Influence of Protein – Micelle Ratios and Cysteine Residues on the Kinetic Stability and Unfolding Rates of Human Mitochondrial VDAC-2

Svetlana Rajkumar Maurya,Radhakrishnan Mahalakshmi,Eugene A Permyakov

doi:10.1371/journal.pone.0087701

Svetlana Rajkumar Maurya, Radhakrishnan Mahalakshmi + Show 1 more

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https://doi.org/10.1371/journal.pone.0087701

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Abstract

Delineating the kinetic and thermodynamic factors which contribute to the stability of transmembrane β-barrels is critical to gain an in-depth understanding of membrane protein behavior. Human mitochondrial voltage-dependent anion channel isoform 2 (hVDAC-2), one of the key anti-apoptotic eukaryotic β-barrel proteins, is of paramount importance, owing to its indispensable role in cell survival. We demonstrate here that the stability of hVDAC-2 bears a strong kinetic contribution that is dependent on the absolute micellar concentration used for barrel folding. The refolding efficiency and ensuing stability is sensitive to the lipid-to-protein (LPR) ratio, and displays a non-linear relationship, with both low and high micellar amounts being detrimental to hVDAC-2 structure. Unfolding and aggregation process are sequential events and show strong temperature dependence. We demonstrate that an optimal lipid-to-protein ratio of 2600∶1 – 13000∶1 offers the highest protection against thermal denaturation. Activation energies derived only for lower LPRs are ∼17 kcal mol−1 for full-length hVDAC-2 and ∼23 kcal mol−1 for the Cys-less mutant, suggesting that the nine cysteine residues of hVDAC-2 impart additional malleability to the barrel scaffold. Our studies reveal that cysteine residues play a key role in the kinetic stability of the protein, determine barrel rigidity and thereby give rise to strong micellar association of hVDAC-2. Non-linearity of the Arrhenius plot at high LPRs coupled with observation of protein aggregation upon thermal denaturation indicates that contributions from both kinetic and thermodynamic components stabilize the 19-stranded β-barrel. Lipid-protein interaction and the linked kinetic contribution to free energy of the folded protein are together expected to play a key role in hVDAC-2 recycling and the functional switch at the onset of apoptosis.

Highlights

The stability of a folded protein is largely a consequence of either thermodynamic or kinetic factors [1,2,3,4,5]
It has been demonstrated earlier that these cysteines exist in the reduced state, are not necessary for barrel activity [31], and are not located at the predicted barrel dimerization interface [32]. Human mitochondrial voltage-dependent anion channel isoform 2 (hVDAC-2), on the other hand, possessed nine cysteine residues, which exist in the reduced state in in vitro refolded protein [22]
Biophysical characterization of the 19-stranded hVDAC-2 bbarrel, which is indispensable for cell survival [28], can provide us with key insight into the factors influencing barrel folding and stability

Summary

Introduction

The stability of a folded protein is largely a consequence of either thermodynamic or kinetic factors [1,2,3,4,5]. The occurrence of irreversibility, in thermal denaturation experiments, leading to protein aggregation, suggests the existence of a kinetic barrier to the (un)folding process (N«URD), and in turn reflects kinetic stabilization of the refolded protein [3,15]. The notion that membrane proteins, in particular, can exist in a kinetically stable state, rather than possessing thermodynamic stability, has gained popularity over the past few years [3,4,5,16]. Unfolding of such proteins can proceed only with sufficient lowering of the large energy barrier separating the native form from the unfolded state(s). The necessary existence of reversible equilibrium between protein (folded) « protein (unfolded), protein-lipid and empty lipid moieties adds greater complexity to thermodynamic studies of transmembrane (TM) proteins [5]

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