Predicting the flavodoxin secondary and tertairy structure

1. Predicting the flavodoxin secondary and tertairy structure • Flavodoxins are electron-transfer proteins involved in a variety of photosynthetic and non-photosynthetic reactions • The redox activity of flavodoxin derives from its bound flavin mononucleotide cofactor (FMN), whose intrinsic properties are profoundly modified by the host protein. • In the last decade of flavodoxin research, the following has been revealed: • the folding pathway • the structure and stability of the apoprotein, • the mechanism of FMN recognition, • the interactions that stabilize the functional complex and tailor the redox potentials • many details of the binding and electron transfer to partner proteins

2. Predicting the flavodoxin secondary and tertairy structure • The next decade should witness an even deeper understanding of the flavodoxin molecule and a greater comprehension of its many physiological roles. • The fact that flavodoxin is essential for the survival of some human pathogens could make it a drug target on its own.

3. Predicting the flavodoxin secondary and tertairy structure • Predict using a multiple alignment of 13 flavodoxin sequences • Redox protein • Involved in photosynthesis and other crucial processes • The 14th sequence on the bottom of the alignment is a VERY distantly related protein cheY • Chemotaxis protein • For example, it interacts with proteins at the base of the flagellar apparatus of E. coli and promotes clockwise flagellar rotation

4. Flavodoxin-cheY multiple sequence alignment: • 1fx1 -PKALIVYGSTTGNT-EYTAETIARQLANAG-YEVDSRDAASVEAGGLFEGFDLVLLGCSTWGDDSI------ELQDDFIPLF-DSLEETGAQGRKVACF • FLAV_DESDE MSKVLIVFGSSTGNT-ESIaQKLEELIAAGG-HEVTLLNAADASAENLADGYDAVLFgCSAWGMEDL------EMQDDFLSLF-EEFNRFGLAGRKVAAf • FLAV_DESVH MPKALIVYGSTTGNT-EYTaETIARELADAG-YEVDSRDAASVEAGGLFEGFDLVLLgCSTWGDDSI------ELQDDFIPLF-DSLEETGAQGRKVACf • FLAV_DESSA MSKSLIVYGSTTGNT-ETAaEYVAEAFENKE-IDVELKNVTDVSVADLGNGYDIVLFgCSTWGEEEI------ELQDDFIPLY-DSLENADLKGKKVSVf • FLAV_DESGI MPKALIVYGSTTGNT-EGVaEAIAKTLNSEG-METTVVNVADVTAPGLAEGYDVVLLgCSTWGDDEI------ELQEDFVPLY-EDLDRAGLKDKKVGVf • 2fcr --KIGIFFSTSTGNT-TEVADFIGKTLGA---KADAPIDVDDVTDPQALKDYDLLFLGAPTWNTG----ADTERSGTSWDEFLYDKLPEVDMKDLPVAIF • FLAV_AZOVI -AKIGLFFGSNTGKT-RKVaKSIKKRFDDET-MSDA-LNVNRVS-AEDFAQYQFLILgTPTLGEGELPGLSSDCENESWEEFL-PKIEGLDFSGKTVALf • FLAV_ENTAG MATIGIFFGSDTGQT-RKVaKLIHQKLDG---IADAPLDVRRAT-REQFLSYPVLLLgTPTLGDGELPGVEAGSQYDSWQEFT-NTLSEADLTGKTVALf • FLAV_ANASP SKKIGLFYGTQTGKT-ESVaEIIRDEFGN---DVVTLHDVSQAE-VTDLNDYQYLIIgCPTWNIGEL--------QSDWEGLY-SELDDVDFNGKLVAYf • FLAV_ECOLI -AITGIFFGSDTGNT-ENIaKMIQKQLGK---DVADVHDIAKSS-KEDLEAYDILLLgIPTWYYGE--------AQCDWDDFF-PTLEEIDFNGKLVALf • 4fxn -MK--IVYWSGTGNT-EKMAELIAKGIIESG-KDVNTINVSDVNIDELL-NEDILILGCSAMGDEVL-------EESEFEPFI-EEIS-TKISGKKVALF • FLAV_MEGEL MVE--IVYWSGTGNT-EAMaNEIEAAVKAAG-ADVESVRFEDTNVDDVA-SKDVILLgCPAMGSEEL-------EDSVVEPFF-TDLA-PKLKGKKVGLf • FLAV_CLOAB -MKISILYSSKTGKT-ERVaKLIEEGVKRSGNIEVKTMNLDAVD-KKFLQESEGIIFgTPTYYAN---------ISWEMKKWI-DESSEFNLEGKLGAAf • 3chy ADKELKFLVVDDFSTMRRIVRNLLKELGFN--NVEEAEDGVDALNKLQAGGYGFVI---SDWNMPNM----------DGLELL-KTIRADGAMSALPVLM • T • 1fx1 GCGDS-SY-EYFCGA-VDAIEEKLKNLGAEIVQD---------------------GLRIDGD--PRAARDDIVGWAHDVRGAI-------- • FLAV_DESDE ASGDQ-EY-EHFCGA-VPAIEERAKELgATIIAE---------------------GLKMEGD--ASNDPEAVASfAEDVLKQL-------- • FLAV_DESVH GCGDS-SY-EYFCGA-VDAIEEKLKNLgAEIVQD---------------------GLRIDGD--PRAARDDIVGwAHDVRGAI-------- • FLAV_DESSA GCGDS-DY-TYFCGA-VDAIEEKLEKMgAVVIGD---------------------SLKIDGD--PE--RDEIVSwGSGIADKI-------- • FLAV_DESGI GCGDS-SY-TYFCGA-VDVIEKKAEELgATLVAS---------------------SLKIDGE--PD--SAEVLDwAREVLARV-------- • 2fcr GLGDAEGYPDNFCDA-IEEIHDCFAKQGAKPVGFSNPDDYDYEESKS-VRDGKFLGLPLDMVNDQIPMEKRVAGWVEAVVSETGV------ • FLAV_AZOVI GLGDQVGYPENYLDA-LGELYSFFKDRgAKIVGSWSTDGYEFESSEA-VVDGKFVGLALDLDNQSGKTDERVAAwLAQIAPEFGLS--L-- • FLAV_ENTAG GLGDQLNYSKNFVSA-MRILYDLVIARgACVVGNWPREGYKFSFSAALLENNEFVGLPLDQENQYDLTEERIDSwLEKLKPAV-L------ • FLAV_ANASP GTGDQIGYADNFQDA-IGILEEKISQRgGKTVGYWSTDGYDFNDSKA-LRNGKFVGLALDEDNQSDLTDDRIKSwVAQLKSEFGL------ • FLAV_ECOLI GCGDQEDYAEYFCDA-LGTIRDIIEPRgATIVGHWPTAGYHFEASKGLADDDHFVGLAIDEDRQPELTAERVEKwVKQISEELHLDEILNA • 4fxn G-----SY-GWGDGKWMRDFEERMNGYGCVVVET---------------------PLIVQNE--PDEAEQDCIEFGKKIANI--------- • FLAV_MEGEL G-----SY-GWGSGEWMDAWKQRTEDTgATVIGT----------------------AIVNEM--PDNA-PECKElGEAAAKA--------- • FLAV_CLOAB STANSIAGGSDIA---LLTILNHLMVKgMLVYSG----GVAFGKPKTHLGYVHINEIQENEDENARIfGERiANkVKQIF----------- • 3chy VTAEAKK--ENIIAA---------AQAGAS-------------------------GYVV-----KPFTAATLEEKLNKIFEKLGM------ • G • Iteration 0 SP= 136944.00 AvSP= 10.675 SId= 4009 AvSId= 0.313

5. Rules of thumb when looking at a multiple alignment (MA) • Hydrophobic residues are internal • Gly (Thr, Ser) in loops • MA: hydrophobic block -> internal -strand • MA: alternating (1-1) hydrophobic/hydrophilic => edge -strand • MA: alternating 2-2 (or 3-1) periodicity => -helix • MA: gaps in loops • MA: Conserved column => functional? => active site

6. Rules of thumb when looking at a multiple alignment (MA) … cont. • Active site residues are together in 3D structure • Helices often cover up core of strands • Helices less extended than strands => more residues to cross protein • -- motif is right-handed in >95% of cases (with parallel strands) • MA: ‘inconsistent’ alignment columns and match errors! • Secondary structures have local anomalies, e.g. -bulges

7. Amino acid properties

8. Amino acid hydrophobicity scale hydrophobic hydrophilic

11. Burried and Edge strands Parallel -sheet Anti-parallel -sheet

12. Periodicity patterns within secondary structures Burried -strand Edge -strand -helix = hydrophilic = hydrophobic

13. TOPS diagrams Circle = helix Triangle = strand

14. -- motif is right-handed in >95% of cases LH RH

15. Building flavodoxin RH