ABSTRACT
Binding of the hydrophobic fluorescent probe, 1-anilino-naphthalene-8-sulfonate (ANS), to synthetic polypeptides and proteins with a different structural organization has been studied. It has been shown that ANS has a much stronger affinity to the protein "molten globule" state, with a pronounced secondary structure and compactness, but without a tightly packed tertiary structure as compared with its affinity to the native and coil-like proteins, or to coil-like, alpha-helical, or beta-structural hydrophilic homopolypeptides. The possibility of using ANS for the study of equilibrium and kinetic molten globule intermediates is demonstrated, with carbonic anhydrase, beta-lactamase, and alpha-lactalbumin as examples.
Subject(s)
Anilino Naphthalenesulfonates , Fluorescent Dyes , Protein Conformation , Proteins/chemistry , Animals , Humans , KineticsABSTRACT
The kinetics of alpha-lactalbumin refolding were studied by the stopped-flow method with the registration of CD and intrinsic fluorescence at several wavelengths. It was shown that the early kinetic intermediate forms during the dead-time of the experiment (20 ms). This intermediate has a considerable amount of secondary structure and unpolar clusters in its molecular structure but has no rigid tertiary structure.
Subject(s)
Lactalbumin , Animals , Cattle , Circular Dichroism , Kinetics , Protein Conformation , Protein Denaturation , Spectrometry, Fluorescence , Spectrophotometry, UltravioletABSTRACT
We describe a novel physical state of a protein molecule which is nearly as compact as the native state and has pronounced secondary structure, but differs from the native state by the large increase of thermal fluctuations (in particular, by the large mobility of side groups). This state has been characterized in detail for the acid form of bovine alpha-lactalbumin as a result of the study of physical properties of this state by a large variety of different methods (hydrodynamics, diffuse X-ray scattering, circular dichroism and infrared spectra, polarization of the luminescence, proton magnetic resonance, deuterium exchange and microcalorimetry). It has been shown that bovine alpha-lactalbumin can be transformed into a similar state by thermal denaturation. This process is thermodynamically two state (i.e. all-or-none transition), which means that this state differs from the native one by a phase transition of the first order.