Computational design of a water-soluble analog of phospholamban

Abstract

Membrane proteins and water-soluble proteins share a similar core. This similarity suggests that it should be possible to water-solubilize membrane proteins by mutating only their lipid-exposed residues. We have developed computational tools to design water-soluble variants of helical membrane proteins, using the pentameric phospholamban (PLB) as our test case. To water-solublize PLB, the membrane-exposed positions were changed to polar or charged amino acids, while the putative core was left unaltered. We generated water-soluble phospholamban (WSPLB), and compared its properties to its predecessor PLB. In aqueous solution, WSPLB mimics all of the reported properties of PLB including oligomerization state, helical structure, and stabilization upon phosphorylation. We also characterized the truncated mutant WSPLB (21-52) comprising only the former transmembrane segment of PLB. This peptide shows a decreased specificity for forming a pentameric oligomerization state.

Figure 1.

The sequence of canine wild-type PLB compared to several soluble mutants: WSPLB, ADA-FULL, SIMM-FULL (Li et al. 2001), PLB-COMP-1, and PLB-COMP-2 (Sabine et al. 2000). In human PLB, Asn27 is substituted by Lys. Positions S16 and T17 are phosphorylated by PKA and PKC. The differences between WSPLB and PLB are shown in red, the differences between SIMM-FULL and WSPLB are shown in blue, and the differences between PLB-COMP-1 (and 2) and SIMM-FULL are shown in green.

Figure 2.

CD spectra of 125 μM WSPLB and WSPLB (21–52). WSPLB spectra taken in 10 mM sodium phosphate pH 7.5, 50 mM NaCl, 1 mM TCEP-HCl. WSPLB (21–52) spectra taken in 15 mM MOPS pH 7.0, 50 mM NaCl, 1 mM EDTA, and 1 mM TCEP-HCl.

Figure 3.

Sedimentation equilibrium of WSPLB in 100 mM NaCl, 25 mM MOPS pH 7.5, 1 mM EDTA, 1 mM TCEP-HCl at 35,000 rpm. Equilibrium A₂₈₀-radius profiles for three cell compartments containing peptide concentrations of 15, 39, and 113 μM are shown. (A) Shows the data fit to a monomer–pentamer equilibrium, while (B) shows a less satisfactory data fit to a monomer–tetramer equilibrium.

Figure 3.

Sedimentation equilibrium of WSPLB in 100 mM NaCl, 25 mM MOPS pH 7.5, 1 mM EDTA, 1 mM TCEP-HCl at 35,000 rpm. Equilibrium A₂₈₀-radius profiles for three cell compartments containing peptide concentrations of 15, 39, and 113 μM are shown. (A) Shows the data fit to a monomer–pentamer equilibrium, while (B) shows a less satisfactory data fit to a monomer–tetramer equilibrium.

Figure 4.

Sedimentation equilibrium of WSPLB (21–52) in 15 mM MOPS pH 7, 50 mM NaCl, 1 mM EDTA, 1 mM TCEP-HCl at 48,000 rpm. Equilibrium A₂₈₀-radius profile for three cells containing 14, 46, and 97 μM. (A) Shows the data fit to a monomer–tetramer, (B) a monomer–pentamer, and (C) a monomer–tetramer–pentamer equilibrium. Although all three schemes provide a good fit to the data, the presence of multiple peaks on size-exclusion chromatography requires the use of a monomer–tetramer–pentamer scheme.

Figure 4.

Sedimentation equilibrium of WSPLB (21–52) in 15 mM MOPS pH 7, 50 mM NaCl, 1 mM EDTA, 1 mM TCEP-HCl at 48,000 rpm. Equilibrium A₂₈₀-radius profile for three cells containing 14, 46, and 97 μM. (A) Shows the data fit to a monomer–tetramer, (B) a monomer–pentamer, and (C) a monomer–tetramer–pentamer equilibrium. Although all three schemes provide a good fit to the data, the presence of multiple peaks on size-exclusion chromatography requires the use of a monomer–tetramer–pentamer scheme.

Figure 4.

Sedimentation equilibrium of WSPLB (21–52) in 15 mM MOPS pH 7, 50 mM NaCl, 1 mM EDTA, 1 mM TCEP-HCl at 48,000 rpm. Equilibrium A₂₈₀-radius profile for three cells containing 14, 46, and 97 μM. (A) Shows the data fit to a monomer–tetramer, (B) a monomer–pentamer, and (C) a monomer–tetramer–pentamer equilibrium. Although all three schemes provide a good fit to the data, the presence of multiple peaks on size-exclusion chromatography requires the use of a monomer–tetramer–pentamer scheme.

Figure 5.

Thermal denaturation of WSPLB, measuring the loss of signal at 222 nm with increasing temperature by circular dichroism. Scans taken with 60-s signal averaging time and equilibration of 4 min at each temperature. Concentrations of peptide are 50 and 125 μM. Data were fit to a two-state model (theoretical curves are shown) with a monomer–pentamer equilibrium.

Figure 6.

Thermal denaturation of WSPLB and pWSPLB from 2 to 94 °C, normalized to % unfolded peptide. Conditions are identical to those in Figure 2 ▶. Scans taken with 60-s signal averaging time and equilibration of 4 min at each temperature. Concentrations of both peptides are 50 μM.

Figure 7.

Thermal denaturation of WSPLB (21–52) from 2 to 94°C. Scans taken with 60-s signal averaging time and equilibration of 4 min at each temperature. Conditions and peptide concentrations are identical to those in Figure 2 ▶.