Folding and stability of the leucine-rich repeat domain of internalin B from Listeri monocytogenes.
Internalin B (InlB), a surface protein of the human pathogen Listeria monocytogenes, promotes invasion into various host cell types by inducing phagocytosis of the entire bacterium. The N-terminal half of InlB (residues 36-321, InlB321), which is sufficient for this process, contains a central leucine-rich repeat (LRR) domain that is flanked by a small alpha-helical cap and an immunoglobulin (Ig)-like domain. Here we investigated the spectroscopic properties, stability and folding of InlB321 and of a shorter variant lacking the Ig-like domain (InlB248). The circular dichroism spectra of both protein variants in the far ultraviolet region are very similar, with a characteristic minimum found at approximately 200 nm, possibly resulting from the high 3(10)-helical content in the LRR domain. Upon addition of chemical denaturants, both variants unfold in single transitions with unusually high cooperativity that are fully reversible and best described by two-state equilibria. The free energies of GdmCl-induced unfolding determined from transitions at 20 degrees C are 9.9(+/-0.8)kcal/mol for InlB321 and 5.4(+/-0.4)kcal/mol for InlB248. InlB321 is also more stable against thermal denaturation, as observed by scanning calorimetry. This suggests, that the Ig-like domain, which presumably does not directly interact with the host cell receptor during bacterial invasion, plays a critical role for the in vivo stability of InlB.
Potsdam University, Physical Biochemistry, Karl-Liebknecht-Str. 24-25, Haus 25, D-14476 Potsdam-Golm, Germany.
Alexander
A
Freiberg
Matthias P
MP
Machner
Wolfgang
W
Pfeil
Wolf-Dieter
WD
Schubert
Dirk W
DW
Heinz
Robert
R
Seckler
D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002942:Circular Dichroism; D007930:Leucine / Q000737:chemistry; D008089:Listeria monocytogenes / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D020449:Repetitive Sequences, Amino Acid; D013816:Thermodynamics
D001426:Bacterial Proteins; D008565:Membrane Proteins; D010446:Peptide Fragments; C095768:inlB protein, Listeria monocytogenes; D007930:Leucine; D002118:Calcium