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

Cytochrome c Oxidase Subunit I [Homo sapiens] NCBI Reference Sequence: YP_003024028 MFADRWLFSTNHKDIGTLYLLFGAWAGVLGTALSLLIRAELGQPGNLLGNDHIYNVIVTAHAFVMIFFMV MPIMIGGFGNWLVPLMIGAPDMAFPRMNNMSFWLLPPSLLLLLASAMVEAGAGTGWTVYPPLAGNYSHPG ASVDLTIFSLHLAGVSSILGAINFITTIINMKPPAMTQYQTPLFVWSVLITAVLLLLSLPVLAAGITMLL TDRNLNTTFFDPAGGGDPILYQHLFWFFGHPEVYILILPGFGMISHIVTYYSGKKEPFGYMGMVWAMMSI GFLGFIVWAHHMFTVGMDVDTRAYFTSATMIIAIPTGVKVFSWLATLHGSNMKWSAAVLWALGFIFLFTV GGLTGIVLANSSLDIVLHDTYYVVAHFHYVLSMGAVFAIMGGFIHWFPLFSGYTLDQTYAKIHFTIMFIG VNLTFFPQHFLGLSGMPRRYSDYPDAYTTWNILSSVGSFISLTAVMLMIFMIWEAFASKRKVLMVEEPSM NLEWLYGCPPPYHTFEEPVYMKS

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!!!

>gi|11128019|ref|NP_061820.1| cytochrome c [Homo sapiens] MGDVEKGKKIFIMKCSQCHTVEKGGKHKTGPNLHGLFGRKTGQAPGYSYTAANKNKGIIWGEDTLMEYLENPKKYIPGTKMIFVGIKKKEERADLIAYLKKATNE

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.

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) 363 70.760Polar (D+E+H+K+N+Q+R+S+T) 150 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.biomedcentral.com/1471-2164/9/119The adaptive evolution of the mammalian mitochondrial genomeFonseca 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 cMGDVEKGKKIFIMKCSQCHTVEKGGKHKTGPNLHGLFGRKTGQAPGYSYTAANKNKGIIWGEDTLMEYLENPKKYIPGTKMIFVGIKKKEERADLIAYLKKATNE

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 105Molecular 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 Number Mole%Small (A+B+C+D+G+N+P+S+T+V) 45 42.857Aliphatic (I+L+V) 17 16.190Aromatic (F+H+W+Y) 12 11.429Non-polar (A+C+F+G+I+L+M+P+V+W+Y) 55 52.381Polar (D+E+H+K+N+Q+R+S+T+Z) 50 47.619Charged (B+D+E+H+K+R+Z) 34 32.381Basic (H+K+R) 23 21.905Acidic (B+D+E+Z) 11 10.476

In humans there are about 52% hydrophobic amino acids in Cytochrome c. How many in your organism???

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.

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…

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.

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