Protein Folding. Cell/mol bio lab. Proteins are like a long spaghetti noodle, folded back upon itself over and over

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1 Protein Folding Cell/mol bio lab Proteins are like a long spaghetti noodle, folded back upon itself over and over Why study the 3-D 3 D shape of a protein? (go to Cell Biology web site from my home page)

2 First though, what is Protein Folding? Proteins are very rickety; their shape is easily distorted. Mother Nature uses this to control enzymes (bind something to an enzyme, and distort the enzyme, turn it off or on) Proteins are rickety because their 3-D 3 D shape is largely due to weak bonds (not strong covalent bonds) Biomolecules/drugs bind to proteins through weak bonds (only rare poisons bind through covalent bonds) to distort enzymes Pages 30-33; 33; , 134, 696, (6 th ed of World of Cell)

3 Protein Folding Some day (as in Star Trek), we hope to design new proteins to cure disease But bad protein folding can CAUSE disease

4 Protein Folding Diseases page 697 in text (6 th ed) Alzheimer patients have extracellular amyloid plaques made up of amyloid-β (Aβ) Because protein A-βA is not folded properly ApoE is a protein that may cause normal A-A β to misfold APP (a membrane protein) is cut (cleaved) to form A-β-A so many study APP cleavage; prevent the cleavage and prevent amyloid plaques (?)

5 Prions induce scrapie,, & mad cow disease and, in humans, Creutzfeldt- Jakob disease (vcjd( vcjd) Prions do not contain DNA or RNA Prions (PrP SC ) are believed to be misfolded versions of normal proteins (PrP( C ) When Prions (PrP SC ) bind to folding protein, they cause the protein to fold abnormally and then clump (aggrate( aggrate) why? What weak bond is involved? This is how the Prions (PrP SC ) reproduces- causes more misfolded proteins without DNA

6 Protein Folding Diseases page 697 in text (6 page 697 in text (6 th ed) Fig. 22A-1

7 View the member societies

8 Alzheimer's disease. Cystic fibrosis. Mad Cow disease. An inherited form of emphysema. Even many cancers. Recent discoveries show that all these apparently unrelated diseases result from protein folding gone wrong. As though that weren't enough, many of the unexpected difficulties biotechnology companies encounter when trying to produce human proteins in bacteria also result from something amiss when proteins fold.

9 Figure The Spontaneity of Polypeptide Folding rare Proteins loose their 3-D shape (denature) when the weak bonds that hold together the structure are broken. Weak bonds are broken when the temperature is raised from body temp (37 C) to about 60 C Or by changing the ph Or by using chemical agents

10 Why is ribonuclease weird? It can renature renature (most proteins unravel, and aggregate and precipitate because they reveal their nonpolar amino acids) most proteins cannot amino acids)- most proteins cannot renature It can fold without chaperones (proteins that help other proteins fold) Chaperones do not provide info, they merely bind to the folding protein to make sure that the wrong weak bonds are not formed Heat up a cell, you could denature proteins, so more Chaperones are made to protect proteins (Chaperones were originally called heat shock proteins)

11 Figure The Structures of the 20 Amino Acids Found in Proteins Don t memorize The structures Just know that there are 3 types of Amino acids Polar (polar cov bond, no full charge but partial charge) Charged Amino acid Nonpolar amino acid (hidden inside spherical protein)

12 Oil in Swimming pool Figure 3-53 Bonds and Interactions Involved in Protein Folding and Stability- which are weak bonds and which strong? Strong covalent bonds are about 50 times stronger than weak bonds

13 Figure The Four Levels of Organization of Protein Structure- memorize this crucial slide Disulfide bridge, all Weak bonds stabilize 3 and 4 th deg structure List of Amino acids From N terminus To C terminus Covalent bond Connects amino acids Small loops: Helix or B pleated Sheet (only H bonding) Big loops subunits

14 Figure The Primary Structure of Insulin

15 Figure The α Helix and β Sheet H bond only stabilizes This level of structure

16 Figure Common Structural Motifs Combinations Of helix and Beta pleated Sheets (purple arrows Point to the end or C Terminus) Helix turn helix Found in Transcriptions Factors (they bind DNA and turn off/on Genes) So motifs often do Same thing but are found in Different proteins

17 Figure Hemoglobin: what levels are present?

18 Figure The Structure of Hair KERATIN What levels of protein Are in the hair protein KERATIN? Proteins are spherical (globular) or Long and thin (called fibrous)

19 Figure The Three-Dimensional Structure of Ribonuclease- A GLOBULAR protein Ball and stick model Spiral and Ribbon model--where are B pleated sheets? Helices?

20 Figure Structures of Several Globular Proteins- Domains make up proteins Domains are about , have similar function in different proteins, a unit of Tertiary structure (made up with secondary structures)

21 Figure An Example of a Protein Containing Two Functional Domains- each domain binds something different

22 Figure Molecular Structures of Lysozyme and Carboxypeptidase A

23 We will be looking at this movement in the enzyme Hexokinase with a program called MAGE. You click and animate the binding of substrate and the movement of the two domains (along the hinge; where is the hinge and 2 domains?) Figure The Conformational Change in Enzyme Structure Induced by Substrate Binding (rickety protein distorts due to the new weak bonds forming between substrate and protein)

24 Calmodulin binds 4 Calcium ions, causing the calmodulin to bend and wrap around other proteins this turns We will View this In MAGE on other proteins Fig Page globular Ends Joined by A helix

25 Calcium makes new weak ionic bonds to the R groups of amino acids of the protein calmodulin this really changes the protein; calmodulin breaks in the middle and hinges to bind another protein (and turn this other protein on)

26 With 4 Ca bound to Calmodulin (from Introduction to Protein Structure, also in lab exercise file)

27 Maybe most impt protein in cell: cdk -an enzyme that turns on cell division (or G1 to S phase). Another protein called cyclin builds up in the cell, and then binds to and activates cdk. Cdk then causes progression to cell division We will be studying cdk s later (another kinase called map kinase can turn it on) We will be using MAGE to view the binding of cyclin and cdk HOW DOES THE CYCLIN BINDING TO CDK TURN ON THE CDK KINASE ACTIVITY?

28 CYCLIN BINDING FORMS NEW WEAK BONDS THAT CAUSE A BIT OF PEPTIDE CALLED THE T LOOP TO MOVE OUT OF THE ACTIVE SITE OF Cdk; THIS UNBLOCKS THE ACTIVE SITE. (from Mol Bio of the Cell; Alberts et al.)

29 CDK2 is off, no cell division PSTAIRE is not in the active site, and T Loop Blocks the binding of substrate

30 CYCLIN (GREEN) BINDS AND MOVES PSTAIRE AND T LOOP TO CORRECT LOCATIONS CELL DIVISION STARTED OR CANCER?

31 Space filling model of CDK2 No Cyclin binding Cyclin binding

32 Membrane proteins are different from soluble globular proteins Fig Globular arrangement of amino acids at the ends in the cytoplasm and outside the cell but.. Nonpolar amino acids found in the center (the Alpha helix that goes through the hydrophobic section of the membrane

33 Porins are membrane proteins that travese the membrane not with alpha helix but with Beta pleated sheets

34 Chaperones Page 696, Chaperones are of two general types (with different mechanism, but do the same thing): Hsp70 and Hsp 60 (heat shock protein) Chaperones bind to a folding protein to help the protein fold properly As protein is made, it begins to fold properly and bind other subunits of the protein (if the protein has 4 o structure)

35 Chaperones bind to a folding protein at the folding protein s hydrophobic region (nonpolar( amino acids), prevents the folding protein from aggregating/precipitating Releases protein by use of ATP breakdown (nonspontaneous( reaction without ATP) Remember: in a typical soluble protein, the nonpolar amino acids are hidden on the inside of the protein so the protein will not precipitate