Perturbation of protein tertiary structure in frozen solutions revealed by 1-anilino-8-naphthalene sulfonate fluorescence.
Although freeze-induced perturbations of the protein native fold are common, the underlying mechanism is poorly understood owing to the difficulty of monitoring their structure in ice. In this report we propose that binding of the fluorescence probe 1-anilino-8-naphthalene sulfonate (ANS) to proteins in ice can provide a useful monitor of ice-induced strains on the native fold. Experiments conducted with copper-free azurin from Pseudomonas aeruginosa, as a model protein system, demonstrate that in frozen solutions the fluorescence of ANS is enhanced several fold and becomes blue shifted relative free ANS. From the enhancement factor it is estimated that, at -13 degrees C, on average at least 1.6 ANS molecules become immobilized within hydrophobic sites of apo-azurin, sites that are destroyed when the structure is largely unfolded by guanidinium hydrochloride. The extent of ANS binding is influenced by temperature of ice as well as by conditions that affect the stability of the globular structure. Lowering the temperature from -4 degrees C to -18 degrees C leads to an apparent increase in the number of binding sites, an indication that low temperature and /or a reduced amount of liquid water augment the strain on the protein tertiary structure. It is significant that ANS binding is practically abolished when the native fold is stabilized upon formation of the Cd(2+) complex or on addition of glycerol to the solution but is further enhanced in the presence of NaSCN, a known destabilizing agent. The results of the present 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, 56100 Pisa, Italy.
Strambini
Giovanni B
GB
D000817:Anilino Naphthalenesulfonates / Q000737:chemistry*; D001059:Apoproteins / Q000737:chemistry*; D001400:Azurin / Q000737:chemistry*; D003461:Crystallography / Q000379:methods*; D005456:Fluorescent Dyes; D005615:Freezing; D007053:Ice; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000737:chemistry; D012996:Solutions; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods*; D014867:Water / Q000737:chemistry
D000817:Anilino Naphthalenesulfonates; D001059:Apoproteins; D005456:Fluorescent Dyes; D007053:Ice; D012996:Solutions; C038778:apoazurin; D014867:Water; D001400:Azurin; C027132:1-anilino-8-naphthalenesulfonate