Copper binding before polypeptide folding speeds up formation of active (holo) Pseudomonas aeruginosa azurin.
Cofactors often stabilize the native state of the proteins; however, their effects on folding dynamics remain poorly understood. To uncover the role of one cofactor, we have examined the folding kinetics of Pseudomonas aeruginosa azurin, a small blue-copper protein with a copper cofactor uniquely coordinated to five protein residues. Copper removal produces apo-azurin which adopts a folded structure identical to that of the holo-form. The folding and unfolding kinetics for apo-azurin follow two-state behavior. The extrapolated folding time in water, tau approximately 7 ms, is in good agreement with the topology-based prediction. Copper uptake by folded apo-azurin, to govern active (holo) protein, is slow (tau approximately 14 min, 50:1 copper-to-protein ratio). In contrast, the formation of active (holo) azurin is much faster when copper is allowed to interact with the unfolded polypeptide. Refolding in the presence of 10:1, 50:1, and 100:1 copper:protein ratios yields identical time-trajectories: active azurin forms in two kinetic phases with folding times, extrapolated to water, of tau = 10 +/- 2 ms (major phase) and tau = 190 +/- 30 ms (minor phase), respectively. Correlating copper-binding studies, with a small peptide derived from the metal-binding region of azurin, support that initial cofactor binding is fast (tau approximately 3.7 ms) and thus not rate-limiting. Taken together, introducing copper prior to protein folding does not speed up the polypeptide-folding rate; nevertheless, it results in much faster (> 4000-fold) formation of active (i.e., holo) azurin. Living systems depend on efficient formation of functional biomolecules; attachment of cofactors prior to polypeptide folding appears to be one method to achieve this.
Pozdnyakova
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Department of Chemistry, Tulane University, New Orleans, Louisiana 70118, USA.
Wittung-Stafshede
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P
D001400:Azurin / Q000737:chemistry / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D003300:Copper / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D017510:Protein Folding; D011550:Pseudomonas aeruginosa / Q000737:chemistry* / Q000378:metabolism
D001426:Bacterial Proteins; D010455:Peptides; D001400:Azurin; D003300:Copper