PUMA
Istituto di Biofisica     
Strambini G. FLUORESCENCE AND PHOSPHORESCENCE METHODS TO PROBE PROTEIN STRUCTURE AND STABILITY IN ICE: THE CASE OF AZURIN. Jameel and Hershenson (eds.). United States: John Wiley and sons, 2010.
 
 
Abstract
(English)
Freezing of protein solutions may result in irreversible protein aggregation and severe loss of catalytic activity of enzymes, reasons for which many proteins cannot be stored in ice or lyophilized from it without partial inhibition of their function [1]. Notwithstanding the relevance of the phenomenon and the commercial importance for the growing polypeptide-based pharmaceutical industry, little is known on the structure and thermodynamic stability of proteins in ice. To date, the hypotheses advanced on the freeze damage mechanism are based on indirect evidence and are construed mainly around the presumed action of cryoprotectants, compounds that at relatively high concentrations prevent these alterations. The paucity of structural information on proteins in the frozen state is due primarily to the insensitivity or the poor resolution of ordinary spectroscopic methods in a highly scattering and anisotropic medium as ice. For example, fourier transform infrared (FTIR) spectroscopy experiments, when conducted on freeze-labile proteins, have not detected significant changes in the secondary structure of the native fold presumably because they are carried out at relatively large protein concentrations, a . Q1 condition in which the perturbation is often attenuated as if the protein itself acted as a stabilizer. For the same reason, little is known on the effect of freezing on the thermodynamic stability of proteins. Indeed, formulations and protocols for the stabilization of pharmacoproteins during industrial freeze-drying processes are still largely worked out empirically, case by case, drawing mostly on long-time experience and guided by thermodynamic data pertaining to stabilizing additives in liquid solutions [3-5]. The more recent introduction of fluorescence and phosphorescence techniques capable of probing the protein tertiary structure and the thermodynamic stability of the native fold in ice holds promise for new insights on the nature of the ice perturbation as well as on the cryoprotection mechanisms of various additives. This chapter reviews recent (as of 2009) results obtained by these techniques on a model protein, azurin (and some appositely prepared mutants), on the effect of freezing on the integrity of the native fold structure and on its thermodynamic stability (G◦). Perturbations of the tertiary structure are probed directly both by alterations in the lifetime of tryptophan (Trp) phosphorescence emission and from freeze-induced binding of the fluorescence probe ANS (1-anilino-8-aminonaphthalene), whereas G◦ is derived from guanidinium chloride denaturation of the protein, monitored by the change in Trp fluorescence spectrum and yield. Azurin from Pseudomonas aeruginosa is a small (14-kDa), monomeric copperbinding protein well characterized in terms of its structure [6,7] and thermodynamic stability [8,9]. Other suitable features of azurin are 1. The macromolecule has a single Trp residue (W48) located within the rigid inner core of the globular structure. Copper-free azurin exhibits the most blueshifted Trp fluorescence spectrum known in proteins [10] and intense longlived phosphorescence even at ambient temperature [11]. 2. Residue W48 is wrapped inside a tight β-barrel motif, and its exposure to the solvent requires no less than global unfolding of the polypeptide as confirmed by the superposition of fluorescence and circular dichroism (CD) denaturation profiles [9]. 3. The denaturation of Cu-free azurin by guanidinium chloride (Gdn) is a fully reversible process and the equilibrium is well represented by a two- state model
Subject PHOSPHORESCENCE PROTEIN STRUCTURE


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