• 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:o the folding pathwayo the structure and stability of the apoprotein, o the mechanism of FMN recognition, o the interactions that stabilize the functional complex and
tailor the redox potentialso many details of the binding and electron transfer to partner
proteins
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.
Predicting the flavodoxin secondary and tertairy structure
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
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
T1fx1 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------
GIteration 0 SP= 136944.00 AvSP= 10.675 SId= 4009 AvSId= 0.313
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
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
Amino acid properties
Amino acid hydrophobicity scale
hydrophobic
hydrophilic
Burried and Edge strands
Parallel -sheet
Anti-parallel -sheet
Periodicity patterns within secondary structures
Burried -strand
Edge -strand
-helix
= hydrophilic = hydrophobic
TOPS diagrams
Circle = helix
Triangle = strand
-- motif is right-handed in >95% of cases
RH LH
Building flavodoxin
RH