Structural Study Of Haemophilus Influenzae BAM Complex

Haemouphilus influenzae is a gram-negative bacterium. It is thought that outer membrane protein biogenesis requires the nascent protein to secrete across inner membrane via Sec pathway. Subsequently, further assisted mainly by an outer membrane protein complex known as β-barrel Assembly Machinery(BAM), outer membrane protein finishes its final membrane insertion and folding in the lipid bilayer. BAM, together with its homologues, is a ubiquitous system widespread in both procaryotic and eucaryotic cells. In E. coli, BAM complex consists of 5 partner proteins:BamA, the outer membrane core/channel, and the other four lipoproteins,BamB, BamC, BamD and BamE. Interestingly, Haemouphilus influenzae BAM complex only have four components: Bam A, BamC, BamD and BamE.Until now, very little structural information is available for the Haemouphilus influenzae BAM complex. In my master study, I want to digdeeper into this barren area and this will help to better understand this ancestorial system not only exist in Haemouphilus influenzae. Thus far, I have cloned BamD 29-262 in pET-15 b, and BamC18-215,BamC45-215 and a series of BamA POTRA fragments in pET-32 m, expressed them in BL21(DE3) or B834(for SelenoMet protein). The construct BamD 29-262 has yielded crystals that diffracts to 4 ?. Furthermore, in order to improve the quality of the crystal, I have re-designed a new experimental strategy involving the co-crystallisation of BamC and BamD. The BamD 29-262/BamC 18-215 complex has yielded crystals that diffracts up to 3.8 ? and BamD 29-262 /BamC 45-215 complex to 4.5 ?. Dehydration cannot inverse the resolution of both the BamD crystal or BamCD complex. MAD strategy,with the incorporation of SelenoMet, failed to phase BamCD complex.His-tag BamD 29-262 yielded crystal that achieved no improvement in resolution.