Transcript Integrative Assignment

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 b found?
What does Cytochrome b do?
See your texbook, Freeman, Biology, 2005.
Cytochrome b is a protein in Complex
Three of the Electron Transport Chain…
would that make it an integral membrane
protein?
• Using the amino acid single letter code,
write the primary structure of the
Cytochrome b found in your organism.
(citation)
• How many amino acids are contained in
Cytochrome b from your organism?
To Answer Those Questions You Could:
Google “cytochrome
b” amino acid sequence Aedes
aegypti 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
sequences.
Click
RefSeq
to
Narrow
the
Results
from
7,629
to 64!
This is the one
you want!
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 b is displayed in COURIER FONT
below and consists of 380 amino acids.
Other fonts are a mess for displaying sequence data.
Reference Sequence: YP_003024038.1 cytochrome b [Homo sapiens]
>gi|251831119|ref|YP_003024038.1| cytochrome b [Homo sapiens]
MTPMRKTNPLMKLINHSFIDLPTPSNISAWWNFGSLLGACLILQITTGLFLAMHYSPDASTAFSSIAHIT
RDVNYGWIIRYLHANGASMFFICLFLHIGRGLYYGSFLYSETWNIGIILLLATMATAFMGYVLPWGQMSF
WGATVITNLLSAIPYIGTDLVQWIWGGYSVDSPTLTRFFTFHFILPFIIAALATLHLLFLHETGSNNPLG
ITSHSDKITFHPYYTIKDALGLLLFLLSLMTLTLFSPDLLGDPDNYTLANPLNTPPHIKPEWYFLFAYTI
LRSVPNKLGGVLALLLSILILAMIPILHMSKQQSMMFRPLSQSLYWLLAADLLILTWIGGQPVSYPFTII
GQVASVLYFTTILILMPTISLIENKMLKWA
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
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.
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
http://bioweb.pasteur.fr/seqanal/interfaces/pepstats.html
PEPSTATS of AP_000651.2 from 1 to 378 Human Cytochrome b
Molecular weight = 42460.18
Residues = 378
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
Property
Small
Aliphatic
Aromatic
Non-polar
Polar
Charged
Basic
Acidic
Number
24
2
11
4
24
24
12
38
9
64
15
15
23
8
7
29
31
10
11
17
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)
Mole%
6.349
0.529
2.910
1.058
6.349
6.349
3.175
10.053
2.381
16.931
3.968
3.968
6.085
2.116
1.852
7.672
8.201
2.646
2.910
4.497
Number
169
112
64
252
126
43
28
15
66.7% of Human
Cytochrome b
residues are
hydrophobic.
Mole%
44.709
29.630
16.931
66.667
33.333
11.376
7.407
3.968
Provide the best illustration you can for the
tertiary structure of human Cytochrome b.
Because it is so difficult to get enough absolutely pure Complex III from human
tissue to crystalize… this is formally all we can say about the structure of
Cytochrome b 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 III are carefully crystallized and x-ray
diffraction data collected.
Obtaining vast
amounts of yeast
mitochondria from
breweries is also an
option!
Complex III
from beef
heart
mitochondria.
Another view of the
two copies of
Cytochrome b in
Complex III of the
mitochondrial
electron transport
chain.
All of these heme
groups stuck into the
proteins are perfect
for binding and
donating electrons…
in exchange for
moving protons
across the
membrane.
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