Membrane Insertion Pathway of Annexin B12: Thermodynamic and Kinetic Characterization by Fluorescence Correlation Spectroscopy and Fluorescence Quenching

Abstract

Experimental determination of the free energy stabilizing the structure of membrane proteins in their native lipid environment is undermined by the lack of appropriate methods and suitable model systems. Annexin B12 (ANX) is a soluble protein which reversibly inserts into lipid membranes under mildly acidic conditions, which makes it a good experimental model for thermodynamic studies of folding and stability of membrane proteins. Here we apply fluorescence correlation spectroscopy for quantitative analysis of ANX partitioning into large unilamellar vesicles containing either 25% or 75% anionic lipids. Membrane binding of ANX results in changes of autocorrelation time and amplitude, both of which are used in quantitative analysis. The thermodynamic scheme describing acid-induced membrane interactions of ANX considers two independent processes: pH-dependent formation of a membrane-competent form near the membrane interface and its insertion into the lipid bilayer. Our novel fluorescence lifetime topology method demonstrates that the insertion proceeds via an interfacial refolded intermediate state, which can be stabilized by anionic lipids. Lipid titration measurements are used to determine the free energy of both transmembrane insertion and interfacial penetration, which are found to be similar, approximately -10-12 kcal/mol. The formation of the membrane-competent form, examined in a lipid saturation experiment, was found to depend on the local proton concentration near the membrane interface, occurring with pK = 4.3 and involving the protonation of two residues. Our results demonstrate that fluorescence correlation spectroscopy is a convenient tool for the quantitative characterization of the energetics of transmembrane insertion and that pH-triggered ANX insertion is a useful model for studying the thermodynamic stability of membrane proteins.

Membrane Association of the Diphtheria Toxin Translocation Domain Studied by Coarse-Grained Simulations and Experiment

“…LUV (large unilamellar vesicles) containing the quencher LysoUB (to allow monitoring the binding process via quenching of the bimane probe (Ladokhin et al, 2002; Posokhov & Ladokhin, 2006; Posokhov et al, 2008b)) were composed of a mixture of POPC:POPG with a 9:1 molar ratio, which has been demonstrated to stabilize the interfacial intermediate state and prevent further insertion into the bilayer (Kyrychenko et al, 2009). In order to further exclude the possibility of transmembrane insertion and limit the observation to the interfacial intermediate, the measurements were performed at the mildly acidic pH of 6.0.…”

Fluorescence Spectroscopy in Thermodynamic and Kinetic Analysis of pH-Dependent Membrane Protein Insertion

“…To circumvent this limitation one has to switch from peptides to more complex protein systems to study the energetics of membrane insertion such as annexin B12 and diphtheria toxin T-domain. Both of these proteins undergo a reversible pH-dependent membrane insertion (Ladokhin and Haigler, 2005; Ladokhin et al , 2004) which makes them attractive models for thermodynamic and kinetic studies using fluorescence correlation spectroscopy (FCS) and other spectroscopic approaches (Ladokhin et al , 2002; Posokhov et al , 2008a,b). …”

“…Due to recent technical developments, FCS is gaining popularity in biological studies (Haustein and Schwille, 2003; Hess et al , 2002; Schwille et al , 1999). Several groups have utilized it to study interfacial binding and transmembrane insertion of peptides and proteins (Posokhov et al , 2008a,b; Rhoades et al , 2006; Rusu et al , 2004). Rusu et al (2004) compared FCS results to those available from other measurements and found them to be in excellent agreement, opening the doors for quantitative application of FCS to membrane protein binding.…”

“…Here, we present several aspects of FCS measurements specific to thermodynamic studies of protein–membrane interactions. Our experiments were conducted using commercially available instrumentation utilizing confocal microscopy technology (e.g., MicroTime 200 from PicoQuant, Berlin, Germany or Alba FCS workstation from ISS, Champaign, IL) as described in detail before (Posokhov et al , 2008a). For a model membrane system we highly recommend the use of large unilamellar vesicles (LUV) with a diameter of ~0.1 μ m, which can be prepared by extrusion (Mayer et al , 1986).…”

“…In our studies of membrane binding we have labeled membrane-inserting proteins ANX (Posokhov et al , 2008a) and diphtheria toxin T-domain (Posokhov et al , 2008b) with Alexa fluorophores which are rather convenient, as their quantum yield is normally insensitive to the changes in environment so that q i = 1 (at least for the systems we have measured so far). Although not crucial, this further simplifies quantitative analysis as the amplitudes in Eq.…”