Apo-azurin folds via an intermediate that resembles the molten-globule

Protein Science : a Publication of the Protein Society
Anders SandbergB Göran Karlsson

Abstract

The folding of Pseudomonas aeruginosa apo-azurin was investigated with the intent of identifying putative intermediates. Two apo-mutants were constructed by replacing the main metal-binding ligand C112 with a serine (C112S) and an alanine (C112A). The guanidinium-induced unfolding free energies (DeltaG(U-N)(H2O)) of the C112S and C112A mutants were measured to 36.8 +/- 1 kJ mole(-1) and 26.1 +/- 1 kJ mole(-1), respectively, and the m-value of the transition to 23.5 +/- 0.7 kJ mole(-1) M(-1). The difference in folding free energy (DeltaDeltaG(U-N)(H2O)) is largely attributed to the intramolecular hydrogen bonding properties of the serine Ogamma in the C112S mutant, which is lacking in the C112A structure. Furthermore, only the unfolding rates differ between the two mutants, thus pointing to the energy of the native state as the source of the observed Delta DeltaG(U-N)(H2O). This also indicates that the formation of the hydrogen bonds present in C112S but absent in C112A is a late event in the folding of the apo-protein, thus suggesting that formation of the metal-binding site occurs after the rate-limiting formation of the transition state. In both mutants we also noted a burst-phase intermediate. Because this intermediate was c...Continue Reading

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Citations

Aug 4, 2015·Nature Communications·Amy E M BeedleSergi Garcia-Manyes
Dec 26, 2006·Biophysical Journal·Giovanni B Strambini, Margherita Gonnelli
Jul 31, 2008·The Journal of Physical Chemistry. B·Giovanni B Strambini, Margherita Gonnelli
Feb 26, 2008·Biochemistry·Giovanni B Strambini, Margherita Gonnelli
Mar 18, 2008·The Journal of Physical Chemistry. B·Giovanni B StrambiniMargherita Gonnelli
Jan 20, 2009·Biochimica Et Biophysica Acta·Margherita Gonnelli, Giovanni B Strambini

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