Integrative Assignment

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Integrative Assignment

• Part I

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You can use wikipedia but you cant cite it.
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Give a description of your organism.
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Homo sapiens is the primate species to which all
modern humans belong. The closest relatives are
chimpanzees from which they are distinguished by
greater size, a peculiar distribution of hair,
and a larger carbon footprint. Humans are
primarily terrestrial animals and can be found on
all continents. They are possibly the most
thoroughly studied organism although mice, E.
coli, and Drosophila are also in the running.
Henrik Kibak, Professor of Biology at CSU
Monterey Bay. Figure below is from the National
Geographic Genographic Project.
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Where in the cell is Cytochrome c Oxidase
found? What does Cytochrome c Oxidase do?
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See your texbook, Freeman, Biology, 2011
Cytochrome c Oxidase is Complex IV of the
Electron Transport Chain the subunit we will be
looking at is Subunit I. Would that make it an
integral membrane protein?
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After checking your textbook, wikipedia might in
fact be your first step, just to get an idea of
what you are looking for Or better yet go to the
NCBI Bookshelf and search for Cytochrome c Oxidase
Bottom Line Your protein (yellow) is about 500
amino acids long, found in the mitochondrial
inner membrane and reduces diatomic oxygen to
make water.
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• Using the amino acid single letter code, write
  the primary structure of the Cytochrome c Oxidase
  I found in your organism. (citation)
• How many amino acids are contained in Cytochrome
  c Oxidase I from your organism?

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To Answer Those Questions You Could

• Google cytochrome c oxidase i amino acid
  sequence Homo sapiens to find information or use
  Google Scholar

But you will probably find too much advanced
material to wade through
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NCBI

• Another approach is to use the National Center
  for Biotechnology Information (NCBI) website.
• NCBI is essentially Google for genes and
  sequences.

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Click RefSeq to Narrow the Results from 7,629 to
64!
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Select FASTA Display in order to get the correct
format for listing the primary sequence.
Note All Cytochromes b have about 370-390 aas
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The primary structure of Homo sapiens Cytochrome
c Oxidase I is displayed in COURIER FONT below
and consists of 513 amino acids. Other fonts are
a mess for displaying sequence data!!!
Cytochrome c Oxidase Subunit I Homo sapiens
NCBI Reference Sequence YP_003024028
MFADRWLFSTNHKDIGTLYLLFGAWAGVLGTALSLLIRAELGQPGNLLGN
DHIYNVIVTAHAFVMIFFMV MPIMIGGFGNWLVPLMIGAPDMAFPRMNN
MSFWLLPPSLLLLLASAMVEAGAGTGWTVYPPLAGNYSHPG
ASVDLTIFSLHLAGVSSILGAINFITTIINMKPPAMTQYQTPLFVWSVLI
TAVLLLLSLPVLAAGITMLL TDRNLNTTFFDPAGGGDPILYQHLFWFFG
HPEVYILILPGFGMISHIVTYYSGKKEPFGYMGMVWAMMSI
GFLGFIVWAHHMFTVGMDVDTRAYFTSATMIIAIPTGVKVFSWLATLHGS
NMKWSAAVLWALGFIFLFTV GGLTGIVLANSSLDIVLHDTYYVVAHFHY
VLSMGAVFAIMGGFIHWFPLFSGYTLDQTYAKIHFTIMFIG
VNLTFFPQHFLGLSGMPRRYSDYPDAYTTWNILSSVGSFISLTAVMLMIF
MIWEAFASKRKVLMVEEPSM NLEWLYGCPPPYHTFEEPVYMKS
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• This is the FASTA format for human Cytochrome c.
• It is displayed in a non-proportional font called
  courier.
• All your sequence data MUST BE DISPLAYED in
  COURIER.
• FASTA format has two requirements.
• The first line has a gt
• The second is sequence.

gtgi11128019refNP_061820.1 cytochrome c Homo
sapiens MGDVEKGKKIFIMKCSQCHTVEKGGKHKTGPNLHGLFGRKT
GQAPGYSYTAANKNKGIIWGEDTLMEYLENPKKYIPGTKMIFVGIKKKEE
RADLIAYLKKATNE
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How many amino acids are contained in Cytochrome
b from your organism? What proportion of these
amino acids are hydrophobic?
We already know how many amino acids are in your
sequences from the GenPept pages. To get the
proportion that is hydrophobic you can simply use
the chart on page 47 of your book, tally the
hydrophobic ones, and divide by the total number
of amino acids in the protein. Should take about
10 minutes. There is also a website that does the
calculations for you (see next slide), but it is
frequently more trouble than it is worth.
http//mobyle.pasteur.fr/cgi-bin/portal.py?formp
epstats
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363/513 71 Non-polar (ACFGILMPVWY)
363 70.760 Polar (DEHKNQRST) 150 2
9.240
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Provide the best illustration you can for the
tertiary structure of human Cytochrome c Oxidase.
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Because it is so difficult to get enough
absolutely pure Complex IV from human tissue to
crystalize this is formally all we can say about
the structure of Cytochrome c Oxidase from human
mitochondria. However, it is less challenging to
obtain a large amount of beef heart mitochondria
from slaughter houses. The mitochondria are
first purified away from other cellular
components. Then their membrane proteins are
gently solubilized by detergents and separated
into the various complexes by a combination of
centrifugation and column chromatography.
Finally membranes containing only Complex IV are
carefully crystallized and x-ray diffraction data
collected.
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Complex IV from beef heart mitochondria. http//ww
w.biomedcentral.com/1471-2164/9/119 The adaptive
evolution of the mammalian mitochondrial
genome Fonseca et al.
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Directions to Find the Cytochrome c for Your
Organism

• Google NCBI
• In the NCBIs front page search box type in
  species name and Cytochrome c
• To the right, in the drop down menu, switch from
  all databases to protein and then click
  search.
• Look through the search results starting at the
  top. You need to find a cytochrome C that is
  about 105aa long. It will say near the top of
  each article. Your protein WILL NOT BE
  Cytochrome c Oxidase.
• Once you find the right protein (about 100-120
  aas long and correct species), switch GenPept
  to FASTA, you now have a more useable format.
• Copy and paste to a notebook document. Save this
  file for for later. Copy and past the nicely
  formatted courier font sequence into your WORD
  document.

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Where in the cell is Cytochrome c found? What
does Cytochrome c do?
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Cytochrome c is a peripheral membrane protein
found on the inner mitochondrial membrane as a
part of the Electron Transport Chain. Cytochrome
c accepts electrons from Cytochrome c1 of Complex
III to Cytochrome a of Complex IV (also know as
Cytochrome c oxidase).
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Cytochrome c diffuses back and forth between
Complexes III and IV, shuttling electrons. See
texbook, Freeman, Biology, 2005.
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Using the amino acid single letter code, write
the primary structure of the Cytochrome c found
in your organism.
gtgi42560196spP99999CYC_HUMAN Cytochrome
c MGDVEKGKKIFIMKCSQCHTVEKGGKHKTGPNLHGLFGRKTGQAPGYS
YTAANKNKGIIWGEDTLMEYLE NPKKYIPGTKMIFVGIKKKEERADLIA
YLKKATNE
http//www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db
proteinqty1c_start1list_uids42560196uidsd
optfastadispmax5sendtofrombegintoendextr
afeatpresent1ef_CDD8ef_MGC16ef_HPRD32ef_ST
S64ef_tRNA128ef_microRNA256ef_Exon512
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How many amino acids are contained in Cytochrome
c from your organism? What proportion of these
amino acids are hydrophobic?
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PEPSTATS of CYC_HUMAN from 1 to 105 Molecular
weight 11748.69 Residues
105 Residue Number Mole A Ala 6 5.714
C Cys 2 1.905 D Asp 3 2.857
E Glu 8 7.619 F Phe 3 2.857
G Gly 13 12.381 H His 3 2.857
I Ile 8 7.619 K Lys 18 17.143
L Leu 6 5.714 M Met 4 3.810
N Asn 5 4.762 P Pro 4 3.810
Q Gln 2 1.905 R Arg 2 1.905
S Ser 2 1.905 T Thr 7 6.667
V Val 3 2.857 W Trp 1 0.952
Y Tyr 5 4.762 Property Residues Num
ber Mole Small (ABCDGNPSTV) 45 42.857
Aliphatic (ILV) 17 16.190 Aromatic (FHWY)
12 11.429 Non-polar (ACFGILMPVWY) 55
52.381 Polar (DEHKNQRSTZ) 50 47.619 Cha
rged (BDEHKRZ) 34 32.381 Basic (HKR) 2
3 21.905 Acidic (BDEZ) 11 10.476
In humans there are about 52 hydrophobic amino
acids in Cytochrome c. How many in your
organism???
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Provide the best illustration you can for the
tertiary structure of human Cytochrome c.

• Strategy
• Take the human Cytochrome c sequence and search
  the structure (PDB) database in NCBI for similar
  sequences for which the structure has been
  solved.
• Download Cn3D to display the structure.
• Export the structure as a PNG file.

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• Select protein blast at NCBI BLAST
  http//www.ncbi.nlm.nih.gov/BLAST/
• 2. Paste sequence. 3. Select Protein Data
  Bank 4. Select blastp

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Be PATIENT! The page will sit here for a while
updating several times until suddenly the results
page appears.
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Click on the first structure link and see what it
is If it makes sense, then click on the S.
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First you will see this page.
Then you will see this page
If you dont have Cn3D installed, download it.
You wont need admin privileges.
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It is a lot easier to work with peripheral
membrane proteins than integral membrane
proteins. Crystal structures have been solved
for Cytochrome c from many organisms.
http//www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv
.cgi?dbtform6doptsuid27574
If link doesnt work, go to next page
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Just like Cytochrome b, Cytochrome c has a heme
group that is good at binding and releasing
electrons.
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Mitochondrial ATP Synthase http//classes.csumb.ed
u/BIO/BIO241-01/campus/Lecture10respiration/ATPase
.flv http//multimedia.mcb.harvard.edu/anim_ATPase
_flv.html http//carbon.cudenver.edu/bstith/1418m
.mov