Helix formation in enzymically ligated peptides as a driving force for the synthetic reaction: example of alpha-globin semisynthetic reaction.
The alpha-globin semisynthetic reaction, namely, the ligation of the complementary fragments of alpha-globin, alpha 1-30 and alpha 31-141, in the presence of 30% l-propanol that is catalyzed by V8 protease is distinct as compared with the previously studied protease-catalyzed splicing of the discontinuity sites of the fragment complementing systems [Sahni et al. (1989) Biochemistry 28, 5456]. The complementary fragments of alpha-globin do not exhibit noncovalent interaction between them even in the presence of l-propanol, the organic cosolvent used to facilitate the alpha-globin semisynthetic reaction. Besides, a significant portion of the fragment alpha 31-141 does not contribute to the protease-catalyzed splicing reaction. Alpha 1-30 and alpha 31-40 are ligated by V8 protease to yield alpha 1-40 in much the same way as the splicing of alpha 1-30 with either alpha 31-141 or alpha 31-47 to yield alpha-globin or alpha 1-47, respectively. An equimolar mixture of alpha 1-30 and alpha 31-40 does not show any 'complexation' in the presence of 30% l-propanol, the medium used for the synthetic reaction. The splicing junction, i.e., Glu30-Arg31 peptide bond, is located in the middle of the B-helix (residues 20-35) of the parent protein. Most of the residues from the A-helix of the protein could also be deleted from segment alpha 1-30 without influencing the V8 protease-catalyzed splicing reaction.(ABSTRACT TRUNCATED AT 250 WORDS)
Roy
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RP
Department of Medicine, Albert Einstein College of Medicine, Bronx, New York 10461.
Khandke
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Manjula
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Acharya
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D000433:1-Propanol; D002850:Chromatography, Gel; D002942:Circular Dichroism; D005914:Globins / Q000138:chemical synthesis / Q000378:metabolism*; D006441:Hemoglobin A / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011487:Protein Conformation; D012697:Serine Endopeptidases / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014357:Trypsin / Q000378:metabolism*
D010446:Peptide Fragments; D005914:Globins; D006441:Hemoglobin A; D000433:1-Propanol; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin