BCH3052 Lecture Notes - Lecture 10: Circular Dichroism, Protein Folding, Phenylalanine
25 May 2018
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Lecture 10 – Mechanisms of Protein Folding
Protein Folding Landscapes
• Multiple unfolded states, which are highly heterogenous
• Number of routes to native state
• Native interactions more stable than non-native
o No. of available conformation is reduced driving the chain towards
native state
How does as Protein Assemble into its Unique 3D Conformation?
• Aim to determine the ensemble of structures found in each conformational
state (N, I, D)
o 2 intermediates
• Experimental approaches are either kinetic (rate of sth: fast or slow) or
equilibrium
o Protein folding is rapid and intermediates are only transiently
populated: difficult to characterise
▪ Look at it over time – kinetics
o Or look at fraction unfolded when denatured – equilibrium
Techniques to Follow Protein Folding
• Absorbance (Tyr)
• Fluorescence Spectroscopy (Trp)
o In proteins, major fluorescent groups are Trp, Tyr and
Phe
o Trp fluorescence is the most dominant
o The environment around Trp residue greatly effects
the intensity and emission wavelength from the Trp
residue
▪ Trp can report the changes in a protein
as it unfolds
• Circular Dichroism (CD)
o Tells us on secondary structure
o Light in instrument is polarised
o Natural light is unpolarised
o CD measures the rotation of light, α-helices and
β-sheets rotate light differently
o Far-UV CD measures the 2ary structure of
protein
▪ Any conformational change in a protein results in 2ary structure
changes will alter the CD signal – protein folding
o α-helices (non-toxic) → β-sheets (toxic)
• NMR
• Differential Scanning calorimetry (enthalpy)
• Gel filtration (Size)
• Light scatter
• Fluorescence Probes
Measuring Kinetics
• Refolding
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