Lecture 5 Phi Analysis

rate

instantaneous change in quantity as a function of time

what does the order of the rate law tell us

the dependence on concentration e.g in second order two molecules have to collide

how can first order reactions be characterized

first order reactions can be characterized by their constant doubling times

on circular dichroism graphs when do you have a faster rate

a faster rate or a higher K has a steeper curve

what is titin

the largest known protein that is a component of muscle sarcomeres

what do IgG domains do

provide elasticity through folding/unfolding

what do optical tweezers do

use light to grab individual molecules to pull to measure force by moving one bead relative to another

what are the steps for chi analysis

measure the folding rate

generate a mutations and then remeasure the folding rate

calculate the ∆∆G+ folding

what happens to the rate of folding as denaturant concentration increases

at lower concentrations K folding dominates and usually has a steep curve because the K folding is ususally faster than the K unfolding

eventually it becomes dominated by K unfolding but the slope remains shallow

what happens to the ∆∆G+ if the mutation affects the transition state

it increases

what makes proteins unfold spontaneously

random thermal energy

what is a chevron plot

a graphical representation of protein folding data that reveals the folding and unfolding rate constants

what type of experiment is a chevron plot

equilibrium experiment

what experiment is used to get a chevron plot

stopped flow apparatus

how does a stopped flow apparatus work

the stop syringe allows the cell to fill to a certain point when the piston hits the block and the flow stops, the mixed reagents continue to react in the cell

what type of experiment is a stopped flow apparatus

kinetic

why is tryptophan intrinsic fluorescence increased when buried away from solvent

the solven quenches the fluorescence if they bump into each other

what part of the stop flow apparatus graph determines the rate of folding

the initial, linear portion

why do stop flow graphs have a dip at the start

an artifact due to mixing solutions

where does each point on a chevron plot come from

one or more kinetic experiments

on a chevron plot what does Cm represent

the midpoint concentration of denaturant which gives 50% unfolded, the Kf and Ku contribute equally to the observed rate

what does it mean when there’s a huge change in unfolding rates between a wildtype and mutant protein

the amino acid that was changed is important for stability of the protein

what does phi measure

the ratio of the change in transition state energy to the change in the energy of folding

formular for phi

change in TS energy (∆∆G⁺_f) / change in stability (∆∆Gf)

what does a phi close to 1 indicate

the mutation affected the transition state

what happens to the energy diagram when the mutation only affects stability

final free energy decreases

what happens when the mutation affects stability and folding rate

transition state free energy and final free energy decrease

are high phi values typically in the core or edges of the protein

core