Protein folding 2021.pdf

Protein folding and modification Dr Mark Carlile Dale 1.06 [email protected] Dr Mark Carlile: Protein Trafficking 1 Overview of my lectures on PHA111 All of my lectures are broadly focused on molecular biology: Wk 25: Nucleic acid biochemistry Wk 26: DNA replication Wk 26: Transcription...

Protein folding and modification Dr Mark Carlile Dale 1.06 [email protected] Dr Mark Carlile: Protein Trafficking 1 Overview of my lectures on PHA111 All of my lectures are broadly focused on molecular biology: Wk 25: Nucleic acid biochemistry Wk 26: DNA replication Wk 26: Transcription Wk 27: Translation (protein synthesis) (Recording) Wk 27: Protein sorting (Recording) Wk 27 : Protein folding (Live) Wk 28: Biotechnology (Live) Wk 28: Recap and some biological therapeutics stuff Protein folding biochemistry : The big picture Protein Folding| Mark Carlile 3 gles) about the C!¬N bond (%) and the C!¬C bond (&) the size of this energy barrier due to their greater bulk. https://www.youtube.com/watch?v=Kewhg5spUjs Folding possibilities Phi = N- C⍺ bond Psi = C⍺-C bond Main chain Side chain Figure 8-3 A polypeptide chain in its fully extended conformation showing the planarity of each of its peptide groups. [Illustration, Irving Geis. Image from the Irving Geis Collection, Howard Hughes Medical Institute. Reprinted with permission.] Remember the peptide bond can’t rotate but the bonds on each side of it can 77% of psi/phi angles are not seen because their interactions are unfavorable For allow rotations around the peptide bond : phi is nearly always negative (except in left-hand helix) psi is nearly can be positive or negative Protein Folding| Mark Carlile 4 Geometry associated with the peptide bond Clockwise rotation = +ve angle Anticlockwise rotation = -ve angle A A D If you look down the B-C bond B B C Peptide bond (B-C) (C behind B) A-B-D angle A A B B phi and psi = 0O prohibited D A D D B phi = 0O, psi = +90O phi = 0O, psi = -90O prohibited prohibited Protein Folding| Mark Carlile 5 Geometry associated with the peptide bond Clockwise rotation = +ve angle Anticlockwise rotation = -ve angle A A B B A D phi = -90O, psi = +90O phi = -90O, psi = -90O Right-handed helix Beta sheet B AA phi = +90O, psi = +90O (roughly) Left-handed helix Protein Folding| Mark Carlile 6 Dipeptide Alanine-Glycine N C⍺ C⍺ N Alanine Glycine Protein Folding| Mark Carlile 7 Dipeptide Alanine-Glycine H Glycine R-group CH3 Ala R-group Planar (flat) structure Side-on view (Alanine nearest to you) Protein Folding| Mark Carlile 8 Tripeptide Alanine-Glycine-Leucine Planar (flat) structure Alanine Glycine Protein Folding| Mark Carlile Leucine 9 Tripeptide Alanine-Glycine-Leucine -CH2-CH-(CH3)2 Leucine R-group Leucine H Glycine R-group Glycine Planar (flat) structure Alanine CH3 Ala R-group Side-on view (Alanine nearest to you) Protein Folding| Mark Carlile 10 Tetrapeptide Alanine-glycine-leucine-tryptophan (alanyl-glycyl-leucyl-tryptophan) Tryptophan Glycine Leucine Alanine Protein Folding| Mark Carlile 11 At the single protein molecule / single sequence level Unfolded (U) Diverse population of structures (No structure preference) Native (N) One structure 2 state model Work by Christian Anfinsen on Ribonuclease (Anfinsen hypothesis) Protein Folding| Mark Carlile 12 The three-dimensional structure of proteins Tertiary structure Protein folding – the thermodynamic sink Folding goes from a high energy state to a lower energy state Movement from disorder to order (acquisition of native conformation) More accurately: Disorder Order Thermodynamic problem: Movement down a thermodynamic landscape Protein Folding| Mark Carlile 13 The three-dimensional structure of proteins Tertiary structure Protein folding – the thermodynamic sink Unfolded (U) Native (N) What we know/assume: 1. The native state is the lowest energy conformation 2. Unfolded state = random coil 3. Folding path and native state are directed by side chain interactions 4. The hydrophobic effect is the largest driver of protein folding 5. Organising interactions are maintained in the native structure Protein Folding| Mark Carlile 14 Protein folding Unfolded (U) Diverse population of structures (No structure preference) Native (N) One structure Native state is the preferential conformation (its selected) Favourable vs Unfavourable interactions between side chains When we unfold proteins (temp/[denaturant]) we get all of the unfolded intermediates falling along the same curve = highly cooperative process What is actually represents is two distinct states Fraction unfolded at 0.5: 50% of molecules unfolded and 50% of molecules still folded rather than all molecules 50% unfolded x or Temperature Protein Folding| Mark Carlile 15 Folding summary: Unfolded (U) Diverse population of structures (No structure preference) Native (N) One structure 2 state model Random coil Folded state △S loss (entropy) : remember nature works to increase energy – protein folding actually increases the whole system entropy by disorganising the water molecules around the unfolded state Water is in fact more ordered around unfolded proteins Form an energetics point of view: △G = △H-T△S Protein Folding| Mark Carlile 16 Folding summary: Unfolded (U) Diverse population of structures (No structure preference) Native (N) One structure 2 state model No stabilising interactions in the unfolded state – described as a featureless landscape For the most part the peptide backbone is the same and so only the sidechains discriminate (maybe Glycine and proline are exceptions here) Folding is thermodynamically driven △GO = -RT ln[N / U] △G = 0 → [N] = [U] : at the 0.5 fraction unfolded point IMPORTANT: If we start to introduce in mutations we move up on the unfolding curve At midpoint 50% of the molecular population still knows how to fold Remember you are not making or breaking bonds as you fold Protein Folding| Mark Carlile 17 Linking the folded and unfolded state with the peptide bond: We can now think about the phi and psi bonds as being native or unfolded Example: a 100 AA protein We will have each phi and psi bond angle either native or unfolded = 2100 different conformers = 1030 conformers (accessible conformations in the unfolded state) So its likely that no two proteins will have the same conformation unless the concentration is > 1030 molecules per unit volume Each phi-psi pairs fold independently of all other phi-psi pairs (except the ones immediately next to it) Protein Folding| Mark Carlile 18 Protein secondary structures Covered this with Dr Gray – A direct link to his material Protein Folding| Mark Carlile 19 Summary This week we have gone into quite a bit of detail on: protein folding Protein modifications (in vivo, and a little on in vitro) the thermodynamic driving protein folding and dynamics In some form this material will be represented on the examination Protein Folding| Mark Carlile 20 Associated essay-style questions (week 27) – I WILL NOT MARK THESE 1. Explain how and why proteins that are active in the endoplasmic reticulum are generated and processed in the cell 2. Explain how membrane spanning proteins are inserted into the plasm membrane 3. Give a detailed overview of how proteins fold and what biochemical properties of a protein sequence direct the folding pathway 4. If proteins can spontaneously fold into their native conformation, why does the cell have specific folding and structure monitoring pathways. Protein Folding| Mark Carlile 21