H3 Molecular Biology

H3 Molecular Biology
Tutorial
12 March 2012
Protein Biochemistry
Alex Law, PhD

Professor
School of Biological Sciences
Nanyang Technological University
Q 1a
uhe haemoglobin sequences
Hb: VLSPA DKuNV KAAWG KVGAH

VHLTP EEKSA VTALW GKVNV
Hb:
V D A G
E W
You will not be able to deduce the sequence of the

i di id l chains,
individual h i you will
ill need
d to
t separate
t the
th chains
h i
and perform the sequencing separately.
Chains may be separated by treatment of detergents or

denaturants followed by some kind of chromatography
Detergent: sodium dodecyl sulphate
Denaturants: guanidinium hydrochloride, urea

Charge
Size
Hydrophobicity
Affi it to
Affinity t chemical
h i l groups.
Proteins may be separated by size, charge,
hydrophobicity,
y p y, or the ability
y uo bind to certain
chemical groups.
Sickle Cell Anemia
Linus Pauling (1945) postulated that sickle cell anemia was a
molecular disease. The subtle difference is that the patients had
haemoglobin, but a variant that made the red blood cell sickle.
Pauling predicted a specific change.
Carriers are not 100% healthy. Under

transmission: typical
severe stress conditions, they do show
autosomal recessive
signs of discomfort e.g. at high altitude.
Q 1b
If you perform the sequencing experiment again, the 6th step would
yield an equal amount of aspartic acid (from the subunit), and
valine from the s subunit.
Q 1c
A A A A
B B S S
Normal Sickle Cell Anemia
Sickle Cell trait : 100% A; 50% B; 50% S
A A A A A A A A
B B S B B S S S
Q 1d
If you perform the sequencing experiments on the carrier, the 6th step will
give you aspartic acid
acid, glutamic acid,
acid and valine in the ratio of 2:1:1.
2:1:1
Obligatory
through red
blood cell
infection
Valine replacing
glutamic acid makes
HbS less (negatively)
charged. The
haemoglobin solution
inside the cell is
therefore more viscous.
The malaria parasite
does not like it.
People with the sickle-cell trait are more resistant to malaria. The
sickle-cell allele confer genetic advantage over the normal allele.
Thus the allele is preserved.

Q 1e
Hb: VLSPA DKuNV KAAWG KVGAH

VHLTP EEKSA VTALW GKVNV
Hb:
Hb: V-LSP ADKuN VKAAW GKVGAH

Hb: VHLTP EEKSA VTALW GKV-NV
This type
t pe of alignment is used
sed to assess the similarities between
bet een proteins
proteins.
50% identity is considered very significant, and indeed, it is supported by
the 3-d structure of the two subunits, they are almost identical.
We may conclude thau the and subunits of haemoglobin have

a common ancestral molecule, from which they evolved.
The two
Th t proteins
t i are saidid to
t be
b homologous.
h l These
Th day,
d it has
h been
b
evolved to quantitate the degree of similarity between proteins.
SequenceofthehumanAHPgene.
1 gtctcaaggg gccgctctct gacatcagag ctgctgtaga gcggagaggg gcaggggtga
61 agggccacgg tggtgcaacc caccacttcc tccaaggagg agctgagagg aacaggaagt
121 gtcaggactt tacgacccgc gcctccagct gaggtttcta gacgtgaccc agggcagact
181 ggtagcaaag cccccacgcc cagccaggag caccgccgag gactccagca caccgagggt
241 gagtgtgcag aggccccgct atgtccacgg tgatttttgg agggaagcac tctcctgccc
301 gaggggagct tcatgggtgg tcagccccag gggttgggcc tgagaccgtc accaagaccc
361 cttccctcca caggacatgc tgggcctgcg ccccccactg ctcgccctgg tggggctgct
421 ctccctcggg tgcggtgagt tctgtgttcc acggagggga ctccacgcgt gacattccaa
481 tcagggagaa cagggcctgc cctcactttc ccatagacag aagttcccgg gaggctgggg
541 ggggtctcct cctggaggct gcctccctgt ctttttgggc ctcctctgac cttcctgctc
601 tactccccct tgggtgtggg cagggcggcc cagagcaccc actcaccagc cggcctcgtc
661 cctcagtcct
g ctctcaggag
gg g tgcacgaagt
g g g tcaaggtcag
gg g cagctgccgg
g g gg gaatgcatcg
g g g
721 agtcggggcc cggctgcacc tggtgccaga agctggtaag tgcctcctgg acccctcccc
781 tgacctcgga cctgtgggca cacggctcag tgagatgggg ctggactgac gctcaggtgt
841 gctgcttctt cctaggagtg tgtggcaggc cccaacatcg ccgccatcgt cgggggcacc
901 gtggcaggca tcgtgctgat cggcattctc ctgctggtca tctggaaggc tctgatccac
961 ctgagcgacc tccgggagta caggcgcttt gagaaggaga agctcaagtc ccagtggaac
1021 aatgtaagtg gccgtccttg ggggtcccac gcagaggggc acgtgcgtcc cacactatgc
1081 gacctcctgc tgcgggaggc cgtggacacc cgtgtgtggc tgcaccccgg cctccccagg
1141 ctcagggaga aagattctgc tctgaaaacc tcccacactc acaagcccct gtttgcattt
1201 cccaccagga taatcccctt ttcaagagcg ccaccacgac ggtcatgaac cccaagtttg
1261 ctgagagtta ggagcacttg gtgaagacaa ggccgtcagg acccaccatg tctgccccat
1321 cacgcggccg agacatggct tgccacagct cttgaggatg tcaccaatta accagaaatc
1441 cagttatttt ccgccctcaa aatgacagcc atggccggcc gggtgcttct gggggctcgt
1501 cggggggaca gctccactct gactggcaca gtctttgcat ggagacttga ggagggaggg
1561 cttgaggttg gtgaggttag gtgcgtgttt cctgtgcaag tcaggacatc agtctgatta
1621 aaggtggtgc caatttattt acatttaaac ttgtcagggt ataaaatgac atcccattaa
1681 g
ttatattgtt aatcaatcac g
gtgtatagaa
g g aaaaaataaa acttcaatac aggctgtcca
gg g
1741 tggaaactgg gcactgtgtc cgctgtattc ccccgactgg caaggtggcc aggcacacgt
1801 gggccctgtc ctgcccgctg acctttgcca cacaggcaca cagctggctt cagaccatgg
SequenceofthehumanAHPgene.:changettou,andhighlightingtheexons.
1 gucucaaggg gccgcucucu gacaucagag cugcuguaga gcggagaggg gcagggguga
61 agggccacgg uggugcaacc caccacuucc uccaaggagg agcugagagg aacaggaagu
121 gucaggacuu uacgacccgc gccuccagcu gagguuucua gacgugaccc agggcagacu
181 gguagcaaag cccccacgcc cagccaggag caccgccgag gacuccagca caccgagggu
241 gagugugcag aggccccgcu auguccacgg ugauuuuugg agggaagcac ucuccugccc
301 gaggggagcu ucaugggugg ucagccccag ggguugggcc ugagaccguc accaagaccc
361 cuucccucca caggacaugc ugggccugcg ccccccacug cucgcccugg uggggcugcu
421 cucccucggg ugcggugagu ucuguguucc acggagggga cuccacgcgu gacauuccaa
481 ucagggagaa cagggccugc ccucacuuuc ccauagacag aaguucccgg gaggcugggg
541 ggggucuccu ccuggaggcu gccucccugu cuuuuugggc cuccucugac cuuccugcuc
601 uacucccccu uggguguggg cagggcggcc cagagcaccc acucaccagc cggccucguc
661 ccucaguccu cucucaggag ugcacgaagu ucaaggucag cagcugccgg gaaugcaucg
721 agucggggcc cggcugcacc uggugccaga agcugguaag ugccuccugg accccucccc
781 ugaccucgga ccugugggca cacggcucag ugagaugggg cuggacugac gcucaggugu
841 gcugcuucuu ccuaggagug uguggcaggc cccaacaucg ccgccaucgu cgggggcacc
901 guggcaggca ucgugcugau cggcauucuc cugcugguca ucuggaaggc ucugauccac
961 cugagcgacc uccgggagua caggcgcuuu gagaaggaga agcucaaguc ccaguggaac
1021 aauguaagug gccguccuug ggggucccac gcagaggggc acgugcgucc cacacuaugc
1081 gaccuccugc ugcgggaggc cguggacacc cguguguggc ugcaccccgg ccuccccagg
1141 cucagggaga aagauucugc ucugaaaacc ucccacacuc acaagccccu guuugcauuu
1201 cccaccagga uaauccccuu uucaagagcg ccaccacgac ggucaugaac cccaaguuug
1261 cugagaguua ggagcacuug gugaagacaa ggccgucagg acccaccaug ucugccccau
1321 cacgcggccg agacauggcu ugccacagcu cuugaggaug ucaccaauua accagaaauc
1441 caguuauuuu ccgcccucaa aaugacagcc auggccggcc gggugcuucu gggggcucgu
1501 cggggggaca gcuccacucu gacuggcaca gucuuugcau ggagacuuga ggagggaggg
1561 cuugagguug gugagguuag gugcguguuu ccugugcaag ucaggacauc agucugauua
1621 aagguggugc caauuuauuu acauuuaaac uugucagggu auaaaaugac aucccauuaa
1681 uuauauuguu aaucaaucac guguauagaa aaaaaauaaa acuucaauac aggcugucca
1741 uggaaacugg gcacuguguc cgcuguauuc ccccgacugg caagguggcc aggcacacgu
1801 gggcccuguc cugcccgcug accuuugcca cacaggcaca cagcuggcuu cagaccaugg
AdditionofthepolyAtail
1741 ugg aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa.... (poly-a tail)
Identificationofthestartcodonaugformethionine
1741 uggaaacugg gcacuguguc cgcuguauuc ccccgacugg caagguggcc aggcacacgu
1801 gggcccuguc cugcccgcug accuuugcca cacaggcaca cagcuggcuu cagaccaugg
Translatingthefirstexon.Notethatthereisaleftovernucleotideattheend.
M L G L R P P L L A L V G L L
S L G C
Protein sequence of AHP: note the split codon for valine in exons 2 and 3.
M L G L R P P L L A L V G L L
S L G C
V L S Q E C T K F D V S S C R E C I
E S G P G C T W C Q K L
D N P L F K S A T T T V M N P K F
A E S *
Fig. 15.11.a
Q 4a
You cannot end with an intron! 17

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H3 Molecular Biology

Alex Law, PhD

Hb: VLSPA DKuNV KAAWG KVGAH

You will not be able to deduce the sequence of the

Chains may be separated by treatment of detergents or

Detergent: sodium dodecyl sulphate

Denaturants: guanidinium hydrochloride, urea

Carriers are not 100% healthy. Under

Normal Sickle Cell Anemia

Sickle Cell trait : 100% A; 50% B; 50% S

Thus the allele is preserved.

Hb: VLSPA DKuNV KAAWG KVGAH

Hb: V-LSP ADKuN VKAAW GKVGAH

We may conclude thau the and subunits of haemoglobin have

You cannot end with an intron! 17

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