Nicked Apomyoglobin: A Noncovalent Complex of Two Polypeptide Fragments Comprising the Entire Protein Chain

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• 作者：Valeria Musi ; Barbara Spolaore ; Paola Picotti ; Marcello Zambonin ; Vincenzo De Filippis ; and Angelo Fontana
• 刊名：Biochemistry
• 出版年：2004
• 出版时间：May 25, 2004
• 年：2004
• 卷：43
• 期：20
• 页码：6230 - 6240
• 全文大小：221K
• 年卷期：v.43,no.20(May 25, 2004)
• ISSN：1520-4995

文摘

Limited proteolysis of the 153-residue chain of horse apomyoglobin (apoMb) by thermolysinresults in the selective cleavage of the peptide bond Pro88-Leu89. The N-terminal (residues 1-88) andC-terminal (residues 89-153) fragments of apoMb were isolated to homogeneity and their conformationaland association properties investigated in detail. Far-UV circular dichroism (CD) measurements revealedthat both fragments in isolation acquire a high content of helical secondary structure, while near-UV CDindicated the absence of tertiary structure. A 1:1 mixture of the fragments leads to a tight noncovalentprotein complex (1-88/89-153, nicked apoMb), characterized by secondary and tertiary structures similarto those of intact apoMb. The apoMb complex binds heme in a nativelike manner, as given by CDmeasurements in the Soret region. Second-derivative absorption spectra in the 250-300 nm region providedevidence that the degree of exposure of Tyr residues in the nicked species is similar to that of the intactprotein at neutral pH. Also, the microenvironment of Trp residues, located in positions 7 and 14 of the153-residue chain of the protein, is similar in both protein species, as given by fluorescence emissiondata. Moreover, in analogy to intact apoMb, the nicked protein binds the hydrophobic dye 1-anilinonaphthalene-8-sulfonate (ANS). Taken together, our results indicate that the two proteolytic fragments1-88 and 89-153 of apoMb adopt partly folded states characterized by sufficiently nativelikeconformational features that promote their specific association and mutual stabilization into a nicked proteinspecies much resembling in its structural features intact apoMb. It is suggested that the formation of anoncovalent complex upon fragment complementation can mimic the protein folding process of the entireprotein chain, with the difference that the folding of the complementary fragments is an intermolecularprocess. In particular, this study emphasizes the importance of interactions between marginally stableelements of secondary structure in promoting the tertiary contacts of a native protein. Considering thatapoMb has been extensively used as a paradigm in protein folding studies for the past few decades, thenovel fragment complementing system of apoMb here described appears to be very useful for investigatingthe initial as well as late events in protein folding.