1. AGR2451 Lecture 2 - M. Raizada -Pick-up questionnaire at the front; results from last week -Did you review your notes within 24 hours?? -15 minute meetings -Reading for this week on reserve in the library: Introduction to Protein Structure (pp. 3-12) Review of Previous Lecture 1. Definition of science.. 2. What is the chemical basis of life and why? -water -hydrophilic and hydrophobic atoms and their functions (eg. membrane layer) 3. Why were N, O, P, S used? Unpaired electrons are critical to Hydrogen bonding, which is critical for proteins, DNA and RNA to function. 4. What is life? Slide 2.1

2. Lecture 2 - An Introduction to Proteins How is life organized? 1. evolution chose proteins to do the work of life. (DNA is only the set of instructions to make proteins.) 2. What do proteins do? -A. Structural proteins make large structures (eg. microtubule cables to pull chromosomes apart during mitosis/meiosis) -B. Enzymes - catalyze biochemical reactions - the key to life demo Rather than 2 reactive molecules trying to “find each other” by random diffusion, an enzyme binds both molecules in close proximity at its active site. The enzyme positions the two molecules in place, thus decreasing the activation energy required for the chemical reaction to proceed. protein cables Slide 2.2 From Biochemistry and Molecular Biology of Plants (W.Gruissem, B. Buchanan and R.Jones p.236 ASPP, Rockville MD, 2000 From Biology of Plants p. 80 (P.Raven, R.Evert, S. Eichhorn) Worth Publishers, New York, 1992 From Introduction to Protein Structure p.206 C. Branden and J. Tooze Garland Publishing, New York, 1999

3. How does an enzyme function? Slide 2.3 Enzymes - Because biochemical molecules come in different sizes, shapes, with different surface charges (charged, polar, hydrophobic), then in order for proteins to grab onto them, they must form a "glove", a pocket at the active site containing the appropriate charges. It must have a second pocket to grab onto a second molecule. + - - + - ++ - hydro- phobic Molecule 1 Molecule 2 Enzyme Enzyme + molecules hydro- phobic hydro- phobic Change in Protein Conformation After binding the two substrates, the enzyme may need to change its shape in order to position them closer together. In addition, the chemistry may need to be protected from the aqueous environment -- for example, a charged molecule may be more attracted to water than to the second molecule involved in the biochemical reaction. In such a case, the charged molecule needs to be hidden away from the outside of the protein into a hydrophobic pocket inside the protein. Because the binding site of the molecule must be near the surface of the protein, the binding must cause a change in conformation of the protein such that the bound molecule is rotated into a cavity inside the protein. hydro- phobic charged H20H20H20H20 protective cavity H20H20 H20H20 Pictures from M. Raizada

4. How do herbicides, pesticides or pharmaceuticals work?: 1. The chemical mimics the real substrate and competes for the enzyme active site. 2. The chemical binds elsewhere to the enzyme, and because of its charge, alters the conformation of the enzyme, causing it to be no longer functional. native substrate (eg. nitrogen metabolism) herbicide + - + - Enzyme-herbicide binding Enzyme Native enzyme Enzyme + herbicide In addition, other molecules (phosphate groups, sugars, lipids) can bind onto proteins and alter its conformation, thus either activating its function or preventing its function. Inactive enzymeCharged phosphateActivated enzyme **Hence, small molecules can be used to switch on/off enzymes**. Slide 2.4Source of pictures: M. Raizada

5. How does an enzyme form loops or change its shape? Slide 2.5 -Parts of the protein interact with other parts of the protein (eg. plus to negative, hydrophobic to hydrophobic) to create loops. -After substrate binding, the local charge might be altered, causing the active site to be more attracted to another internal region of the protein, hence causing a change in protein conformation. demo + - Hydrophobic stretches + - Hydrophobic stretches + - Substrate-binding alters local protein charge *+ uncharged region + - + Positive attracted to negative, causes change in conformation Source of cartoonss: M. Raizada From Introduction to Protein Structure p.56 C. Branden and J. Tooze Garland Publishing, New York, 1999

6. To facilitate the binding of molecules and changes in protein conformation, proteins have an arsenal of 20 amino acid building-blocks, each with a unique size, shape and charge. Introduction to Amino Acids Slide 2.6 HHH H H H H + + - - - PP PP P P PP * What charges can amino acids have? + positive charged - negative P polar H hydrophobic * very flexible From Biochemistry and Molecular Biology of Plants (W.Gruissem, B. Buchanan and R.Jones p.360 ASPP, Rockville MD, 2000

7. Slide 2.7 Amino acids join together through peptide bonds that can rotate. Why is this useful? rotate From An Introduction to Genetic Analysis (6th ed) A.J. Griffiths et al., page346 W.H. Freeman and Co., New York, 1996

8. By placing these at particular places relative to each other in a 3-dimensional chain, they can form the binding sites necessary to bind molecules for biochemistry or bind one another to form large structures. Specific amino acids bond to specific regions of the reactant molecule. Slide 2.8 From Introduction to Protein Structure p.60-61 C. Branden and J. Tooze Garland Publishing, New York, 1999

9. Protein enzymes can adopt multiple shapes by folding. Horsheshow - RNasinTIM Barrel - Rubisco Beta roll - transcription factorBeta barrell - GFP Slide 2.9 Protein Folding To review, what are the molecular functions of an enzyme? Therefore, why are the shapes of proteins important? How many different protein shapes (unique folds) are there in all of life? Is this a surprise?

10. Bonds between amino acids can create elaborate secondary and higher order scaffolds upon which or within which the biochemistry can be performed. alpha-helix scaffold beta-sheet scaffold Alpha/beta scaffold structures create pocket for enzyme active site Slide 2.10 From Introduction to Protein Structure p.73 C. Branden and J. Tooze Garland Publishing, New York, 1999 From Biochemistry and Molecular Biology of Plants (W.Gruissem, B. Buchanan and R.Jones) p.347-348 ASPP, Rockville MD, 2000

11. Post-Translation Correct 3-D protein folding: demo -to create correct enzyme active site and shape -only <1000 folds in all of life!!! -DNA is rigid, but amino acid peptide bonds can rotate, so many combinations -other protein complexes (chaperones) assist in folding in a destabilizing aqueous environment Slide 2.11 From Biochemistry and Molecular Biology of Plants (W.Gruissem, B. Buchanan and R.Jones p.438 --chaperone

12. What has to be the function of the genetic code? *Somehow in evolution,DNA “code” had to interact with amino acids & control how the proteins were assembled.* Proteins do not contain the genetic code. Why not?? What features must be possessed by the molecule selected by evolution to encode the genetic code? Slide 2.12 DNA RNA Proteins

13. Lecture 2 - Key Concepts 1. Because of the variety of amino acids available, evolution selected proteins to be the main enzymes of life. 2. Enzymes increase the probability that two reactive molecules will form or break a bond at an active site. 3. Local amino acid charges interact with nucleotides, other amino acids, chemicals very precisely. Any change in the local charge or size can cause changes in protein conformation or binding. 4. The addition or loss of small molecules (phosphates, lipids, glucose) can be used as an “on/off” switch for protein activity. 5. Proteins are basically a carbon scaffold upon which charged or hydrophobic surfaces exist to do biochemistry. 6. Proteins do NOT carry the genetic code, but must interact with the genetic code. Slide 2.13