Determination of the Interface of a Large Protein Complex by Transferred Cross-saturation Measurements

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Background:The CERT PH domain is indispensable for the ER-to-Golgi ceramide transport. Results: The three-dimensional structure and interaction study revealed the Golgi recognition mode of the CERT PH domain. Conclusion:The basic groove in the CERT PH domain plays a critical role in the Golgi recognition. Significance: Conservation of the basic groove within lipid transporters uncovers functional significance of the structural motif.

Section: Methodsmentioning

confidence: 99%

Structural Basis for the Golgi Association by the Pleckstrin Homology Domain of the Ceramide Trafficking Protein (CERT)

Sugiki

Takeuchi

Yamaji

et al. 2012

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“…In (19,20) we prepared a sample containing LolA or mLolB labeled with 2 H and 15 N and the unlabeled binding partner. The molar ratio of the unlabeled protein to the labeled protein was set to 4:1.…”

Section: Methodsmentioning

confidence: 99%

Structural Investigation of the Interaction between LolA and LolB Using NMR

Nakada

Sakakura

Takahashi

et al. 2009

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Lipoproteins that play critical roles in various cellular functions of Gram-negative bacteria are localized in the cells inner and outer membranes. Lol proteins (LolA, LolB, LolC, LolD, and LolE) are involved in the transportation of outer membrane-directed lipoproteins from the inner to the outer membrane. LolA is a periplasmic chaperone that transports lipoproteins, and LolB is an outer membrane receptor that accepts lipoproteins. To clarify the structural basis for the lipoprotein transfer from LolA to LolB, we examined the interaction between LolA and mLolB, a soluble mutant of LolB, using solution NMR spectroscopy. We determined the interaction mode between LolA and mLolB with conformational changes of LolA. Based upon the observations, we propose that the LolA⅐LolB complex forms a tunnel-like structure, where the hydrophobic insides of LolA and LolB are connected, which enables lipoproteins to transfer from LolA to LolB.

“…Determination of the Site Buried in the Membrane on Sapecin-To identify the site buried in the membrane (membraneburied site), we performed TCS experiments (12)(13)(14). The TCS experiments were carried out under conditions with an excess amount of uniformly 2 H-and 15 N-labeled sapecin relative to the unlabeled phospholipid vesicle.…”

Section: Resultsmentioning

confidence: 99%

“…1C and 6A. Transferred Cross-saturation (TCS) Experiments-TCS experiments were carried out as previously described (12)(13)(14). All experiments were performed at pH 5.5 on a Bruker Avance 600 spectrometer.…”

Section: Expression and Purification Of Sapecin-sapecin Was Expressed Inmentioning

confidence: 99%

Channel-forming Membrane Permeabilization by an Antibacterial Protein, Sapecin

Takeuchi

Takahashi

Sugai

et al. 2004

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The action mechanism of sapecin, an antibacterial peptide with membrane permeabilization activity, was investigated. The dose dependence of the membrane permeabilization caused by sapecin was sigmoidal, suggesting that sapecin oligomerization leads to the membrane permeabilization. Solution nuclear magnetic resonance analysis of the sapecin-phospholipid vesicle complex revealed the surface buried in the membrane and oligomerization surface on the sapecin molecule. The membrane-buried surface of sapecin was determined by observing the transferred cross-saturation phenomena from the alkyl chains of the phospholipid vesicle to the amide protons of sapecin. The membraneburied surface contains basic and highly exposed hydrophobic residues, which are suitable for interacting with the acidic bacterial membrane. The oligomerization surface was also identified by comparisons between the results from hydrogen-deuterium exchange experiments and transferred cross-saturation experiments. On the basis of the results from the NMR experiments we built a putative model of sapecin oligomers, which provides insights into the membrane permeabilization caused by insect defensins.