Stability and structure of the membrane protein transporter Ffh is modulated by substrates and lipids.
The cytosolic protein Ffh transports membrane proteins from the ribosome to the inner membrane in complex with 4.5S RNA. Here we show that native Ffh binds to the hydrophobic probe ANS in a 1 Ffh:3 ANS stoichiometry, revealing a hydrophobic binding site. Thermal precipitation of Ffh is shifted upwards by approximately 10 degrees C by ANS or substrate protein, suggesting that the hydrophobic binding site makes the protein aggregation prone. Chemical denaturation confirm that Ffh is a rather unstable protein. 4.5S RNA destabilizes Ffh further, suggesting it keeps the protein in a more open conformation than the apoprotein. Escherichia coli lipid and DOPG (and to a smaller extent DOPC) increase Ffh's alpha-helical content, possibly related to Ffh's role in guiding membrane proteins to the membrane. Binding is largely mediated by electrostatic interactions but does not protect Ffh against trypsinolysis. We conclude that Ffh is a structurally flexible and dynamic protein whose stability is significantly modulated by the environment.
Department of Life Sciences, Aalborg University, Sohngaardsholmsvej 49, DK-9000 Aalborg, Denmark.
Marika E
ME
Reinau
Daniel E
DE
Otzen
D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D005960:Glucosides / Q000737:chemistry / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D050356:Lipid Metabolism / Q000502:physiology*; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011489:Protein Denaturation; D055550:Protein Stability; D018271:Signal Recognition Particle / Q000737:chemistry* / Q000378:metabolism; D013379:Substrate Specificity / Q000502:physiology; D013816:Thermodynamics; D013824:Thermus / Q000737:chemistry
D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C082931:Ffh protein, E coli; D005960:Glucosides; D008565:Membrane Proteins; D018271:Signal Recognition Particle; C040358:dodecyl maltoside