Transcript Integrative Assignment - California State University

Integrative Assignment
Part I
You can use
wikipedia but
you can’t cite
it.
Give a description of your organism.
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.
Where in the cell is Cytochrome c Oxidase found?
What does Cytochrome c Oxidase do?
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?
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.
• 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?
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…
NCBI
• Another approach is to use the National
Center for Biotechnology Information
(NCBI) website.
• NCBI is essentially “Google” for genes and
sequences.
Click
RefSeq
to
Narrow
the
Results
from
7,629
to 64!
Select FASTA Display in order to get the correct format for listing the primary sequence.
Note: All Cytochromes b have about 370-390 aa’s
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
MFADRWLFSTNHKDIGTLYLLFGAWAGVLGTALSLLIRAELGQPGNLLGNDHIYNVIVTAHAFVMIFFMV
MPIMIGGFGNWLVPLMIGAPDMAFPRMNNMSFWLLPPSLLLLLASAMVEAGAGTGWTVYPPLAGNYSHPG
ASVDLTIFSLHLAGVSSILGAINFITTIINMKPPAMTQYQTPLFVWSVLITAVLLLLSLPVLAAGITMLL
TDRNLNTTFFDPAGGGDPILYQHLFWFFGHPEVYILILPGFGMISHIVTYYSGKKEPFGYMGMVWAMMSI
GFLGFIVWAHHMFTVGMDVDTRAYFTSATMIIAIPTGVKVFSWLATLHGSNMKWSAAVLWALGFIFLFTV
GGLTGIVLANSSLDIVLHDTYYVVAHFHYVLSMGAVFAIMGGFIHWFPLFSGYTLDQTYAKIHFTIMFIG
VNLTFFPQHFLGLSGMPRRYSDYPDAYTTWNILSSVGSFISLTAVMLMIFMIWEAFASKRKVLMVEEPSM
NLEWLYGCPPPYHTFEEPVYMKS
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.
1. The first line has a “>”
2. The second is sequence.
>gi|11128019|ref|NP_061820.1| cytochrome c [Homo sapiens]
MGDVEKGKKIFIMKCSQCHTVEKGGKHKTGPNLHGLFGRKTGQAPGYSYTAANKNKGIIWGE
DTLMEYLENPKKYIPGTKMIFVGIKKKEERADLIAYLKKATNE
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?form=pepstats
363/513 = 71%
Non-polar
(A+C+F+G+I+L+M+P+V+W+Y)
Polar
(D+E+H+K+N+Q+R+S+T)
363
150
70.760
29.240
Provide the best illustration you can for the
tertiary structure of human Cytochrome c
Oxidase.
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.
Complex IV from beef
heart mitochondria.
http://www.biomedcentr
al.com/1471-2164/9/119
The adaptive evolution
of the mammalian
mitochondrial genome
Fonseca et al.
Directions to Find the Cytochrome c for
Your Organism
•
Google NCBI
•
In the NCBI’s 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 aa’s 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.
Where in the cell is Cytochrome c found?
What does Cytochrome c do?
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).
Cytochrome c diffuses back and forth between Complexes III and IV, shuttling
electrons. See texbook, Freeman, Biology, 2005.
Using the amino acid single letter code, write the
primary structure of the Cytochrome c found in
your organism.
>gi|42560196|sp|P99999|CYC_HUMAN Cytochrome c
MGDVEKGKKIFIMKCSQCHTVEKGGKHKTGPNLHGLFGRKTGQAPGYSYTAANKNKGIIWGEDTLMEYLE
NPKKYIPGTKMIFVGIKKKEERADLIAYLKKATNE
http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=protein&qty=1&c_start=1&list_uids=42560196&uids=
&dopt=fasta&dispmax=5&sendto=&from=begin&to=end&extrafeatpresent=1&ef_CDD=8&ef_MGC=16&ef_
HPRD=32&ef_STS=64&ef_tRNA=128&ef_microRNA=256&ef_Exon=512
How many amino acids are contained in
Cytochrome c from your organism?
What proportion of these amino acids are
hydrophobic?
PEPSTATS of CYC_HUMAN from 1 to 105
Molecular weight = 11748.69
Residues = 105
Residue
A = Ala
C = Cys
D = Asp
E = Glu
F = Phe
G = Gly
H = His
I = Ile
K = Lys
L = Leu
M = Met
N = Asn
P = Pro
Q = Gln
R = Arg
S = Ser
T = Thr
V = Val
W = Trp
Y = Tyr
Mole%
5.714
1.905
2.857
7.619
2.857
12.381
2.857
7.619
17.143
5.714
3.810
4.762
3.810
1.905
1.905
1.905
6.667
2.857
0.952
4.762
Property
Small
Aliphatic
Aromatic
Non-polar
Polar
Charged
Basic
Acidic
Number
6
2
3
8
3
13
3
8
18
6
4
5
4
2
2
2
7
3
1
5
Residues
(A+B+C+D+G+N+P+S+T+V)
(I+L+V)
(F+H+W+Y)
(A+C+F+G+I+L+M+P+V+W+Y)
(D+E+H+K+N+Q+R+S+T+Z)
(B+D+E+H+K+R+Z)
(H+K+R)
(B+D+E+Z)
Number
45
17
12
55
50
34
23
11
In humans there are about
52% hydrophobic amino
acids in Cytochrome c. How
many in your organism???
Mole%
42.857
16.190
11.429
52.381
47.619
32.381
21.905
10.476
Provide the best illustration you can for the
tertiary structure of human Cytochrome c.
Strategy
1. Take the human Cytochrome c sequence and search the structure
(PDB) database in NCBI for similar sequences for which the
structure has been solved.
2. Download Cn3D to display the structure.
3. Export the structure as a PNG file.
1. Select protein blast at NCBI BLAST http://www.ncbi.nlm.nih.gov/BLAST/
2. Paste sequence. 3. Select “Protein Data Bank” 4. Select “blastp”
Be PATIENT! The page will sit here for a while… updating several times until
suddenly the results page appears.
Click on the first structure link and see what it is… If it
makes sense, then click on the S.
First you will see this
page.
Then you will see
this page
If you don’t have
Cn3D installed,
download it. You
won’t need admin
privileges.
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?db=t&form=6&dopt=s&uid=27574
If link doesn’t work, go to next page…
Just like Cytochrome b,
Cytochrome c has a heme
group that is good at binding
and releasing electrons.
Mitochondrial ATP Synthase
http://classes.csumb.edu/BIO/BIO241-01/campus/Lecture10respiration/ATPase.flv
http://multimedia.mcb.harvard.edu/anim_ATPase_flv.html
http://carbon.cudenver.edu/~bstith/1418m.mov