Calorimetric determination of the energetics of the molten globule intermediate in protein folding: apo-alpha-lactalbumin.
High-sensitivity differential scanning calorimetry has been used to characterize the energetics of the molten globule state of apo-alpha-lactalbumin. This characterization has been possible by performing temperature scans at different guanidine hydrochloride (GuHCl) concentrations in order to experimentally define the temperature-GuHCl stability surface of the protein. Multidimensional analysis of the heat capacity surface has allowed simultaneous resolution of the energetics of the unfolded and molten globule states. These experiments indicate that the intrinsic enthalpy difference (i.e., excluding additional contributions such as those arising from differential GuHCl binding) between the unfolded and native states is 31.8 kcal/mol at 25 degrees C whereas that of the molten globule and native states is only 7.7 kcal/mol. At the same temperature, the entropy changes are 99.2 and 23.7 cal/K.mol and the heat capacity changes are 1821 and 326 cal/K.mol, respectively. Analysis of the thermodynamic data indicates that in passing from the native to the molten globule state only approximately 19% of the hydrogen bonds are broken. In addition, the magnitude of delta Cp for the molten globule suggests that water does not largely penetrate into the interior of the molten globule, implying that significant hydrophobic interactions are still present in this state. These parameters provide precise energetic constraints to the allowed structural conformations of the molten globule.
Xie
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D
Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218.
Bhakuni
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V
Freire
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E
D001059:Apoproteins / Q000737:chemistry*; D002152:Calorimetry, Differential Scanning; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006860:Hydrogen Bonding; D007768:Lactalbumin / Q000737:chemistry*; D011487:Protein Conformation; D013816:Thermodynamics
D001059:Apoproteins; D006146:Guanidines; C041966:apo-alpha-lactalbumin; D007768:Lactalbumin; D019791:Guanidine