6BICH002W Coursework Part 2 – designing oligonucleotide primers for PCR and site-directed mutagenesis / DNA sequences.
Shown below is a typical L-Glutamate Binding Domain (GBD) protein sequence (underlined). This binding domain occurs as part of a larger receptor protein subunit sequence (not fully shown). The key L-glutamate interacting residues are highlighted. The L-Glutamate Binding Domain (underlined) has been engineered to be expressed as an independent, soluble protein for crystallisation studies (see Figure 1).
360 370 380 390 400QVRIQGLTGN VQFDHYGRRV NYTMDVFELK STGPRKVGYW NDMDKLVLIQ
410 420 430 440 450
DMPTLGNDTA AIENRTVVVT TIMESPYVMY KKNHEMFEGN DKYEGYCVDL
460 470 480 490 500
ASEIAKHIGI KYKIAIVPDG KYGARDADTK IWNGMVGELV YGKAEIAIAP
510 520 530 540 550
LTITLVREEV IDFSKPFMSL GISIMIKKPQ KSKPGVFSFL DPLAYEIWMC
560 570 580 590 600
IVFAYIGVSV VLFLVSRFSP YEWHTEEPED GKEGPSDQPP NEFGIFNSLW
610 620 630 640 650
FSLGAFMQQG CDISPRSLSG RIVGGVWWFF TLIIISSYTA NLAAFLTVER
660 670 680 690 700
MVSPIESAED LAKQTEIAYG TLDSGSTKEF FRRSKIAVYE KMWTYMRSAE
710 720 730 740 750
PSVFTRTTAE GVARVRKSKG KFAFLLESTM NEYTEQRKPC DTMKVGGNLD
760 770 780 790 800
SKGYGVATPK GSSLGNAVNL AVLKLNEQGL LDKLKNKWWY DKGECGSGGG
810 820 830 840 850
DSKDKTSALS LSNVAGVFYI LVGGLGLAML VALIEFCYKS RAEAKRMKLT
GBD structure
Residues critical for the binding ot L-Glutamate to GBD are highlighed in yellow in the above protein sequence and are shown highlighted in the coresponding motifs below:
R-E-x-V-[I,V]-D-F
G-[S,A]-T-x(2)-F-F
A-[Y,F]-L-x-E-[S,A]-[T,A]
DNA sequence for a putative GBD protein:
SEQUENCE 1
TCAACGAGAAAGGGCGCCGGACCAATTACACCCTCCACGTGATCGAAATGAAACATGATG
GAATCCGAAAGATTGGTTACTGGAATGAAGACGATAAATTTGTCCCCGCAGCCACCGACG
CTCAGGCTGGAGGGGACAACTCAAGCGTCCAGAATAGGACCTACATCGTCACTACTATCC
TCGAAGATCCTTACGTGATGCTTAAAAAGAATGCCAACCAGTTTGAGGGCAATGACCGCT
ATGAGGGCTACTGTGTGGAGCTGGCTGCAGAGATCGCCAAGCACGTGGGCTACTCCTACC
GACTTGAGATTGTCAGCGACGGCAAATATGGAGCCCGGGATCCCGACACAAAGGCTTGGA
ATGGCATGGTGGGAGAACTGGTCTATGGAAGAGCAGACGTGGCTGTGGCTCCCTTGACCA
TAACCTTGGTCCGGGAGGAAGTCATCGACTTCTCCAAGCCATTCATGAGTTTGGGAATCT
CCATTATGATTAAGAAGCCACAGAAGTCCAAGCCAGGTGTCTTCTCCTTTCTTGACCCTT
TGGCCTATGAGATCTGGATGTGTATAGTGTTTGCCTACATTGGAGTGAGCGTCGTCCTCT
TCCTGGTCAGCCGTTTCAGCCCCTACGAATGGCACAGCGAAGAGTTTGAAGAGGGACGAG
ACCAGACAACCAGTGACCAGTCAAATGAGTTTGGCATATTCAACAGCCTGTGGTTCTCCC
TGGGGGCCTTCATGCAGCAAGGATGTGACATTTCCCCCAGGTCCCTGTCCGGACGCATCG
TCGGCGGCGTCTGGTGGTTCTTCACTTTGATCATCATCTCCTCGTACACAGCCAACCTGG
CTGCCTTCCTGACTGTGGAGAGGATGGTGTCTCCCATTGAGAGTGCAGAGGACCTGGCAA
AGCAGACGGAAATTGCTTATGGGACATTGGAAGCAGGCTCCACTAAGGAGTTCTTCAGGA
GATCTAAAATCGCTGTGTTTGAGAAGATGTGGACATACATGAAGTCTGCAGAACCATCCG
TGTTTGTTCGGACCACAGAGGAAGGCATGATCAGAGTGAGAAAATCTAAAGGCAAATACG
CCTACCTCCTGGAGTCCACCATGAATGAGTATATTGAGCAACGAAAGCCCTGTGACACCA
TGAAAGTGGGAGGTAACTTGGATTCCAAAGGCTATGGCATTGCGACACCCAAGGGGTCCG
CCCTGAGAAATCCAGTAAACCTGGCAGTGTTAAAACTGAACGAGCAGGGGCTTTTGGACA
AATTGAAAAACAAATGGTGGTACGACAAGGGCGAGTGCGGCAGCGGGGGAGGTGACTCCA
AGGACAAGACCAGCGCTTTGAGCCTCAGCAATGTGGCAGGCGTGTTCTACATCCTGATTG
GAGGGCTGGGACTGGCCATGCTGGTTGCCTTAATCGAGTTCTGCTACAAATCCCGTAGCG
SEQUENCE 2
GAGCTCCCATCAGGAAATGACACGTCTGGGCTTGAAAACAAGACTGTGGTGGTCACCACA
ATATTGGAATCTCCATATGTTATGATGAAGAAAAATCATGAAATGCTTGAAGGGAATGAG
CGTTACGAGGGCTACTGTGTTGACTTAGCTGCAGAAATTGCCAAACACTGTGGGTTCAAG
TACAAGCTGACTATTGTTGGGGATGGCAAGTATGGGGCCAGGGATGCCGACACCAAAATT
TGGAATGGTATGGTTGGAGAGCTTGTCTACGGGAAAGCTGACATTGCAATTGCTCCATTA
ACTATCACTCTCGTGAGAGAAGAGGTGATTGACTTCTCCAAGCCCTTCATGAGTCTTGGA
ATCTCTATCATGATCAAGAAGCCTCAGAAGTCCAAACCAGGAGTGTTTTCCTTTCTTGAT
CCTTTAGCCTATGAGATCTGGATGTGCATTGTGTTTGCCTACATTGGGGTCAGTGTAGTT
TTATTCCTGGTCAGCAGATTTAGCCCCTACGAGTGGCACACTGAGGAATTTGAAGATGGA
AGAGAAACACAAAGTAGTGAATCAACTAATGAATTTGGGATTTTTAATAGTCTCTGGTTT
TCCTTGGGTGCCTTTATGCGGCAGGGATGCGATATTTCGCCAAGATCCCTCTCTGGGCGC
ATTGTTGGAGGTGTGTGGTGGTTCTTTACCCTGATCATAATCTCCTCCTACACGGCTAAC
TTAGCTGCCTTCCTGACTGTAGAGAGGATGGTGTCTCCCATCGAAAGTGCTGAGGATCTG
TCTAAGCAAACAGAAATTGCTTATGGAACATTAGACTCTGGCTCCACTAAAGAGTTTTTC
AGGAGATCTAAAATCGCAGTGTTTGATAAAATGTGGACTTATATGAGGAGTGCAGAGCCC
TCTGTGTTTGTGAGGACTACCGCAGAAGGAGTAGCCAGAGTCCGGAAATCCAAAGGAAAG
TATGCCTACTTGCTGGAGTCCACAATGAACGAGTACATCGAGCAGAGGAAGCCTTGTGAC
ACCATGAAAGTGGGAGGAAACTTGGATTCCAAAGGCTACGGCATCGCCACACCTAAAGGA
TCCTCATTAGGAAATGCGGTTAACCTCGCAGTACTAAAACTGAATGAACAAGGCCTGTTG
GACAAATTGAAAAACAAATGGTGGTACGACAAAGGAGAGTGCGGCAGCGGGGGAGGTGAT
TCCAAGGAAAAGACCAGTGCCCTCAGTCTGAGCAACGTTGCTGGAGTATTCTACATCCTT
SEQUENCE 3
GAAAGTCAGAAAGGAAAGCCAGCAAATATCACAGACTCATTGTCTAATCGTTCTTTGATT
GTTACCACCATTTTGGAAGAACCGTATGTTCTGTTTAAGAAGTCTGACAAACCACTCTAT
GGGAATGATCGATTTGAAGGCTACTGTATTGATCTCCTACGAGAGTTATCTACAATCCTT
GGCTTTACATATGAGATTAGGCTTGTGGAGGATGGGAAATATGGAGCCCAGGATGATGTG
AACGGACAATGGAATGGAATGGTTCGTGAACTAATCGATCATAAAGCTGACCTTGCAGTT
GCTCCACTGGCTATAACCTATGTTCGTGAGAAGGTCATCGACTTTTCAAAGCCGTTTATG
ACACTTGGAATAAGTATTTTGTACCGCAAGCCCAATGGTACAAACCCAGGCGTCTTCTCC
TTCCTGAATCCTCTCTCCCCTGATATCTGGATGTATGTTCTGCTGGCTTGCTTGGGTGTC
AGTTGTGTGCTCTTTGTCATAGCCAGGTTTAGTCCCTATGAGTGGTATAACCCACACCCT
TGCAACCCTGACTCAGACGTGGTGGAAAACAATTTTACCTTGCTAAATAGTTTCTGGTTT
GGAGTTGGAGCTCTCATGCAGCAAGGTTCTGAGCTCATGCCCAAAGCACTCTCCACCAGG
ATAGTGGGAGGCATTTGGTGGTTTTTCACACTTATCATCATTTCTTCGTATACCGCTAAC
CTAGCCGCCTTTCTGACTGTGGAACGCATGGAGTCGCCCATTGACTCTGCTGACGATTTA
GCTAAGCAAACCAAGATAGAGTATGGAGCAGTGGAGGACGGCGCAACCATGACGTTTTTT
AAGAAATCAAAAATTTCAACGTATGATAAAATGTGGGCGTTTATGAGCAGCAGGAGACAG
TCTGTGCTTGTCAAAAGCAATGAGGAAGGGATCCAACGAGTCCTCACCTCGGATTATGCT
TTCTTAATGGAGTCAACAACCATCGAGTTTGTTACACAGCGGAACTGTAACCTCACGCAG
ATTGGCGGCCTTATAGACTCCAAAGGCTATGGCGTTGGCACTCCTATGGGCTCTCCATAT
CGAGACAAAATCACCATAGCAATTCTTCAGCTGCAGGAGGAAGGCAAGCTGCACATGATG
AAGGAGAAATGGTGGCGGGGCAATGGCTGCCCAGAGGAGGAGAGCAAAGAGGCCAGTGCT
CTGGGGGTGCAGAATATTGGTGGTATCTTCATTGTCCTGGCAGCCGGCTTGGTGCTCTCA
SEQUENCE 4
TCTGGTGGTCACAACAATCCTGGAGAACCCGTATGTCATGCGCCGGCCCAACTTCCAGGC
CCTGTCAGGGAACGAGCGCTTTGAGGGCTTCTGCGTGGACATGCTTCGGGAGCTGGCCGA
GCTGCTGCGCTTCCGGTATCGCCTGCGGTTGGTGGAGGACGGACTGTACGGGGCACCTGA
GCCCAATGGTTCCTGGACAGGAATGGTTGGCGAGCTCATCAACCGGAAGGCAGACCTGGC
TGTGGCAGCCTTCACCATCACCGCGGAGAGAGAGAAGGTCATCGACTTCTCCAAGCCATT
CATGACCCTGGGGATCAGCATCCTCTACAGGGTGCACATGGGCCGCAAGCCTGGCTACTT
CTCCTTCCTGGACCCTTTCTCCCCTGCCGTGTGGCTCTTCATGCTTCTTGCCTACCTGGC
TGTCAGCTGTGTCTTGTTTCTGGCTGCCAGGCTGAGCCCTTATGAGTGGTATAACCCTCA
CCCGTGTCTTCGGGCGCGTCCCCATATCCTGGAGAACCAGTACACGCTGGGTAACAGCCT
TTGGTTCCCTGTGGGTGGCTTCATGCAGCAGGGCTCGGAGATCATGCCCCGGGCGCTGTC
CACACGCTGTGTCAGCGGAGTCTGGTGGGCCTTCACCTTGATCATCATCTCCTCCTACAC
GGCCAACCTGGCTGCCTTCCTCACGGTGCAGCGCATGGAGGTGCCGGTGGAGTCTGCAGA
CGACTTGGCGGATCAGACCAACATTGAGTACGGCACTATCCACGCTGGCTCCACCATGAC
CTTCTTTCAGAACTCACGGTACCAGACGTACCAGCGGATGTGGAATTACATGCAGTCGAA
GCAGCCCAGCGTGTTCGTCAAGAGCACAGAGGAGGGGATCGCCCGTGTCCTCAACTCACG
TTACGCCTTCCTGCTGGAGTCCACCATGAACGAGTACCACAGGCGCCTCAATTGCAACCT
CACCCAGATCGGGGGCCTCCTCGACACCAAGGGCTACGGCATCGGCATGCCGCTGGGCTC
CCCATTCCGGGATGAGATCACGCTGGCCATCCTGCAGCTCCAGGAGAACAACAGGCTGGA
GATCCTGAAGCGCAAGTGGTGGGAGGGCGGCCGGTGCCCCAAGGAGGAGGACCACAGGGC
CAAAGGTTTGGGCATGGAGAACATTGGCGGTATTTTTGTCGTGCTGATCTGTGGCCTCAT
SEQUENCE 5
TTCAAAAGAAGTATTAGCGTGGCGACAAGGTGTTGGATTCTTCTCAAATCGAACAGTTGC
TCAACATTCGAGAAAGAGTCAAAATGATCATAAGGATAATCAAGTGATAGTGCTCACGAA
TCTAGTTGCTCCATTTGTGATGATAAAACGAGAATGTTTGGAAATGGCAAATTTGACAGA
ATGTCAGGGGAACAATAAGTTTGAAGGATTTTGTATCGACTTGCTGAAACTGTTGGCTGA
CAAAATTGAGGAGTTCAATTATGAGATCAAATTAGGAACAAAGGCGGGATCAAAACAAGC
TGACGGAAGCTGGGATGGAATGATTGGAGAACTTCTGAGTGGAAGAGCCCATGCTGTAGT
TGCATCACTTACAATAAATCAAGAAAGAGAACGAGTCGTGGATTTTTCAAAACCATTTAT
GACAACTGGAATTTCAATTATGATCAAGAAGCCTGACAAACAAGAGTTTTCAGTATTTTC
CTTTATGCAACCGCTTAGTACAGAGATTTGGATGTACATCATATTCGCGTATATTGGGGT
GTCAGTAGTCATCTTTCTTGTTTCAAGGTTTTCACCGTATGAATGGAGAGTCGAGGAGAC
TTCCAGAGGAGGATTCACTATTTCCAACGATTTTTCTGTTTATAATTGCTTATGGTTTAC
ACTAGCAGCTTTCATGCAGCAAGGAACTGATATTTTACCAAGATCAATTTCGGGTCGTAT
CGCATCTTCTGCTTGGTGGTTTTTCACAATGATCATCGTTTCTTCTTACACTGCTAACTT
GGCTGCGTTCCTAACACTTGAAAAAATGCAAGCTCCTATAGAAAGTGTTGAAGACTTGGC
GAAACAGTCGAAGATTAAATACGGAATTCAAGGTGGCGGATCGACAGCTTCTTTCTTCAA
ATATTCCTCCGTTCAAATATATCAGAGAATGTGGAGATATATGGAATCGCAAGTTCCGCC
AGTTTTTGTGGCTAGTTATGCGGAAGGAATTGAAAGAGTTCGAAGTCATAAAGGAAGATA
TGCATTTCTTTTAGAAGCAACTGCAAATGAATATGAGAATACAAGAAAACCATGTGATAC
AATGAAAGTTGGAGCAAATCTCAACAGTATCGGATATGGAATAGCGACACCTTTCGGCTC
CGACTGGAAGGATCATATCAACTTGGCAATTTTAGCCCTCCAAGAACGTGGTGAACTGAA
AAAGTTGGAAAACAAATGGTGGTATGATAGAGGACAATGCGATGCGGGTATTACTGTTGA
CGGGTCATCTGCTAGTTTGAATCTATCAAAAGTTGCTGGAA
SEQUENCE 6
CCCTTCTCAGATCAACAAATCAGCAATGACAGCTCATCCTCAGAGAACCGGACCATTGTA
GTGACTACCATTCTGGAATCACCATATGTGATGTATAAAAAGAATCATGAGCAGCTGGAA
GGAAATGAGCGCTATGAAGGCTACTGTGTTGATTTAGCCTATGAAATAGCCAAACACGTA
AGGATCAAATACAAATTGTCCATTGTCGGTGATGGGAAATATGGCGCCAGAGATCCAGAG
ACTAAAATATGGAATGGCATGGTTGGGGAACTTGTCTATGGAAGAGCTGATATAGCTGTT
GCTCCACTCACTATAACATTGGTCCGTGAAGAAGTCATAGATTTCTCAAAGCCATTTATG
AGCCTGGGAATCTCCATCATGATAAAGAAGCCTCAGAAATCAAAGCCAGGCGTCTTTTCA
TTCCTGGATCCTTTGGCTTATGAAATCTGGATGTGCATTGTCTTCGCTTACATTGGAGTC
AGTGTAGTTCTCTTCCTAGTCAGCAGATTTAGCCCTTATGAATGGCACTTGGAAGACAAC
AATGAAGAACCTCGTGACCCACAAAGCCCTCCTGATCCTCCCAATGAATTTGGAATATTT
AACAGTCTTTGGTTTTCCTTGGGTGCTTTCATGCAGCAAGGATGTGATATTTCTCCAAGA
TCACTTTCTGGGCGCATTGTTGGAGGGGTTTGGTGGTTCTTCACCCTGATCATAATCTCT
TCCTACACTGCAAACCTTGCTGCTTTCCTGACTGTGGAGAGGATGGTGTCCCCTATAGAG
AGCGCTGAAGACTTAGCCAAGCAGACTGAAATTGCATATGGGACCCTGGACTCTGGTTCA
ACAAAAGAATTTTTCAGACGATCCAAAATTGCTGTGTATGAGAAAATGTGGTCTTACATG
AAATCCGCAGAGCCATCTGTGTTTACCAAAACAACAGCTGACGGGGTAGCCCGAGTTCGG
AAGTCCAAGGGAAAGTTCGCCTTCCTGCTGGAGTCGACCATGAACGAGTACATTGAGCAG
AGAAAGCCGTGCGATACGATGAAAGTTGGTGGAAATCTGGATTCCAAAGGCTATGGTGTG
GCAACCCCTAAAGGCTCAGCATTAGGAAATGCTGTTAACCTGGCAGTATTAAAACTGAAT
GAGCAAGGCCTCTTGGACAAATTGAAAAACAAATGGTGGTACGACAAAGGAGAGTGCGGC
AGCGGGGGCGGTGACTCCAAGGACAAGACCAGTGCTCTAAGCCTGAGCAATGTGGCAGGC
GTGTTCTATATACTTGTCGGAGGTCTGGGCCTGGCCATGATGGTGGCTTTGATAGAATTC