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Conversion of phospholamban into a soluble pentameric helical bundle
1Department of Chemistry, Department of Molecular Biophysics and Biochemistry, and Howard Hughes Medical Institute, Yale University, New Haven, Connecticut 06520-8114, USA.
Biochemistry
|May 31, 2001
Summary
Researchers redesigned the membrane protein phospholamban (PLB) for solubility. The soluble PLB variant remained a stable helical pentamer, suggesting membrane protein interiors dictate aqueous folding.
Area of Science:
- Biochemistry
- Structural Biology
- Membrane Protein Research
Background:
- Membrane proteins and soluble proteins fold differently.
- Redesigning membrane protein surfaces may enable soluble folding.
- Phospholamban (PLB) is a membrane protein forming a helical pentamer.
Purpose of the Study:
- To test if redesigning the surface of a membrane protein (PLB) allows it to fold and function in an aqueous environment.
- To investigate if the interior of membrane proteins contains folding determinants for aqueous environments.
Main Methods:
- Surface residues of the PLB transmembrane domain were replaced with charged/polar residues.
- Circular dichroism (CD) spectroscopy assessed alpha-helicity.
- Small-angle X-ray scattering (SAXS) and multiangle laser light scattering (MALLS) determined oligomeric state.
- Nuclear Magnetic Resonance (NMR) spectroscopy probed protein structure.
Main Results:
- The full-length soluble PLB variant was highly alpha-helical.
- Soluble PLB associated as a stable pentamer, mirroring native PLB oligomerization.
- Mutations destabilizing native PLB also disrupted the soluble pentamer.
- NMR suggested molten globule-like properties in the redesigned protein.
Conclusions:
- Converting a membrane protein (PLB) into a soluble helical pentamer is achievable.
- The interior of membrane proteins likely holds determinants for aqueous folding.
- Surface redesign is a viable strategy for studying membrane protein structures and gaining insights into their folding mechanisms.