The structure of denatured alpha-lactalbumin elucidated by the technique of disulfide scrambling: fractionation of conformational isomers of alpha-lactalbumin.
The structure of denatured alpha-lactalbumin (alpha-LA) has been characterized using the method of disulfide scrambling. Under denaturing conditions (urea, guanidine hydrochloride, guanidine thiocyanate, organic solvent or elevated temperature) and in the presence of thiol initiator, alpha-LA denatures by shuffling its four native disulfide bonds and converts to a mixture of fully oxidized scrambled structures. Analysis by reversed-phase HPLC reveals that the denatured alpha-LA comprises a minimum of 45 fractions of scrambled isomers. Among them, six well populated isomers have been isolated and structurally characterized. Their relative concentrations, which represent the fingerprinting of the denatured alpha-LA, vary substantially under different denaturing conditions. These results permit independent plotting of the denaturation and unfolding curves of alpha-LA. Most importantly, unique isomers of partially unfolded alpha-LA were shown to populate at mild and selected denaturing conditions. Organic solvent disrupts preferentially the hydrophobic alpha-helical domain, generating a predominant isomer containing two native disulfide bonds at the beta-sheet domain and two scrambled disulfide bonds at the alpha-helical region. Thermal denaturation selectively unfolds the beta-sheet domain of alpha-LA, producing a prevalent isomer that exhibits structural characteristics of the molten globule state of alpha-LA.
Chang
J Y
JY
Research Center for Protein Chemistry, Institute of Molecular Medicine and the Department of Biochemistry and Molecular Biology, The University of Texas, Houston 77030, USA. Rowen.Chang@uth.tmc.edu
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D000097:Acetonitriles / Q000494:pharmacology; D001671:Biochemistry / Q000379:methods*; D002118:Calcium / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004220:Disulfides; D004305:Dose-Response Relationship, Drug; D019791:Guanidine / Q000494:pharmacology; D006146:Guanidines / Q000494:pharmacology; D007700:Kinetics; D007768:Lactalbumin / Q000737:chemistry*; D008958:Models, Molecular; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012997:Solvents / Q000494:pharmacology; D013237:Stereoisomerism; D013696:Temperature; D013816:Thermodynamics; D013861:Thiocyanates / Q000494:pharmacology; D013997:Time Factors; D014508:Urea / Q000494:pharmacology
D000097:Acetonitriles; D004220:Disulfides; D006146:Guanidines; D012997:Solvents; D013861:Thiocyanates; C054436:guanidine thiocyanate; D014508:Urea; D007768:Lactalbumin; D019791:Guanidine; D002118:Calcium; C032159:acetonitrile