title: Loop anchor modification causes the population of an alternative native state in an SH3-like domain.
abstract: Many stably folded proteins are proposed to contain long, unstructured loops. A series of hybrid proteins (EbE1-4) containing the folded scaffold of photosystem I accessory protein E (PsaE), an SH3-like protein, and the 40-residue heme-binding loop of cytochrome b(5) was created to inspect the dependence of thermodynamic and kinetic parameters on the residues at the interface of folded and flexible regions. Compared to the simplest hybrid (EbE1), the chimeras differed by Gly insertions (EbE2, EbE3) or an asymmetric four-residue restructuring of loop termini (EbE4). NMR spectroscopy indicated that the chimeras retained the PsaE topology; native and unfolded state solubilities, however, were affected to varying degrees. Thermal and chemical denaturation experiments revealed that the EbE2 and EbE1 constructs resulted in a modest destabilization of the PsaE core, whereas apparent stability was increased by >5 kJ/mol in EbE4. EbE3 aggregated at microM concentrations and was not studied in detail. EbE4 populated two native states (N1 and N2), which differed by hydrophobic core packing and C-terminal interactions. At room temperature, the population ratio ( approximately 3-4:1) favored the state whose spectroscopic properties most resembled those of PsaE (N1). EbE4 also demonstrated altered folding kinetics, displaying multiple slow phases related to the population of intermediates and possibly N2. It was concluded that loop anchors can affect protein properties, including stability, via short-range effects on local structure and long-range communication with the packed hydrophobic core. Modification of the attachment points appears to be a possible stepping stone in the transition from one three-dimensional structure to another.
authors: Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
authors: Jane A
authors: JA
authors: Knappenberger
authors: Juliette T J
authors: JT
authors: Lecomte
mesh_terms: D002942:Circular Dichroism; D015786:Cytochromes b5 / Q000737:chemistry*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D045331:Photosystem I Protein Complex / Q000737:chemistry*; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013816:Thermodynamics
chemical_list: D045331:Photosystem I Protein Complex; C079741:photosystem I, psaE subunit; D015786:Cytochromes b5