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syllabus 2013

On doing homework. Typed is nice if you have long answers - otherwise you can write it in by hand. Please print out your answers and bring them to class. (I don't want you to email your answers back to me unless absolutely necessary to get them in on time).

I do not want long sections cut form the web. Shorter and directed to answer the question is better.

The copy of the homework on the web will have active links to various pages. If you find any dead links please email me immediately.


Homework-1: Introduction

Hand in questions 1 and 2
The reference format for journal articles I would like you to use in this class is. Last name, first initials for all authors (year) Full title of paper vol, first page-last page.
For example, Zhang, J., Frerman, F.E. and Kim, J.J. (2006) Structure of electron transfer flavoprotein-ubiquinone oxidoreductase and electron transfer to the mitochondrial ubiquinone pool. Proc. Natl. Acad. Sci. U.S.A. 103, 16212–16217.
If you are using information from a web site than give me the full URL.

1) Use Wikipedia or GOOGLE a biochem or cell bio book. Give me a brief definition. Give me the reference for each piece of information.
DEFINE
-a) Mitochondria and Chloroplast
-b) Heme and quinone
-c) van der Waals forces
-d) pKa of an acid
-e) protein backbone; protein side chain

2) Use: Oxidative Phosphorylation at the fin de siecle to answer questions a-e. Other reviews of mitochondrial proteins that may be of interest are from Rich and from Newmeyer.
a) Write out the reference to the three papers using the form described above.
b) What is the overall reactant and product of oxidative phosphorylation?
c) 5 proteins are described as Complex I-V.
What are the products and reactants for each protein?
The answers for complex 1:
Complex I is known as the NADH: ubiquinone oxidoreductase - It takes electrons from NADH (oxidizing it) and gives them to ubiquinone (reducing that). This process is described by the name. Protons are pumped across the membrane when this happens.

d) List the non-amino acid cofactors shown for each protein.
The protein contains 1 flavin mononucleotide, seven or eight different FeS centers, covalently bound lipid and at least 3 bound quinonl molecules.
e) use in the membrane proteins of known structure to find the 4 letter code for one structure of each of the 5 proteins. When the structure was solved. How many different structures are available.
For complex I: 1M9C solved in 2010. Two structures are available from different organisms.
You may want to go look at one of the proteins in the Protein data bank


3) Ion channels and ion pumps. See Gadsby review.
a) What is the difference between how an ion channel and an ion pump work?
b) Does a channel make a gradient or dissipate a gradient? Answer the same question for a pump.
c) How can channels and pumps work together?

_____________EXTRA WEB SITES TO LOOK FOR INTRODUCTION __________________

How to read scientific papers - while this says it is for non-scientists it's actually very good. You should read several papers at this level for this class. One trick will be looking through many papers to decide which few you will work through. But that's one of the things you will get to pratice here.

Start with the web sites of interest page.
I suggest you download one of the molecular graphics packages (I will use PYMOL VMD, Rasmol, Swiss PDB viewer) are all ok. I will use PYMOL in class.Swiss PDB viewer is probably the easiest.
Look up amino acids in the Wikipedia or GOOGLE ,
Amino acid web sites Indiana State (basic chemistry of Biomolecules page), Image Library ,
Look at the protein chapter in the Indiana State (Protein structure and analysis)
Find a real cell biology or biochemistry text book and look up amino acids and protein synthesis.
Make sure you can download the figure for the amino acid homework.
Go to all the recommended links (Useful web sites ).

A bit more on protein synthesis: RNA>PROTEIN; and a lovely movie.

Papers that connect biology to chemistry
Can biological phenomena be understood by humans? pdf
The Biological Frontier of Physics. pdf

Web and utube pages that I showed in the first class
aquaporins
bacteriorhodopsin and another on bacteriorhodopsin-with a larger view
lipids and md simulation of lipid bilayer in water



Homework 2 Lecture: Amino acid properties; peptide bonds
make use of the Introduction to protein structure chapter for this assignment.


Print out this figure;
(1) Label each side chain with its full name (e.g. Glycine); Three letter name (Gly); and its 1 letter name (G)
- Color each Oxygen red and each Nitrogen blue
- Label which are NP non-polar; P polar; + positively charged; - negatively charged
- Label which are S (small); M (medium); and L (large) in size (this is a bit subjective)

(2) Draw a valine amino acid (backbone and side chain). Draw a dipeptide Ala-Val. You need to combine 2 amino acids to form a peptide bond. Circle the peptide bond; circle each amino acid; and label each side chain.

(3) What distinguishes hydrophobic and hydrophilic amino acids? Give me the reference you used for the definition.

(4) Find a table of side chain physics properties (this can be found in some of the web references provided or the protein chapter I've given you)
4a) What is the molecular volume of and Ala, a Lys, a Val? Is this value for just the side chain or for the whole amino acid? (what reference did you use?). Compare the molecular volume with the hydrophobicity of these residues. Use this hydrophobicity scale.
4b) Look at the Suggested amino acid substitutions. Thre are 4 groups circled. For 2 of the circles say what properties these amino acids have in common.
4c) Make the following graphs. (1) Surface area vs solubility; (2) Volume vs solubility. Which has a better correlation? Use data at: Jena Library site. If you use another data source give me the reference.

5a) Give me a definition of the pK of an acid or base (and the reference you used).
5b) What amino acids have acidic side chains?
5c) Which ones have basic side chains?
5d) What is the pK of the side chain for these acidic and basic groups? (what reference did you use?). [do not give me the n-terminal or c-terminal site pK here].
5e) Every side chain (except proline) has a amino n-terminal and carboxylic acid c-terminal. What is the pK of the n-terminal and the c-terminal.

_____________EXTRA WEB SITES TO LOOK FOR AMINO ACIDS__________________
I will send you a copy of a chapter in preparation for a biophysics book.
Jena Library site
- Individual properties of the 20 standard aa (including pKs);
Look at the ionic equilbria review page
A good list of properties. An introductory page.
pH dependence of residue charge: isolated aa
Membrane proteins. Look at the Experimentally determined hydrophobicity scales.
Look at the amino acids web sites suggested in the reading/homework for homework 1.



HOMEWORK 3 Lecture: Our proteins

Papers describing the protein database are by Rose-2010 and Rose-2013.
If you are interested in the history of PDB you can see papers by Berman and by Goodsell.
It's a good time to get a molecular visualization. If you are using pymol here is a manual

You will use this information in questions 1-8 for your project.
1) Find the right protein
Find your protein at the membrane proteins of known structure web site
What is the 4 letter code for the pdb file for your protein in the protein databank?

2) Find some good references
What is the main reference suggested the membrane proteins of known structure web site?
Give a reference to a review article for the protein (look at pubmed and search for reviews).
You may need to find different papers for the structure and for the function. For some proteins you may find a better review for a protein in your group of protein rather than for your specific protein (eg Bacteriorhodopsin review will help you understand halorhodopsin; cytochrome c oxidase may help with quinol oxidase - these connections are noted in the handout sheet for your assigned proteins).
Give me the URL for a web site on line that is dedicated to your protein.
Find a review at PubMed.

3) What does the protein do?
What is the function of your protein? What are the reactants and the products? (this may be chemical A + electrons go to chamical +B or it may be protons move from the cytoplasm to the periplasm. Draw a sketch of the reaction (giving me the reference you are using).
Does the reaction happen in several steps? If yes what are the intermediates?
Does the protein store energy in the membrane gradient - or does it use the membrane gradient as its energy source?
A nice review of electron transfer 'cofactors'

4) What is important, what is missing and what is extra in your pdb structure file?
What are the non-amino acid groups (cofactors, reactants, products) needed for the function of your protein? Find this in the review articles.
What are the non-amino acids found in the crystal structure? This is in the Ligand Chemical Component and Modified Residue section of the main page for your protein at the protein databank.
Match up the cofactors/reactants/products needed for function and the groups found in the structure. Do you have extra groups or missing groups.
How many polypeptide chains should your protein have (look in a review article). How many do you find in the structure? [Different organisms have different number of polypeptide subunits. Usually mammalian proteins have more subunits. If you have a choice use a smaller protein to start. However some proteins are missing important subunits. Others have extra proteins attached - Look out for proteins that are co-crystalized with an antibody. You do not want to look at the antibody!

5 - not about your protein) Use: The Biological Frontier of Physics. pdf to tell me how many moles ATP you synthesize each day. (You can modify this based on your caloric intake.)

_____________EXTRA WEB SITES TO LOOK FOR ELECTRON TRANSFER CHAINS__________________

Good web sites
general overview for Electron Transfer Chains here

 



Homework 4 Lecture: Protein Motifs; 1°,2°,3° structures; visualization
make use of the Introduction to protein structure chapter for this assignment.

This is a very important excercise for your project - you will use this information for your project.
(1) Go to the The protein data bank (PDB) site..
1a) How many entries are currently in the protein data bank for all proteins?

You have each been given a protein. Find the structures for your protein. Each protein structure has a unique 4-letter code used in all of these sites.
Look up your protein in all these sites.
(1a) membrane protein site. This should be the first site you go to. Use this to find PDB ID (4 letter) codes for your protein. There may be multiple copies of the same protein - from different organisms, with different mutations, or with different bound molecules. In the end you will need to pick one file to work with.
How to choose your PDB file:
--Resolution - smaller is better. Any structure under 2Å will be fine, but many membrane proteins structures can by 3 or even 4. If that's the best you can do use it.
--For your project it is nice to have the right ligands bound. If you have an electron transfer protein there are non-amino acid pieces that are important to your reaction. They are not bound in all structures. You may have an inhibitor bound in the active site. This choice requires some knowledge and may get you to change the structure you are using later. If you are doing a channel you may find a structure with the ion bound. That is a good thing because this shows a place where the ion is interacting with the protein strongly.
--Number of subunits. Too Few: Some proteins have many polypeptides that come together to make it work. For example, many ion channels must have 4 polypeptide chains to make the channel - some the PDB structures only has one. This one must be combined with 3 other to make the full channle - you need the biological assembly not the protein in the unit cell.
-- Number of subunits. Too Many: Bacterial proteins often have many fewer subunits than their mammalian cousins. They both work and do the same job. Use the simplest structure possible. It will be much easier to manipulate and to see things. Addition of an immunoglobulin peptide. This is added to help crystalization. It is not your protein and should be removed/ignored.
--What's a quick way to figure out if my structure has too much or too little?
For many proteins Wikipedia will have a pricture of the working protein with information about the important bits. In the membrane protein site. there will be a link to a journal article. This will describe the good and bad of this particular structure. You really will need to read this paper carefully. (And you can ask me my email or in person).

(1b) the protein databank search by the name of your protein and by the PDB ID.
(1c) Macromolecular Structure Database search by PDB ID.
(1d) PubMed and use the pull down at the top of the page (default PubMed) and go to structure and seach by PDB ID.
(1e) SCOP Do a Keywork search on your protein name; try again on the PDB ID.

For 1a: Give me the PDB ID you will be using.
For 1b-1e: Write down the link to your file.
--If you can't find your protein in the database than report that.
--Report how many items you get at each web site when you search on the name of your protein. (eg search of cytochrome c (name) vs 2YCC (PDB ID)).
At this point I'd suggest you bookmark these page and look at them throughout the semester. They have some similar information and some unique. And there are big differences in ease of finding things.

You can answer these questions from The protein data bank (PDB) sitee. In each case just type your 4 letter code in and play around.

For the PDB file for your protein you have chosen:
From the SUMMARY page.

2a) What is the pdb id?
2b) What is the 'source'. What's the common name for this organism?
2c) How many atoms in the protein?
2e) How many residues?
2f) How many polypeptide chains (subunits) (use sequence tab)?
2g) What is the resolution? Low values of resolution are better. Below 2 Å is not too bad.
2h) What non-amino acid groups are bound to this protein (list the ligands and prosthetic groups)?
On the Annotations Page
2i) What is the SCOP fold?
From the SEQUENCE Page
2j) How many helices are there? What percent is helical?
From the Sequence Similarity page
2k) How many proteins are 100% similar (identical).
2l) How many proteins are on the list of 30% similarity. Tell me what some of them are.
In the Geometry page.
2m) Find the Ramachandran plot for your protein. MolProbity Ramachadran Plot or Procheck in the Structure Analysis/Geometry section of PDB site.
2n) What percentage of the residues at the optimum position for the 2° structure type?
Use 'SUMMARY tab - chemical ligand component.
2o) What Ligands are found? Make a list of ligands and indicate if they are important for protein function (a cofactor, a substrate/reactant/inhibitor or if you don't know). This is important to get straight now.
Click through the other tabs at the PDB site.

2) Look up the function of your protein.
2a) What does your protein do?
2b) Give me the reference to a review article about your protein.
Try using PubMed, Google Scholar, (or when on campus ISI web of knowledge or Science Direct). You can use the citation for your pdb file or use 'related articles' in pubmed to find some papers. Use keyword 'review' to narrow your seach.

_____________EXTRA WEB SITES TO LOOK FOR PROTEIN STRUCTURES__________________
Biochem book tertiary structure
Peptide bond
helix ; hbd in helix and strand and sheets
Ramachandran space cpk for different angles



Homework 5 Lecture: Protein folds and families

1a) Compare the classification system of SCOP and CATH (SCOP has Class ->Fold -> Superfamily -> Family, CATH starts with Architecture). What levels correspond in the 2 systems. Define what each level describes.
1b) How many topologies are there in CATH? How many folds are in SCOP?

2) Use the pdb file for your protein you started looking at in homework 4.
2a) Compare the classification of your proein in SCOP and CATH.

2b) How many domains are found for each chain of your protein?
2c) What other kinds of proteins are found in the same SCOP superfamily?
2d) What other kinds of proteins are found in the same SCOP family?

A - Evolution of the Protein Repertoire Science (2003) 300 pg 1701
B-One thousand families for the molecular biologist Nature (1992) 357 pg 543-4
C-CATH-a hierarchic classification of protein domain structures Structure (1997) 5 pg 1093-1108. A more recent paper Dessaily et al.
D-From protein sequence to function Curr Opin Struct Biol (1999) 9 pg 363-376.
E- SCOP JMB reference

-3a) What is a protein domain? (Give a definition from ref A and from another source).
-3b) How do CATH and SCOP handle proteins with multiple domains?
-3c) What are the 2 alternative ways for evolution to occur described in ref 1 and which one does Chothia support?
-3d) What do protein families have in common?
-3e) Have all the genomes discussed here in 1992 been sequenced? Give me the URL for the web site for one of them?
-3f) How many proteins are now available at the Protein Data Bank?
-3g) How many protein families did Chothia think there would be in 1992? How good was his prediction?
-3h) How much does the structure tell us about the function? Give 2 examples described in ref 4.



6-(no homework). Membranes and the difference between membrane and soluable proteins.
More information about membrane proteins from Steve White on membrane proteins and on lipids.
A nice review by Engelman.


7. Powerpoint slide for the structure of your protein. template
This is a template for the single slide you should give me with help to make the figures in pymol. If you copy the figures from a web site rathar than do them yourself you must include the URL or paper reference in the slide.
Mail me a powerpoint (.ppt) slide. If you use keynote save it as powerpoint format.
If you don't own powerpoint I'd recomment OpenOffice. Free word/excel/powerpoint programs for mac/pc/linx. They work pretty well. Just make sure you export the slide to powerpoint.
It will help me if you name your slide with LAST-NAME.ppt (i.e. your last name).

 


 

Last revised September 16, 2013