Protein Folding Flashcards
Landscape Theory
- protein folding is mediated by a complex energy landscape directing the unfolded state conformations into the encoded native genome
- energy surface representing conformational energy states available to a polypeptide
- horizontal points represent the conformation/torsion angles and the vertical points represent the free energy associated with this
Protein Folding
A protein folds via a series of conformational adjustments reducing free energy and entropy to reach the native state
Anfinsen Experiment
- showed the thermodynamic minimum of the free energy is the native state
- B-mercaptoethanol allowed reformation of proper disulfide bonds that are most stable and not further reduced
- native ribonuclease was denatured and reduced with urea and B-mercaptoethanol
- removing the denaturant without adding reductant left a scrambled protein with non native disulfide bonds
- trace reductant addition caused a conversion into the native state via shuffling of bonds
- non native bonds are less stable so are broken and reformed until the native bonds form
Protein Stability
structured proteins fold into a native state characterised by a well-defined 3D structure that is more stable / lower free energy
- difference in conformational stability of native state given by free energy difference between native/unfolded states
Chemical Stability
ability to maintain the chemical structure of the native state
Conformational Stability
ability to adopt a well-defined conformation rather than a random coil state
Determinants of Folding
- unfavourable entropy change of folding a flexible polypeptide
- favorable enthalpy conditions from H bonds/salt bridges
- favorable entropy change from burying hydrophobic groups in the molecule
Parameters of Folding
- ph/T/P, ionic strength, crowding
- parameters of the system can affect the free energy and folding
- demonstrated in extremophilic organism’s enzymes
Compactness
- proteins are compact with a high density
- native fold is most stable because of the compactness of helices/sheets
- folding is largely directed by internal residues
evidence: - modification of Lys residues in Rnase A by poly A doesn’t affect folding
Heirarchy
- folding and structures are heirarchal
Adaptability
- structure is adaptive
- packing of apolar side chains in core is unique
- mutagenesis of T4 lysozyme reveals great adaptability in that mutations are accommodated without affecting folding
Sequence Versatility
- conservation of sequence can lead to similar folding
- 20% aa sequence identified between 2 proteins means they have the same overall fold
- once you change 50% of the sequence, you change the fold
Techniques for Measuring Stability
Anything measuring Protein Stability distinguishes between unfolded and folded states
- absorbance
- fluorescence
- CD
- NMR
- catalytic activity
- urea gradient gels
Circular Dichroism
- essentially a spectroscopy giving the molar absorptivity as a function of wavelength
- measures molar absorption difference of left and right handed circularly polarized light
- each secondary structure element produces a characteristic spectra
- proteins are chiral so interaction with left/right polarised light differently
Circularly Polarized Light
Direction of electric field vector rotates about its propagation direction
Forms a helix along the vector of propogation (k)
Ellipticity
CD reported as gamma, which is an angle describing an ellipse
- the angle defined by the large/small vectors of an ellipse, ie. the two axes of light
Circular Dichroism Formula
Reports difference in absorption of left handed vs right handed circularly polarized light
E = El - Er
Cons of CD
over-interpreted & lacks resolution at single amino acid residue level
Pulsed HD Exchange
- follows time course of individual residues in a folding protein
- weakly acidic protons exchange with those of water
- exchange followed by NMR
- protein denatured so all residues exposed (specifically amide/hydroxyl)
- folding initiated in stopped flow analysis and pH increased to initiate H exchange
- peptide nitrogen atoms who haven’t found H bonds will exchange
- H/D ratio determined by NMR to show the time course to H bond formation at each residue
Levinthal Paradox
Too many possible conformations for proteins to fold via random search
- for a 100 amino acid protein with only 3 possible conformations you have 3^100 conformations
- therefore, proteins search for the native conformation must be non-random
2 State Folding Kinetics
- TS theory is a kinetic model used to analyse the 2 state model of folding
- high energy metastable TS between the D/N states
- we use this to study folding kinetics
- fold rate is proportional to the exponential of the negative activation free energy, the free energy difference between the TS and denatured state D
FRET
- used in kinetic analysis to probe conformational transitions in single molecular transitions
- 2 flourophores: one donor and one acceptor
- irradiate the donor emitting light in the absorbance range of the second flourophores which emits light
- if the two molecules are too far away, the acceptor will not emit
- in the folded state the 2 fp become close enough to emit, giving a high FRET efficiency
Stopped Flow Analysis
- rapidly mixes denatured protein with buffer dilutant
- fit results to an exponential curve to calculate the rate constant, giving kinetic data for folding (can be further used in phi value analysis)
Phi Value Analysis
- experimental technique studying TS structure using mutational analysis
- used to study protein domain folding in a 2 state manner
- essentially a kinetic/equilibrium ratio
- folding kinetics/ conformational folding stability are compared with those of point mutants to find phi values shows the mutant residues energetic contribution to the folding TS and revealing the degree of native structure around the residue in the TS