Changes in non-core regions stabilise plastocyanin from the thermophilic cyanobacterium Phormidium laminosum

Abstract

We report a theoretical investigation on the different stabilities of two plastocyanins. The first one belongs to the thermophilic cyanobacterium Phormidium laminosum and the second one belongs to its mesophilic relative Synechocystis sp. These proteins share the same topology and secondary-structure elements; however, the melting temperatures of their oxidised species differ by approximately 15 K. Long-time-scale molecular dynamics simulations, performed at different temperatures, show that the thermophilic protein optimises a set of intramolecular interactions (interstrand hydrogen bonding, salt bridging and hydrophobic clustering) within the region that comprises the strands beta 5 and beta 6, loop L5 and the helix. This region exhibits most of the differences in the primary sequence between the two proteins and, in addition, it is involved in the interaction with known physiological partners. Further work is in progress to unveil the specific structural features responsible for the different thermal stability of the two proteins.

Modulation of copper site properties by remote residues determines the stability of plastocyanins

“…This region shows a low degree of sequence conservation. Its stability is larger in Pho‐WT than in Syn‐WT [30]. According to Molecular Dynamics calculations, loop 5 being highly mobile [19,30].…”

“…Its stability is larger in Pho‐WT than in Syn‐WT [30]. According to Molecular Dynamics calculations, loop 5 being highly mobile [19,30]. Moreover, non‐bonding interactions such as that between Pro49 and Tyr85 (Fig.…”

“…Moreover, non-bonding interactions such as that between Pro49 and Tyr85 (Fig. 1) anchor it to the barrel [30]. Hence, we designed the double substitution P49G/G50P, to prevent this interaction.…”

Key Role of the Local Hydrophobicity in the East Patch of Plastocyanins on Their Thermal Stability and Redox Properties

“…According to their T m values, the oxidized form of Pho -Pc is more stable in solution than the reduced one, whereas the mesophilic variant shows the opposite trend, in agreement with previous works. 6,7,11,15 Interestingly, the V48I mutation increases the thermal stability of Syn -Pc. In particular, the oxidized V48I mutant is characterized by a higher T m value than the oxidized Syn -Pc WT protein, whereas the T m values of the reduced mutant and WT proteins were similar.…”

“…15,16 However, E84 is involved in the network of salt bridges across the east side of the protein. 11 R87 is highly conserved in Pcs, and a 30 mV decrease in E 0 ′ takes place upon the mutation of this residue in Nostoc Pc. 30 This residue points to the exposed region of the so-called east patch and, in Syn -Pc species, forms a salt bridge with E84.…”