Protein Folding kinetics

Rate constants

• k_f → rate constant for folding

• k_u → rate constant for unfolding

How do we measure folding rates?

• Folding (U → N): Start with unfolded protein in high denaturant → dilute into buffer → fit the signal to y = Ae^-k_ft

• Unfolding (N → U): Start with folded protein → add denaturant → fit to y = Ae^k_ut

Chevron plot

Because we usually cannot measure folding rates directly in pure water (it happens too fast or the protein is too unstable), we measure k_obs at various denaturant concentrations and extrapolate.

Transition state and β_T value

• TS is the “mountain peak” the protein must cross to fold or unfold

• We can calculate β_T (a value between 0-1) to see what the TS looks like using the formula

• βT measures how much surface area is buried in the transition state compared to the fully folded state

What does it mean if m_f >> m_u —> β_T is around 1?

TS is compact and looks like the native state!

What does it mean if m_f << m_u —> β_T is around 0?

TS is unstructured and looks like the unfolded state!

How can we see which amino acids have formed their native contacts in the TS?

Through a Φ-value analysis!

• Φ = 0: mutated residue is largely unfolded and does not contribute to stabilising the transition state

• Φ = 1: mutated residue and its local environment are fully structured and native-like in the transition state

• Φ = 0-1: the residue has partially formed contacts in the TS