ANS fluorescence detects widespread perturbations of protein tertiary structure in ice.
Freeze-induced perturbations of the protein native fold are poorly understood owing to the difficulty of monitoring their structure in ice. Here, we report that binding of the fluorescence probe 1-anilino-8-naphthalene sulfonate (ANS) to proteins in ice can provide a general monitor of ice-induced alterations of their tertiary structure. Experiments conducted with copper-free azurin from Pseudomonas aeruginosa and mutants I7S, F110S, and C3A/C26A correlate the magnitude of the ice-induced perturbation, as inferred from the extent of ANS binding, to the plasticity of the globular fold, increasing with less stable globular folds as well as when the flexibility of the macromolecule is enhanced. The distortion of the native structure inferred from ANS binding was found to draw a parallel with the extent of irreversible denaturation by freeze-thawing, suggesting that these altered conformations play a direct role on freeze damage. ANS binding experiments, extended to a set of proteins including serum albumin, alpha-amylase, beta-galactosidase, alcohol dehydrogenase from horse liver, alcohol dehydrogenase from yeast, lactic dehydrogenase, and aldolase, confirmed that a stressed condition of the native fold in the frozen state appears to be general to most proteins and pointed out that oligomers tend to be more labile than monomers presumably because the globular fold can be further destabilized by subunit dissociation. The results of this study suggest that the ANS binding method may find practical utility in testing the effectiveness of various additives employed in protein formulations as well as to devise safer freeze-drying protocols of pharmaceutical proteins.
Gabellieri
Edi
E
Consiglio Nazionale delle Ricerche, Istituto di Biofisica, 56124 Pisa, Italy.
Strambini
Giovanni B
GB
D000817:Anilino Naphthalenesulfonates; D001059:Apoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001400:Azurin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D005456:Fluorescent Dyes; D007053:Ice; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017434:Protein Structure, Tertiary / Q000502:physiology; D013050:Spectrometry, Fluorescence
D000817:Anilino Naphthalenesulfonates; D001059:Apoproteins; D005456:Fluorescent Dyes; D007053:Ice; D001400:Azurin; C027132:1-anilino-8-naphthalenesulfonate