scholarly journals The semisynthesis of fragments corresponding to residues 66-104 of horse heart cytochrome c

1979 ◽  
Vol 179 (1) ◽  
pp. 169-182 ◽  
Author(s):  
C J Wallace ◽  
R E Offord
Keyword(s):  
Biological Activity ◽  
Amino Acid ◽  
Cytochrome C ◽  
Amino Acid Sequences ◽  
Side Chain ◽  
Horse Heart Cytochrome ◽  
Natural Sequence ◽  
C Terminus ◽  
N Terminus ◽  
Horse Heart Cytochrome C

We describe the N epsilon-acetimidylation of horse heart cytochrome c with retention of biological activity, the cleavage of the modified protein by CNBr, the separation of the fragments, and their further side-chain protection. We describe the manipulation of the amino acid sequences of the fragments by stepwise semisynthetic methods. We have prepared fragments corresponding to residues 66-78 and 66-79 of the protein, as well as the [Asp66] analogue of fragment 66-79. We have prepared the natural sequence and the [o-fluoro-Phe82] analogue of the fragment corresponding to residues 81-104 of the protein, and the [N epsilon-trifluoroacetyl-Lys79], the [N epsilon-dinitrophenyl-Lys79] and the [S-acetamidomethyl-Cys79] analogues of fragment 79-104, and the [N epsilon-Cbz-Lys81] analogue of fragment 80-104. We have coupled back the fragments of natural sequence to form a semisynthetic fragment corresponding to residues 66-104 of the protein. Modified fragments were also coupled to give analogues of the 66-104-residue sequence. In every case the homoserine residue representing methionine-80 was removed from the C-terminus of the 66-80-residue fragment and replaced by methionine on the N-terminus of the 81-104 residue fragment during the preparation of the fragments for coupling. The semisynthetic fragments are ready for specific deprotection and further coupling. We have coupled one such fragment to the (1-65)-peptide to produce semisynthetic [Hse65]cytochrome c. The product has satisfactory characteristics on chemical analysis, and on assay of its biological activity.

scholarly journals Enzyme-assisted semisynthesis of polypeptide active esters and their use

1988 ◽  
Vol 249 (1) ◽  
pp. 83-88 ◽  
Author(s):  
K Rose ◽  
C Herrero ◽  
A E I Proudfoot ◽  
R E Offord ◽  
C J A Wallace
Keyword(s):  
Amino Acid ◽  
Cytochrome C ◽  
Amino Acid Ester ◽  
Amino Group ◽  
Horse Heart Cytochrome ◽  
C Terminus ◽  
Active Ester ◽  
Chemical Coupling ◽  
Concentrated Solution ◽  
Horse Heart Cytochrome C

A method is described for the preparation of polypeptides activated uniquely at the C-terminus. The polypeptide is incubated in a concentrated solution of an amino acid active ester, the latter having its amino group free but adequately protected by protonation. The amino acid ester is coupled via its amino group to the C-terminus of the polypeptide by enzymic catalysis (reverse proteolysis). The resulting polypeptide C-terminal active ester is then isolated and coupled to a suitable amino component (generally a polypeptide) in a subsequent chemical coupling. The method appears to be generally applicable; fragments of horse heart cytochrome c, and porcine insulin, are used as examples. Two new analogues of cytochrome c have been prepared by using this method, with yields of up to 60% in the final coupling. Scope and limitations of the method are discussed.

scholarly journals The primary structure of cytochrome c from the nematode Caenorhabditis elegans

1990 ◽  
Vol 271 (3) ◽  
pp. 613-620 ◽  
Author(s):  
J R Vanfleteren ◽  
E A I M Evers ◽  
G Van de Werken ◽  
J J Van Beeumen
Keyword(s):  
Amino Acid ◽  
Caenorhabditis Elegans ◽  
Cytochrome C ◽  
Amino Acid Sequences ◽  
Similar Data ◽  
Amino Acid Residues ◽  
Nematode Caenorhabditis Elegans ◽  
Horse Heart Cytochrome ◽  
Human Cytochrome ◽  
Horse Heart Cytochrome C

The complete amino acid sequence of cytochrome c from the nematode Caenorhabditis elegans was determined. The native protein displays the same spectral properties in the oxidized and reduced states as horse heart cytochrome c. The apoprotein consists of 110 amino acid residues and differs from human cytochrome c by 44 substitutions, one internal deletion, five N-terminal additions and two C-terminal additions. One of the substitutions is the replacement of an ‘invariant’ phenylalanine residue at position 15 by tyrosine. The N-terminal sequence extension contains a short peptide motif, which is highly homologous with a peptide fragment present at the N-terminus of annelid and insect cytochrome c sequences. From the number of amino acid changes and the evolutionary rate of cytochrome c it would appear that nematodes diverged from a line leading to man about 1.4 billion years ago. When similar data based on the amino acid sequences of the histones H1, H2A, H2B and H3 are taken into account, the average estimate is 1.1 +/- 0.1 billion years.

scholarly journals The Extracellular Transport Signal of the Vibrio cholerae Endochitinase (ChiA) Is a Structural Motif Located between Amino Acids 75 and 555

2002 ◽  
Vol 184 (8) ◽  
pp. 2225-2234 ◽  
Author(s):  
Jason P. Folster ◽  
Terry D. Connell
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Vibrio Cholerae ◽  
Structural Information ◽  
Amino Acid Sequences ◽  
Structural Motif ◽  
Terminal Branch ◽  
C Terminus ◽  
N Terminus ◽  
Extracellular Transport

ABSTRACT ChiA, an 88-kDa endochitinase encoded by the chiA gene of the gram-negative enteropathogen Vibrio cholerae, is secreted via the eps-encoded main terminal branch of the general secretory pathway (GSP), a mechanism which also transports cholera toxin. To localize the extracellular transport signal of ChiA that initiates transport of the protein through the GSP, a chimera comprised of ChiA fused at the N terminus with the maltose-binding protein (MalE) of Escherichia coli and fused at the C terminus with a 13-amino-acid epitope tag (E-tag) was expressed in strain 569B(chiA::Kanr), a chiA-deficient but secretion-competent mutant of V. cholerae. Fractionation studies revealed that blockage of the natural N terminus and C terminus of ChiA did not prevent secretion of the MalE-ChiA-E-tag chimera. To locate the amino acid sequences which encoded the transport signal, a series of truncations of ChiA were engineered. Secretion of the mutant polypeptides was curtailed only when ChiA was deleted from the N terminus beyond amino acid position 75 or from the C terminus beyond amino acid 555. A mutant ChiA comprised of only those amino acids was secreted by wild-type V. cholerae but not by an epsD mutant, establishing that amino acids 75 to 555 independently harbored sufficient structural information to promote secretion by the GSP of V. cholerae. Cys77 and Cys537, two cysteines located just within the termini of ChiA(75-555), were not required for secretion, indicating that those residues were not essential for maintaining the functional activity of the ChiA extracellular transport signal.

Structural relationships in the two-zinc insulin hexamer

1979 ◽  
Vol 57 (6) ◽  
pp. 469-479 ◽  
Author(s):  
E. J. Dodson ◽  
G. G. Dodson ◽  
D. C. Hodgkin ◽  
C. D. Reynolds
Keyword(s):  
Crystal Structure ◽  
Biological Activity ◽  
Main Chain ◽  
Side Chain ◽  
C Terminus ◽  
Structural Relationships ◽  
N Terminus ◽  
Independent Molecules ◽  
A Chain ◽  
Resolution Data

The refinement of the crystal structure of two-Zn pig insulin using 1.5-Å (1 Å = 0.1 nm) resolution data by Fourier and fast Fourier least-squares methods allows us to make detailed comparisons between the two independent molecules present in the two-Zn insulin dimer and to describe their interactions in the monomer, dimer, and hexamer. The main chain structures for the two molecules agree well except at the N terminus of the A chain and the C terminus of the B chain. The residues along the line of the local two-fold axes, apart from theB25 side chain, conform extremely closely to the two-fold symmetry, although the discrepancies are much more apparent away from this axis. The ability of the insulin molecule to adopt different conformations may be an important factor in the expression of its biological activity.

Amino-Acid Sequence of Horse Heart Cytochrome C : The Complete Amino-acid Sequence

Nature ◽  
1961 ◽  
Vol 192 (4808) ◽  
pp. 1125-1127 ◽  
Author(s):  
E. MARGOLIASH ◽  
EMIL L. SMITH ◽  
GUNTHER KREIL ◽  
HANS TUPPY
Keyword(s):  
Amino Acid ◽  
Cytochrome C ◽  
Amino Acid Sequence ◽  
Horse Heart Cytochrome ◽  
Complete Amino Acid Sequence ◽  
Horse Heart Cytochrome C
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