Protein folding

Description

Protein Form and Function (Protein folding/misfolding) Mind Map on Protein folding, created by sophie_connor on 26/05/2013.
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Mind Map by sophie_connor, updated more than 1 year ago
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Created by sophie_connor over 11 years ago
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Resource summary

Protein folding
  1. Central dogma
    1. DNA becomes RNA forming a sequence which folds to enable a protein to fulfill its function
      1. Native structure of the protein fulfills its function
        1. Must be in a cellular environment
        2. A protein chain folds in a specific way
          1. Secondary and tertiary structures
            1. Sequence defines fold
              1. Sequence determines 3D structure
                1. From an amino acid sequence you could guess the fold
                  1. A small protein of 101 amino acids has 9^100 possible conformations
                    1. Protein fold defines function
                      1. Free energy of unfolding
                        1. DELTAG(D-N) = GD - GN
                          1. To measure the differences the process needs to be in equilibrium
                            1. The equilibrium needs to be disturbed and determine the fraction folded
                              1. Denaturant urea is best
                                1. Temperature can be used but the process may not be reversible
                                  1. Can cause protein misfolding
                            2. A ribonuclease solution will break all disulphide bonds and denature the protein giving its polypeptide chain structure
                              1. Another solution can reverse this and it will go back to its folded structure
                              2. Environment is an important folding factor
                          2. Proteins are marginally stable
                            1. Stability of denatured and native states is due to sum of non-covalent interactions
                              1. Folding is thermodynamically controlled
                                1. Native state is thermodynamically stable
                                  1. Folding must occur rapidly
                                  2. Levinthal's paradox
                                    1. Sequence tells you what structure is likely to be
                                      1. Folding cannot occur by the protein trying every different conformation
                                        1. Take too long!
                                        2. Folding must occur by intermediates
                                        3. Pathway is needed!
                                          1. Proteins must fold via pathways
                                            1. Cut down the number of conformations the protein needs to search in native state
                                              1. Folding funnels
                                                1. Consider the energy landscape of folding
                                                  1. If protein folding was random then the diagram would be flat with the same energy
                                                    1. Protein would eventually find native state and fall down the middle
                                                      1. Takes too long
                                                2. Hierarchical protein folding mechanism
                                                  1. Simplifies the search for native state
                                                    1. Uncouples formation of secondary and tertiary structure
                                                      1. Framework model
                                                        1. Secondary structure is separated followed by the tertiary structure
                                                          1. Requires intermediates
                                                            1. Reduces size of conformational space a protein has to search through
                                                              1. Denatured state to begin
                                                                1. Intermediate
                                                                  1. High energy state of molten globules
                                                                    1. Much less stable and eventually ends up at low energy native state
                                                        2. Classical nucleation model
                                                          1. An element of secondary structure that nucleates the rest of the structure which allows formation of tertiary structure
                                                          2. Hydrophobic collapse model
                                                            1. All hydrophobic amino acids in the centre of the protein drive folding
                                                              1. Requires intermediates
                                                            2. All models rely on local interactions and sequence
                                                            3. Alternative view: no intermediates
                                                              1. Energy level is so high that you can't see the intermediate
                                                                1. CI2 (random protein)
                                                                  1. Not necessary to have a stable intermediate
                                                                    1. Protein goes straight from denatured to native state
                                                                    2. Intermediates might slow folding
                                                                      1. Intermediates may result in misfolding or an off pathway
                                                                        1. No matter what state the protein is in, it will always fall back to the same native state as it is most energetically favorable
                                                                          1. Folding funnels consist of lots of random highs and lows
                                                                            1. Suggests possibility of multiple folding pathways
                                                                              1. Bumps show that proteins could get stuck which could represent protein misfolding
                                                                          2. Lysosyme
                                                                            1. Small protein
                                                                              1. Contains some disulphide bonds
                                                                                1. Begins denatured and forms a collapsed state
                                                                                  1. There are a number of ways to get to the native state
                                                                                    1. Can be slow or fast pathway
                                                                                  2. Measuring protein folding and stability
                                                                                    1. Need to consider
                                                                                      1. How to monitor population of denatured and native
                                                                                        1. How to change populations of denatured and native
                                                                                          1. How to analyse results
                                                                                          2. Stability of protein is expressed as free energy of unfolding
                                                                                            1. Protein folding probes
                                                                                              1. In vitro assays in cuvette
                                                                                                1. In vivo assays under a microscope
                                                                                                  1. Fluorescence will tell you about chromophores in protein
                                                                                                    1. Can find buried or exposed amino acids in the protein
                                                                                                      1. Unfolded state
                                                                                                        1. Trp is exposed giving a particular fluorescence signal
                                                                                                        2. Folded state
                                                                                                          1. Trp is buried and gives different fluorescence signal
                                                                                                        3. Emission spectra
                                                                                                          1. Fix the excitation wavelength and scan the monochromator across fluorescence wavelengths
                                                                                                          2. Unfolding proteins by denaturants
                                                                                                            1. Urea most commonly used
                                                                                                              1. GuHCL a much strong denaturing condition
                                                                                                                1. Solvents increase the solubility of polar and non-polar side chains and stabilse the denatured state
                                                                                                                  1. Other denaturants
                                                                                                                    1. Temperature
                                                                                                                      1. pH
                                                                                                                        1. Salt
                                                                                                                          1. Pressure
                                                                                                                      2. Differences in protein folding between mutants
                                                                                                                        1. We know if a particular part of the molecule is important for folding
                                                                                                                          1. Mutate this part of protein and measure folding again
                                                                                                                          2. If free energy is different then we can tell the amino acid was important for protein folding
                                                                                                                        2. Experimental timeline and problems
                                                                                                                          1. To map folding reaction all states must be characterised
                                                                                                                            1. Mechanisms must also be characterised
                                                                                                                            2. Characterisation is often difficult
                                                                                                                              1. Dynamic, heterogenous, transient nature of non-native states
                                                                                                                              2. All possible states between denatured and native state must be investigated
                                                                                                                                1. Stable states are measured by equilibrium methods and protein engineering
                                                                                                                                  1. Native state can use crystallography and NMR
                                                                                                                                    1. Denatured state can be determined using NMR
                                                                                                                                      1. Semi stable states must be trapped
                                                                                                                                        1. Hydrogen/deuterium quench flow
                                                                                                                                          1. Traps something as it's changing from denatured to native state
                                                                                                                                          2. Sometimes conditions may be found where partly folded states can be observed by equilibrium methods
                                                                                                                                            1. May not be relevant to pathway
                                                                                                                                            2. Transient events can be followed by kinetic techniques
                                                                                                                                              1. Stopped flow to follow protein folding
                                                                                                                                                1. 2 solutions A and B can be mixed together and monitored during a reaction
                                                                                                                                                  1. A solution fills stopping syringe, plunger hits a block causing flow to be stopped instantaneously
                                                                                                                                                    1. Using appropriate techniques, the kinetics of the reaction can be measured in the cell
                                                                                                                                              2. Representing protein folding
                                                                                                                                                1. Chevron plot
                                                                                                                                                  1. Shows protein folding kinetic data in varying denaturant concentrations
                                                                                                                                                    1. Shows protein folding and unfolding
                                                                                                                                                      1. Cm is the denaturation midpoint
                                                                                                                                                        1. Either side are the limbs
                                                                                                                                                          1. Linear limbs
                                                                                                                                                            1. Straight lines
                                                                                                                                                              1. Suggests a 2 state model
                                                                                                                                                              2. Non-linear limbs
                                                                                                                                                                1. Curving
                                                                                                                                                                  1. Indicates a non-2 state model of folding/unfolding
                                                                                                                                                                    1. Suggests an off pathways intermediate
                                                                                                                                                                2. Chevron/V-shaped kinetics curve
                                                                                                                                                                  1. Proteins are denatured as all parts of the protein are more soluble
                                                                                                                                                                    1. Free energy of transfer to denatured state is nearly linear with concentration of denatured protein
                                                                                                                                                                      1. TS in an intermediate structure between denatured and native states
                                                                                                                                                                        1. TS stabilised by denatured state with respect to native structure
                                                                                                                                                                          1. TS destabilised with respect to denatured state
                                                                                                                                                                          2. Structure of proteins TS for folding can be analysed by combining kinetic and equilbrium data
                                                                                                                                                                            1. Produces a ratio called PHI value
                                                                                                                                                                              1. Value obtained by normalising
                                                                                                                                                                                1. Free energy from folding kinetics
                                                                                                                                                                                  1. Free energy from refolding kinetics
                                                                                                                                                                                    1. Free energy from equilibrium data
                                                                                                                                                                              2. Denaturant lowers activation energy of denaturation and raises activation energy of folding
                                                                                                                                                                                1. 2 state model means intermediate is passed by quickly
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