Partially folded states of equine lysozyme. Structural characterization and significance for protein folding

Abstract

Despite their homologous structure, c-type lysozymes and alpha-lactalbumins have been found to differ profoundly in their unfolding behavior, in that the alpha-lactalbumins readily enter a partially unfolded collapsed state (the "molten globule"), whereas lysozymes unfold cooperatively to a highly unfolded state. The calcium-binding property of lysozyme from equine milk provides an evolutionary link between the two families of proteins. We demonstrate here that equine lysozyme undergoes a two-stage unfolding transition upon heating or in the presence of guanidine hydrochloride that is highly dependent on the state of calcium binding. Differential scanning calorimetry shows the two transitions to be particularly well resolved in the calcium-free protein, where the first transition occurs with a midpoint at 44 degrees C at pH 4.5 or in 0.8 M GdnHCl at pH 7.5, 25 degrees C, and the second occurs near 70 degrees C at pH 4.5 or in 3.7 M GdnHCl at pH 7.5, 25 degrees C. In the presence of calcium, the first transition takes place with a midpoint of 55 degrees C or in excess of 2.5 M GdnHCl, but the parameters for the second transition remain unchanged. Fluorescence emission and UV difference absorption spectroscopy suggest that the first transition generates an intermediate state in which sequestration of some aromatic side chains from solvent has occurred whereas the second represents denaturation to a highly unfolded state. CD and 1H NMR results indicate that the intermediate state possesses extensive secondary and tertiary structure, although the latter is substantially disordered.(ABSTRACT TRUNCATED AT 250 WORDS)

Electrostatic interactions in the acid denaturation of α‐lactalbumin determined by nmr

“…With regard to their unfolding behavior, it appears an important difference between a-LA and lysosymes arises from their calcium binding properties (Haezerbrouck et al, 1993;Nitta et al, 1993;Van Dael et al, 1993;Griko et al, 1995;Pardon et al, 1995;Wu et al, 1996;Kuhlman et al, 1997). Recently, the functional role of the calcium binding residues in a-LA was investigated by sitedirected mutagenesis .…”

Structural Origin of ELOA Toxicity – Implication for HAMLET-Type Protein Complexes with Oleic Acid

“…Calcium is coordinated by a loop positioned at the bottom of the cleft and important for the structural integrity of the protein, yet the physiological function of calcium binding to EL and other calcium-binding lysozymes is still unclear. The calciumbinding usually increases the protein stability against denaturing treatments, however in the case of EL, the significantly lower stability and cooperatively was observed compared to non-calcium-binding lysozymes even in its holo-form, while in the apo-form its thermodynamic stability is closer of -lactalbumins than to c-type lysozymes [15,16]. EL forms a wide range of partially folded states under equilibrium conditions similar to these of -lactalbumins [16,3,17,18].…”

“…The calciumbinding usually increases the protein stability against denaturing treatments, however in the case of EL, the significantly lower stability and cooperatively was observed compared to non-calcium-binding lysozymes even in its holo-form, while in the apo-form its thermodynamic stability is closer of -lactalbumins than to c-type lysozymes [15,16]. EL forms a wide range of partially folded states under equilibrium conditions similar to these of -lactalbumins [16,3,17,18]. However, EL molten globule is much more structured compared to the "classical" molten globules of α-lactalbumins, possessing an extended native-like hydrophobic core stabilised by interactions between three major α-helices (A, B and D-helices) in the α-domain [17,18].…”

Two partially unfolded states oftorpedo californicaacetylcholinesterase

“…The time of mixing did not exceed I O s. An excitation wavelength of 280 nm was employed, and the slit width of the monochromators was 5 nm. The ratio of intensities at fixed wavelengths on the shoulders of the emission peak has been shown to be very sensitive to structural changes and independent of protein concentration (Jiang & London, 1990;Van Dael et al, 1993). The kinetics of the transition were, therefore, followed by monitoring emission intensities at 320 and 375 nm.…”