Short Announcements 1 st Quiz today Homework #2 on web. Due next Monday. Chpt 2 Reading Due next Wednesday (NOT Monday) Today’s Lecture: Protein Folding

Quiz #1 (covering Chpt 1, ECB) 1. What are the three major classes of filaments that make up the cytoskeleton?__________________________________________ 2. All cells are enclosed by a _________________that separates the inside of the cell from the environment. 3. All cells contain _____as a store of genetic information and use it to guide the synthesis of__________. 4. A) List the 3 kingdoms of life. ____________________________ B) You are a member of which kingdom? __________________ 5. The presence of this organelle is the most striking difference between prokaryotic and eukaryotic organisms. _______________________ 6. The ______________is the organelle most responsible for energy production in a eukaryotic cell. Microtubule, actin filaments and intermediate filaments Plasma membrane bacteria, archea, eukarya/eukaryotes eukaryotes mitochondria nucleus, membrane bound organelles DNA proteins

The Protein (Free) Energy Landscape Largely from Martin Gruebele, Chemistry, Physics UIUC

A typical protein folding equilibrium constant K eq ≈ 1000 means a protein is unfolded for 100 sec/day! day AA Not nearly enough chaperones to help re-fold. Tend to do this by itself % are natively unfolded– bind to negatively charged substrate and then folds. Hydrophobic regions become exposed. Become ubiquinated. Reused aa in proteasomes. A+B  A-BK eq = [A-B]/[A][B] K eq = [A folded ]/[A] unfolded = A unfolded  A folded  kfkf k uf k f / k uf 

How does a Protein go from unfolded to folded a) at all; b) in 1 msec; c)with no chaperones Hans Frauenfelder, founder of biological physics. Unfolded  Folded Inactive  Active Main driving force : 1) Shield hydrophobic (black spheres) residues/a.a. from solvent/ water; 2) Formation of intramolecular hydrogen bonds. Active areas: 4 centuries on it and still not solved! Difficulty relating to experimental observations.

In a crowded cell, chaperones are needed, but take a protein assembly under dilute conditions, they fold fine.

Energy and Free Energy Landscapes Amino acid represented as beads  Black bead: hydrophobic (H)  White bead: hydrophilic (P) Bonds represented by straight lines H-H (= -1000J =1/3 kT) and P-P (= -250J) bonds favorable Based on work by N. Go M. Levitt, K. A. Dill, Shakhnovich/Karplus H-H go inside; P-P on outside/solvent exposed Peptides don’t fold because they have too few H-H and P-P to fold stably.

Protein Example 6-mer 2 hydrophobic AA 4 hydrophilic AA

Chirality in Amino acids To avoid issues with chirality, all molecules are made so that the first two amino acids go upwards. Also, the first kink always goes to the right. Although most amino acids can exist in both left and right handed forms, Life on Earth is made of left handed amino acids, almost exlusively. Why? Not really known. Meteorites have left- handed aa. rality_with_hands.jpg

Rotation Rules 2-D model - no rotations allowed. Molecules are only al- lowed to change by a single 90˚ “kink” per time step. Allowed kinetics– one moves only 90 degrees. Kinetic moves by diffusion.

The Journey A trap! Direct folding!

Entropy

Conformation Analysis E Reaction Coordinate Kinetic traps -0.5 kJ x Only nearest neighbors that count Molecular Dynamics has actually taken over No none example s where there are multiple states.

This is the folding funnel: E Entropy k ln1 = 0 k ln14 Entropy : horizonal scale

Entropy vs. Energy correlated monotonic function Ln 14 Ln 1

Entropy vs. Reaction Coordinate

Free Energy (if compressibility is neglected so H ≈ E) G(x) = H(x) - TS(x) ≈ E(x) - TS(x) G is almost always flat.E goes up, S also goes up. They compensate

Free Energy Analysis (200K) x Downhill folding (but in reality, at 200K, nothing moves)

Free Energy Analysis (298K) Downhill folder

Free Energy Analysis (360 K) Two state folder Unfolded state—has some structure This is likely the equilibrium of 50:50 where they are interconverting and equally stable.

Free Energy Analysis (2000K) Downhill unfolder

Free energy x Enthalpy Config. entropy  S<0  G>0  H<0  G<0 Wolynes Bryngelson Onuchic Luthey-Schulten Dill Thirumalai 0 1 Free energy x Energy Funnel and Free Energy Surface  G =  H - T  S

There is a lower energy state which is fibers—e.g. ameloid fibers– mutliple states! Amyloid Fibers…involved in Alzheimers Protein amyloid fibers are often found to have a β-pleated sheet structure regardless of their sequence, leading some to believe that it is the molecule's misfolding that leads to aggregation ~db=medi~order=page Enzymes act on the APP (Amyloid precursor protein) and cut it into fragments of protein, one of which is called beta-amyloid and its crucial in the formation of senile plaques in Alzheimer Enzymes act on the APP (Amyloid precursor protein) and cut it into fragments of protein, one of which is called beta-amyloid and its crucial in the formation of senile plaques in AlzheimerEnzymes act on the APP (Amyloid precursor protein) and cut it into fragments of protein, one of which is called beta-amyloid and its crucial in the formation of senile plaques in Alzheimer

Proteins can fold. Don’t need chaperones. ΔG is always about zero. Therefore can fold fast. Kinetics – fast cause not huge barriers Summary of Protein Folding

Class evaluation 1. What was the most interesting thing you learned in class today? 2. What are you confused about? 3. Related to today’s subject, what would you like to know more about? 4. Any helpful comments. Answer, and turn in at the end of class.