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The interaction of saturated fatty acids of different length (C8:0 to C18:0) with β-lactoglobulin (βLG) was investigated by molecular dynamics simulation and docking approaches. The results show that the presence of such ligands in the hydrophobic central cavity of βLG, known as the protein calyx, determines an enhancement of atomic fluctuations compared with the unliganded form, especially for loops at the entrance of the binding site. Concerted motions are evidenced for protein regions that could favor the binding of ligands. The mechanism of anchoring of fatty acids of different length is similar for the carboxylate head-group, through electrostatic interactions with the side chains of Lys60/Lys69. The key protein residues to secure the hydrocarbon chain are Phe105/Met107, which adapt their conformation upon ligand binding. In particular, Phe105 provides an additional hydrophobic clamp only for the tail of the two fatty acids with the longest chains, palmitic, and stearic acid, which are known to bind βLG with a high affinity. The search of additional external binding sites for fatty acids, distinct from the calyx, was also carried out for palmitic acid. Two external sites with a lower affinity were identified as secondary sites, one consisting in a hydrophobic cavity allowing two distinct binding modes for the fatty acid, and the other corresponding to a surface crevice close to the protein α-helix. The overall results provide a comprehensive picture of the dynamical behavior of βLG in complex with fatty acids, and elucidate the structural basis of the binding of these physiological ligands.

Molecular dynamics simulation has been carried out for the blue copper protein amicyanin from two different sources, Paracoccus denitrificans and Paraccocus versutus, to investigate the structural and dynamical properties common to the two molecules and to identify prominent features shared with proteins of the same family, the monomeric cupredoxins. The two amicyanins have almost identical secondary and tertiary structure. In the simulation, they differ for the number of hydrogen bonds in the main chain and the conformation of some beta-strands. However, they strictly maintain the arrangement of the portions of the beta-barrel that are conserved in the folding architecture of the blue copper proteins. Paracoccus versutus amicyanin equilibrates more rapidly, shows lower atomic deviation values, and is less rigid with respect to Paracoccus denitrificans amicyanin. Principal component analysis reveals that the conformational subspaces corresponding to eigenvectors with the same index for each of the two molecules are not necessarily equivalent. Nevertheless, a core scaffold with constrained dynamics exist for both amicyanins. In addition, two fairly flexible regions that are located on the opposite side with respect to the interaction sites with the partner molecules in the redox process have been evidenced in the protein structure. This description of amicyanin, with a few mobile regions remote from the active site and a rigid scaffold including most of the protein beta-barrel, has a close similarity with that of azurin and plastocyanin, two other cupredoxins previously investigated in simulation. Furthermore, similarities in the distribution of the atomic fluctuations indicate that amicyanin, azurin, and plastocyanin possess common dynamical features, in spite of differences in their structure. On the basis of these findings, we suggest that topological constraints imposed by the folding in correspondence of protein regions that are the most conserved determine the protein dynamics of the cupredoxin family. The dynamical properties of the cupredoxins might be controlled for functional advantages that include the binding mechanism with the biological partners and the collective inner motions of the protein matrix required for the electron transfer, whereas long-range conformational changes in the redox reaction should be excluded.

The contribution of the copper ion to the stability and to the unfolding pathway of pseudoazurin was investigated by a comparative analysis of the thermal unfolding of the Cu(II)-holo and apo form of the protein. The unfolding has been followed by calorimetry, fluorescence, optical density, and electron paramagnetic resonance (EPR) spectroscopy. The thermal transition of Cu(II)-holo pseudoazurin is irreversible and occurs between 60.0 and 67.3°C, depending on the scan rate and technique used. The denaturation pathway of Cu(II)-holo pseudoazurin can be described by the Lumry–Eyring model: N U F; the protein reversibly goes from the native (N) to the unfolded (U) state, and then irreversibly to the final (F) state. The simulation of the experimental calorimetric profiles, according to this model, allowed us to determine the thermodynamic and kinetic parameters of the two steps. The ΔG value calculated for the Cu(II)-holo pseudoazurin is 39.2 kJ·mol−1 at 25°C. The sequence of events in the denaturation process of Cu(II)-holo pseudoazurin emergence starts with the disruption of the copper site and the hydrophobic core destabilization followed by the global protein unfolding. According to the EPR findings, the native type-1 copper ion shows type-2 copper features after the denaturation. The removal of the copper ion (apo form) significantly reduces the stability of the protein as evidenced by a ΔG value of 16.5 kJ·mol−1 at 25°C. Moreover, the apo Paz unfolding occurs at 41.8°C and is compatible with a two-state reversible process N U.© 2006 Wiley Periodicals, Inc. Biopolymers 83: 487–497, 2006 This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com