Sandbox Reserved 310: Difference between revisions
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=Structure= | =Structure= | ||
The BLUF domain is a <scene name='Sandbox_Reserved_310/ | ==Domains== | ||
Each monomer is comprised of 5 β-strands and 4 α-helices in the order of β1α1β2β3α2β4β5α3α4. Specifically, the BLUF domain of the monomer contains β1α1β2β3α2β4β5, while the C-terminal domain contains α3α4<ref name="one" />. The C-terminal domain interacts with the end of the β-sheet of the neighbouring monomer. | [[Image:FADbonding.jpg|thumb|left|300px|Figure 1. The hydrogen bonds the FAD ligand forms with nearby amino acid residues.]] | ||
The BLUF domain is a <scene name='Sandbox_Reserved_310/Decamer/1'>decamer</scene> with a molecular weight of approximately 160kDa<ref name="one" />. There are ten monomers observed in each asymmetric unit. The crystalline structure of the BLUF domain from the T110078 protein was solved by single isomorphous replacement (SIR) method using a mercury derivative. The double ringed decamer has a diameter of approximately 95Å, a thickness of 60Å and a central channel approximately 35Å in diameter<ref name="one" />. | |||
Each monomer is comprised of 5 <scene name='Sandbox_Reserved_310/Monomer/3'>β-strands</scene> and 4<scene name='Sandbox_Reserved_310/Monomer/2'>α-helices</scene> in the order of β1α1β2β3α2β4β5α3α4. Specifically, the BLUF domain of the monomer contains β1α1β2β3α2β4β5, while the C-terminal domain contains α3α4<ref name="one" />. The C-terminal domain interacts with the end of the β-sheet of the neighbouring monomer. | |||
The isoalloxazine ring of FAD is located between α1 and α2 of the BLUF domain, between two highly conserved hydrophobic residues: Ile24 and Ile66<ref name="one" />. FAD forms hydrogen bonds with the following amino acid residues: Asn21, Asn32, Gln50, Arg65 and Asp69<ref name="one" />. More specifically, the side chain of Asn31 binds to O2 of FAD and Asn32 binds to N3 and O4 of FAD. The guanido group of Arg65 contributes to a network between FAD and the apo protein. The amide N of the Gln50 sidechain interacts with N5 and O4 of FAD through hydrogen bonding, while the amide O of the sidechain is closely linked with the hydroxyl oxygen of the highly conserved Tyr8 residue, forming a FAD-Gln50-Tyr8 network<ref name="one" />. This conserved Tyr8 residue is the only residue that has been shown to be essential for light reaction in the BLUF domain containing AppA and Slr1694 proteins<ref name ="seven">PMID: 17042486</ref>. | The isoalloxazine ring of FAD is located between <scene name='Sandbox_Reserved_310/Monomer/4'>α1 and α2</scene> of the BLUF domain, between two highly conserved hydrophobic residues: Ile24 and Ile66<ref name="one" />. FAD forms hydrogen bonds with the following amino acid residues: Asn21, Asn32, Gln50, Arg65 and Asp69 (Figure 1)<ref name="one" />. More specifically, the side chain of Asn31 binds to O2 of FAD and Asn32 binds to N3 and O4 of FAD. The guanido group of Arg65 contributes to a network between FAD and the apo protein. The amide N of the Gln50 sidechain interacts with N5 and O4 of FAD through hydrogen bonding, while the amide O of the sidechain is closely linked with the hydroxyl oxygen of the highly conserved Tyr8 residue, forming a FAD-Gln50-Tyr8 network<ref name="one" />. This conserved Tyr8 residue is the only residue that has been shown to be essential for light reaction in the BLUF domain containing AppA and Slr1694 proteins<ref name ="seven">PMID: 17042486</ref>. | ||
==Further Analyses== | |||
While Asn32, Gln50, Asp69, Arg71, His72 and Ser10 are completely conserved residues, Asn31, Arg65 and Ser28 are only moderately conserved. This network surrounding FAD is thought to be highly conserved in all BLUF domains. In order to study the importance of the interactions between the isoalloxaizine ring of FAD, and Asn31, Asn32 and Gln50, the amino acids were replaced with alanine residues<ref name="one" />. It was observed that the spectra of the mutants were qualitatively similar to that of the wild-type. However, the low energy absorbance peak of N32A (Asn32 replaced with Ala) was blue shifted compared to the wildtype by 6nm<ref name="one" />. This blue shift can be accounted for by the lack of hydrogen bonding between the side-chain and isoalloxazine ring<ref name="one" />. In the wild-type protein, Asn32 forms 2 hydrogen bonds with FAD; the absence of these bonds allows relocation of an electron on FAD, resulting on the spectral blue shift. | While Asn32, Gln50, Asp69, Arg71, His72 and Ser10 are completely conserved residues, Asn31, Arg65 and Ser28 are only moderately conserved. This network surrounding FAD is thought to be highly conserved in all BLUF domains. In order to study the importance of the interactions between the isoalloxaizine ring of FAD, and Asn31, Asn32 and Gln50, the amino acids were replaced with alanine residues<ref name="one" />. It was observed that the spectra of the mutants were qualitatively similar to that of the wild-type. However, the low energy absorbance peak of N32A (Asn32 replaced with Ala) was blue shifted compared to the wildtype by 6nm<ref name="one" />. This blue shift can be accounted for by the lack of hydrogen bonding between the side-chain and isoalloxazine ring<ref name="one" />. In the wild-type protein, Asn32 forms 2 hydrogen bonds with FAD; the absence of these bonds allows relocation of an electron on FAD, resulting on the spectral blue shift. | ||