Goat a-Lactalbumin

Recently, the molten globule state of a-lactalbumin has been shown to possess antitumor activity when complexed with a fatty acid [36,37], and hence the protein may possess secondary biological activity in addition to the primary activity of native a-lactalbumin, i.e., substrate specificity modifier activity in a lactose synthase system [38,39]. The molten globule of a-lactalbumin thus provides an example of the folding intermediate of a protein exhibiting a secondary biological activity. 

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Schewale, J. G., Sinha, S. K. and Brew, K. 1984. Evolution of a-lactalbumins. The complete amino acid sequence of the a-lactalbumin from a marsupial (Macropus rufogri-seus) and corrections to regions of sequence in bovine and goat a-lactalbumins. J. BioL Chem. 259, 4947-4956. 

Experimental and Simulation Studies of the Folding/Unfolding of Goat a-Lactalbumin... 

Fig. 2.1. The backbone structures of authentic and recombinant goat a-lactalbumin in the crystal form. The backbone of Mol A of the authentic protein, represented by a wire model, was superimposed on the backbone of the recombinant protein. Gray and black wires represent the authentic and recombinant proteins, respectively. The C -atom RMSD value between the two proteins was 0.54 A. The PDB codes for the authentic and recombinant proteins are 1HFY and 1HMK, respectively...

Fig. 2.2. GdnHCl-induced unfolding transition curves for authentic and recombinant goat a-lactalbumin [22], The filled diamonds indicate the unfolding transition of the methionine-free recombinant protein produced by CNBr cleavage. The unfolding was carried out at 25° C in the presence of 1 mM CaCB, 50 mM NaCl, and 50 mM sodium cacodylate (pH 7.0). The transitions were monitored by CD measurements at 222 nm (circles and diamonds) and at 270 nm (triangles), and the transition curves were normalized between the native and fully unfolded baselines. The black line with symbols represents the authentic form, and the gray line with symbols represents the recombinant form. Reproduced with permission from [22]...

Considering that the X-ray crystallographic structures of authentic and recombinant goat a-lactalbumin were essentially identical to each other, with the exception of the N-terminal region and a loop region between residues 105 and 110 (Fig. 2.1), the difference in stability between the two proteins was remarkable. 

We therefore studied the unfolding and refolding kinetics of authentic and recombinant goat a-lactalbumin, induced by GdnHCl concentration jumps,... 

Fig. 2.6. The dynamic domains of goat a-lactalbumin, domain 1 (dark gray) and domain 2 (light gray), and the screw axis of the interdomain motion [24]. The C-helix is involved in domain 2 and moves together with the Ca2+-binding site and the 13-domain. Reproduced with permission from [24]...

The unfolding behaviors of the authentic and recombinant forms of goat a-lactalbumin are remarkably different, although both forms have an identical three-dimensional structure. The recombinant form was found to be l.lkcalmol-1 less stable than the authentic form, and the recombinant form unfolded at a ninefold faster rate than the authentic form. The destabilization and unfolding-rate acceleration were due to the presence of an extra methionine residue at the N-terminus in the recombinant protein. 

We carried out two series of unfolding MD simulations, one for the authentic form and the other for the recombinant form of goat a-lactalbumin at 398 K. The unfolding simulations reasonably reproduced the experimentally observed difference between the proteins, i.e., the faster rate of unfolding of the recombinant protein. 

Fig. 2.T. [1BN, 1H]-HSQC spectrum of 1BN-labeled recombinant goat a-lactalbumin at pH 6.3 and 25°C in 95% H20/5% D20 (Nakamura et al., unpublished). Peaks are labeled with their residue-specific assignments...

Fig. 2.8. Histograms showing the distribution of protection factors from amide hydrogen exchange for (a) the authentic form and (b) the recombinant form of goat a-lactalbumin in the MG-state at pHobs 1.7 and 25°C (Nakamura et al., unpublished)...

The MD unfolding simulations at 498 K led to the global unfolding of both authentic and recombinant goat a-lactalbumin, but comparison of the... 

Fig. 2.9. The T-values ((a) experimental 4>-values, and (b) 4>md obtained from MD trajectories) mapped onto the three-dimensional structure of goat a-lactalbumin [25]. Reproduced with permission from [25]...

To further characterize the structural changes of goat a-lactalbumin during unfolding, we examined the probability distributions of the following four structural parameters in each of the nine clusters of the structural ensemble of MD trajectories (1) the fractional native contact (Q) of the entire molecule, (2) the RMSD of C atoms between a pair of structures that belong to the same cluster, (3) the solvent-accessible surface area (SASA) of hydrophobic side chains, and (4) the SASA of hydrophilic side chains [25]. 

We carried out unfolding MD simulations of goat a-lactalbumin at 498 K. The protein structure was represented in the segmental Q-coordinate, and cluster analyses and multiple-trajectory alignments were carried out to obtain the transition-state structure solely from the MD simulation. The structure obtained by this approach was very close to that obtained experimentally, and hence the results of the kinetic unfolding experiments were well reproduced by the simulations. 

Vanhooren, A., Devreese, B., Vanhee, K., VanBeeumen, J., and Hanssens, I. (2002) Photoexcitation of tryptophan groups induces reduction of two disulfide bonds in goat a-lactalbumin. Biochemistry, 41, 11035. 

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