystine or its derivatives by paper chromatography and autoradiography.

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Zusammenfassung Eine Methode für den Nachweis von Cystin oder Peptiden in Keratindigerierungen, die Cystinderivate enhalten, wird beschrieben. Sie besteht in der Herstellung radioaktiver Wolle durch intravenöse Injektion von (³⁵S) Cystin in Schafe, Kultur der keratinolytischen Pilze auf die erzeugte radioaktive Wolle, sowie in dem papierchromatographischen und autoradiographischen Nachweis von löslichen Substanzen in entsprechenden Kulturfiltraten, die Cystin oder Cystinderivate enthalten.

     

Oxidation of tryptophan to oxindolylalanine by dimethyl sulfoxide-hydrochloric acid. Selective modification of tryptophan containing peptides

Tryptophan is readily oxidized to oxindolylalanine (2-hydroxytryptophan) in good yield on treatment in acetic acid solution with a mixture of dimethyl sulfoxide (DMSO) and concentrated aqueous HCl at room temperature. Other sulfoxides can be used in combination with HCl; for example, methionine sulfoxide reacts with an equimolar amount of tryptophan to give high yields of methionine and oxindolylalanine. Methionine and cysteine are quantitatively oxidized by DMSO/HCl to methionine sulfoxide and cystine, respectively. The tryptophan containing peptides LRF (luteinizing hormone-releasing factor), somatostatin, valine-gramicidin A and ACTH 1-24 were each treated with the DMSO/HCl reagent in acetic acid solution and the corresponding oxindolylalanine-derivatives isolated in over 90% yield after chromatography. The identity and purity of the derivatives were established on the basis of ultraviolet spectral characteristics and quantitative amino acid analysis of the oxindolylalanine content of acid hydrolyzates of the oxidized peptides with 3N-p-toluenesulfonic acid at 110 degrees for 24 h. The results indicate that modification of tryptophan peptides with DMSO/HCl provides a useful procedure, which seems superior to previously used reagents. In addition, the method could be well applied to other indoles of biological and pharmacological interest.     

Interaction of indole derivatives and tryptophan peptides with interfaces of sodium dodecyl sulfate micelles.

The free energies of transfer for indole and tryptophan derivatives and pentapeptides having single tryptophan residues from aqueous to sodium dodecyl sulfate (SDS) micellar phases have been systematically studied using the conventional method of ultraviolet absorption spectrophotometry. The free energies for the position isomers of methyl indoles varied depending on the substitution positions. Thus, the contribution of the methyl group to the binding affinity of the 4-methyl indole to the micelle was about twice that of the 2- and 7-methyl indoles. The free energy changes with the introduction of halogen groups to the indole rings were correlated to the nonpolar water-accessible surface area (DeltaA(np)) of the halogen moieties, which were regarded as hydrophobic. The relationships followed straight lines passing through the origins. Position dependence having tendencies similar to the methyl indoles was observed among the magnitudes of the slopes of the straight lines. These results strongly suggest that the indole rings of the derivatives residing in the micellar interface regions direct their imino moieties --NH-- toward the micellar surfaces. Experiments using model tryptophan pentapeptides showed that the magnitude of free energy change per methylene unit of an alkyl amino acid residue in the pentapeptide increased with elongation of the alkyl moiety and was not a constant value as reported for various alkyl compounds. When the peptides distribute to the SDS micelles, the peptide backbones are anchored in aqueous phases and the amino acid side chains in the interfaces extend their alkyl groups toward the micellar centers. Thus, the free energy changes can be connected to the positions of the alkyl groups of the amino acid residues in the micelles.     

Side reactions of hydrogen fluoride on peptides containing glutamic acid in or near the C-terminus in solid phase synthesis

Various peptides containing glutamic acid in or near the C-terminus were synthesized by the solid phase method and treated with anhydrous hydrogen fluoride in the presence of anisole. Compounds, apparently resulting from conversion of glutamic acid to a cyclic derivative, and presumably containing anisole, appeared while glutamic acid disappeared.     

Interactions of the C-terminus of viral protein R with nucleic acids are modulated by its N-terminus.

The basic viral protein R (Vpr) performs several functions during the human immunodeficiency virus HIV-1 retroviral cycle, including G2 mitosis arrest and nuclear import of the preintegration complex allowing lentivirus to replicate in nondividing cells. Accordingly, this protein was found in the nucleus of infected cells. In the virus, Vpr is incorporated through interaction with both nucleocapsid protein 7 (NCp7) and p6, two small proteins encoded by the C-terminal part of the Gag precursor. NCp7 is also involved in genomic RNA encapsidation during the budding process suggesting a possible interaction of Vpr with nucleic acids, either directly or via the NCp7 intermediate. Gel shift experiments were carried out with RNA and DNA using synthetic Vpr and peptide derivatives. The results show that Vpr binds to nucleic-acid inducing aggregates. This process, which requires the C-terminal basic domain of the protein (in particular the helical 70-80 domain), is regulated by the N-terminal region of Vpr. Moreover, NCp7 was shown to enhance RNA recognition by Vpr, a feature that could be required for Vpr encapsidation and during nuclear import of the preintegration complex.     

Study of the time-resolved tryptophan fluorescence of crystalline alpha-chymotrypsin

The tryptophan environments in crystalline alpha-chymotrypsin were investigated by fluorescence. The heterogeneous emission from this multitryptophan enzyme was resolved by time-correlated fluorescence spectroscopy. The fluorescence decays at 296-nm laser excitation and various emission wavelengths could be characterized by a triple-exponential function with decay times tau 1 = 150 +/- 50 ps, tau 2 = 1.45 +/- 0.25 ns, and tau 3 = 4.2 +/- 0.4 ns. The corresponding decay-associated emission spectra of the three components had maxima at about 325, 332, and 343 nm. The three decay components in this enzyme can be correlated with X-ray crystallographic data [Birktoft, J.J., & Blow, D.M. (1972) J. Mol. Biol. 68, 187-240]. Inter- and intramolecular tryptophan-tryptophan energy-transfer efficiencies in crystalline alpha-chymotrypsin were computed from the accurately known positions and orientations of all tryptophan residues. These calculations indicate that the three fluorescence decay components in crystalline alpha-chymotrypsin can be assigned to three distinct classes of tryptophyl residues. Because of the different proximity of tryptophan residues to neighboring internal quenching groups, the decay times of the three classes are different. Decay tau 1 can be assigned to Trp-172 and Trp-215 and tau 2 to Trp-51 and Trp-237, while the tryptophyl residues 27, 29, 141, and 207 all have decay time tau 3.     

MAP-1 and MAP-2 binding sites at the C-terminus of beta-tubulin. Studies with synthetic tubulin peptides

The interaction of microtubule-associated proteins MAP-1 and MAP-2 with different peptides containing sequences covering the C-terminal region of beta-tubulin isoforms has been analyzed. Our results indicate that MAP-1 and MAP-2 bind to a common sequence within the variable C-terminal region of the different beta-tubulin isoforms, while MAP-2 also interacts with the subdomain beta (422-434) of the constant region, in agreement with previous results (Maccioni, R.B., Rivas, C., & Vera, J.C. (1988) EMBO J. 7, 1957-1963). The productive interaction of MAP-2 with the latter domain appears to be involved in the assembly of microtubules.     

Bradykinin analogs containing the 4-amino-2-benzazepin-3-one scaffold at the C-terminus.

High affinity peptide ligands for the bradykinin (BK) B(2) subtype receptor have been shown to adopt a beta-turn conformation of the C-terminal tetrapeptide (H-Arg(1)-Pro(2)-Pro(3)-Gly(4)-Phe(5)-Ser(6)-Pro(7)-Phe(8)-Arg(9)-OH). We investigated the replacement of the Pro(7)-Phe(8) dipeptide moiety in BK or the D-Tic(7)-Oic(8) subunit in HOE140 (H-D-Arg(0)-Arg(1)-Pro(2)-Hyp(3)-Gly(4)-Thi(5)-Ser(6)-D-Tic(7)-Oic(8)-Arg(9)-OH) by 4-amino-1,2,4,5-tetrahydro-2-benzazepin-3-one templates (Aba). Binding studies to the human B(2) receptor showed a correlation between the affinities of the BK analogs and the propensity of the templates to adopt a beta-turn conformation. The L-spiro-Aba-Gly containing HOE140 analog BK10 has the best affinity, which correlates with the known turn-inducing property of this template. All the compounds did not modify basal inositolphosphate (IP) output in B(2)-expressing CHO cells up to 10 microM concentration. The antagonist properties were confirmed by the guinea pig ileum smooth muscle contractility assay. The new amino-benzazepinone (Aba) substituted BK analogs were found to be surmountable antagonists.     

N-Bromosuccinimide modification of tryptophan 241 at the C-terminus of the manganese stabilizing protein of plant photosystem II influences its structure and function

Life on earth depends upon the ability of oxygenic photosynthesis to oxidize water to molecular oxygen. This process is catalyzed by water–plastoquinone oxido-reductase complex. In addition to the photosystem II (PSII) reaction core, it includes a manganese stabilizing protein (MSP) that plays an important regulatory role in the process in plants and algae. Tryptophan 241, located at the carboxyl-terminus of the MSP, is its sole tryptophan. Modification of MSP by N-bromosuccinimide (NBS) was carried out to explore the role of Trp241 in maintaining its structure and function. Data and arguments are presented to show that it is Trp241, not other tyrosines in MSP, that is involved in the modification and changes observed in this study. Further, the pH-dependence of the modification and the comparison of features of fluorescence spectra of MSP suggested that Trp241 is buried in the hydrophobic interior of the protein. Hydropathy analysis revealed that Trp241 is located in the middle of the hydrophobic region at the C-terminus of MSP. Circular dichroism spectroscopy showed that NBS modification of Trp241 dramatically modified the protein structure. The affinity of MSP to PSII decreased greatly after the modification of Trp241, and no oxygen-evolving activity was recovered after its reconstitution. This study provides a novel demonstration that Trp241 at the C-terminus hydrophobic region of the MSP is critical for maintaining appropriate structure and function of MSP.     

Primary structure of human copper-zinc superoxide dismutase. Cysteine - and tryptophan - containing peptides

Analysis of soluble peptides derived from tryptic and chymotryptic digestions of carboxymethylated human superoxide dismutase gives primary structural information for approximately 67% of the protein. Regions so far elucidated appear to be highly homologous to the corresponding bovine enzyme; in particular Cys 6 and the two cysteine residues 55 and 144, which form the intrasubunit disulfide bond of the bovine enzyme, are conserved. A cluster of three substitutions including the fourth cysteine residue unique to the human enzyme has been found in positions 107–109 and may be related to the presence of persulfide groups in the human enzyme. The single tryptophan residue of the human protein is not homologous to the single tyrosine residue of the bovine protein.