RESUMO
Structure and membrane interaction of a 31 amino acid residue fragment of the membrane bound FKBP-like protein twisted dwarf 1 (TWD1) from Arabidopsis thaliana was investigated by solid-state NMR spectroscopy. The studied peptide TWD1(335-365) contained the putative membrane anchor of the protein (residues 339-357) that was previously predicted by sequence hydrophobicity analysis. The TWD1 peptide was synthesized by standard solid phase peptide synthesis and contained three uniformly (13)C- and (15)N-labelled residues (Phe 340, Val 350, Ala 364). The peptide was incorporated into either multilamellar vesicles or oriented planar membranes composed of an equimolar ternary phospholipid mixture (POPC, POPE, POPG), where the POPC was sn-1 chain-deuterated. (31)P NMR spectra of the membrane in the absence and in the presence of the peptide showed axially symmetric powder patterns indicative of a lamellar bilayer phase. Further, the addition of peptide caused a decrease in the lipid hydrocarbon chain order as indicated by reduced quadrupolar splittings in the (2)H NMR spectra of the POPC in the membrane. The conformation of TWD1(335-365) was investigated by (13)C cross-polarization magic-angle spinning NMR spectroscopy. At a temperature of -30 degrees C all peptide signals were resolved and could be fully assigned in two-dimensional proton-driven (13)C spin diffusion and (13)C single quantum/double quantum correlation experiments. The isotropic chemical shift values for Phe 340 and Val 350 exhibited the signature of a regular alpha-helix. Chemical shifts typical for a random coil conformation were observed for Ala 364 located close to the C-terminus of the peptide. Static (15)N NMR spectra of TWD1(335-365) in mechanically aligned lipid bilayers demonstrated that the helical segment of TWD1(335-365) adopts an orientation perpendicular to the membrane normal. At 30 degrees C, the peptide undergoes intermediate time scale motions.
Assuntos
Proteínas de Arabidopsis/química , Membrana Celular/química , Glicosilfosfatidilinositóis/química , Bicamadas Lipídicas/química , Proteínas de Ligação a Tacrolimo/química , Sítios de Ligação , Espectroscopia de Ressonância Magnética , Fusão de Membrana , Ligação ProteicaRESUMO
FKBP42 is a membrane-anchored immunophilin playing a critical role in morphogenesis and development of higher plants. We present the X-ray structure of the cytoplasmic portion of FKBP42 comprising both the FKBP-like domain and the TPR domain at 2.85 A resolution. The data shed light on the probable binding modes of key interaction partners, including HSP90 and two classes of ABC transporters. The resulting models provide a structural background for further investigation of the unique biological properties of this protein.
Assuntos
Proteínas de Arabidopsis/química , Imunofilinas/química , Proteínas de Ligação a Tacrolimo/química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Cristalografia por Raios X , Citoplasma , Proteínas de Choque Térmico HSP90/metabolismo , Estrutura Molecular , Ligação Proteica , Conformação ProteicaRESUMO
We present the three-dimensional structure of the N-terminal FK506-binding protein (FKBP)-like domain of the immunophilin FKBP42 from Arabidopsis thaliana. The data provide the structural background for the explanation of key functional properties reported previously.
Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/química , Proteínas de Ligação a Tacrolimo/química , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Proteínas de Ligação a Tacrolimo/metabolismoRESUMO
Two fragments of FKBP42 from Arabidopsis thaliana covering differing lengths of the molecule have been expressed, purified and crystallized. For each construct, crystals belonging to two different space groups were obtained and subjected to preliminary X-ray analysis.