The Native And Non Native States
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The Native And Non Native States

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The Native And Non Native States Presentation Transcript

  • 1. Protein folding: the native and non-native states Any peptide (in theory) could adopt many different secondary and tertiary structures But in general ALL molecules of a given protein species adopt the SAME 3D-conformation. This structure is called the NATIVE STATE of the protein - the native state is usually (but not always) the most stable (lowest energy) state of the folded protein
    • - disruption of the native structure (by BREAKING the weak bonds responsible for 2 ° and 3° structures) is called denaturation
    • - the result is a protein in a non- native or denatured state
    • Denaturation can be caused by:
      • - raising (or more rarely lowering) the temperature
      • - extremes of pH
      • chaotropes (such as 8M urea or guanidine
      • hydrochloride)
      • - detergents etc.
    Most denatured proteins precipitate
  • 2. Denatured proteins will spontaneously refold in vitro (in the test tube) e.g. folding of RNAse A denaturation renaturation Incubate protein in guanidine hydrochloride (GuHCl) or urea 100-fold dilution of protein into physiological buffer - the amino acid sequence of a polypeptide is sufficient to specify its three-dimensional conformation Thus: “ protein folding is a spontaneous process that does not require the assistance of extraneous factors ” Anfinsen, CB (1973) Principles that govern the folding of protein chains. Science 181 , 223-230.
  • 3. Protein Folding: the Levinthal paradox folding denatured protein: random coil- a very large number of possible extended conformations native protein 1 stable conformation in vitro in vivo folding t = seconds t = seconds or less
  • 4. Levinthal paradox: a new folding view is needed Consider a protein with 100 amino acids - using a very simplified model where there are only 3 possible orientations per residue - assume 1 conformation can form every 10 -13 seconds (100 picoseconds) - then 5 x 10 47 x 10 -13 s = 1.6 x 10 27 years to correctly fold a protein Obviously NOT ALL conformations can be ‘sampled’ during folding!
      • 3 100 different conformations = 5 x 10 47 !!!
  • 5. Resolving the paradox a. there are a limited number of secondary structural elements b. these elements tend to form spontaneously during the co-translational folding of a protein c. proteins fold via so-called “folding landscapes”, where the proteins follow “pathways” of folding that lead to the correct three-dimensional structure d. folding intermediates may be important in such folding landscapes/pathways
  • 6. Protein folding theory • folding can be thought to occur along “ energy surfaces or landscapes” • limited number of secondary structure elements: helices, sheets and turns Dobson, CM (2001) Phil Trans R Soc Lond 356, 133-145
  • 7. A simplified view: A folding funnel unfolded (non-native) states (two of many different conformations are shown) Native state (N) Energy landscape - descent towards Free energy minimum state - A- rapid folding B- secondary energy minima
  • 8. Usually, ∆G for folding is negative i.e. favourable; but this is due to a balance of several thermodynamic factors: - Conformational entropy : works against folding (contributes positively to ∆ G), since unfolded condition = random cycling between many possible states, it involves higher entropy than the single folded state. - Enthalpy contribution : works in favour of folding (contributes negatively to  G) i.e. reduction in Enthalpy due to formation of energetically favourable interactions (e.g. salt bridges, H bonds, van der Waals etc.) between chemical groups in folded state. - Entropy contribution from hydrophobic effect : works in favour of folding (contributes negatively to  G) i.e. the burying of hydrophobic R groups in protein increases entropy of whole system (protein + water). ∆ G = ∆ H - T ∆ S overall free energy change for folding Thermodynamics of protein folding balance = -ve ∆ G
  • 9. Proteins fold in stages
    • Local folding through nucleation of small clusters of residues
    • A General Order of Folding
      • Short regions rapidly form small stable secondary elements; like alpha-helices and beta-sheets etc.
      • These small structure elements interact with other local elements and interact folding into ‘ globular’ units on an intermediate time scale. Associations are through various weak chemical interactions.
      • These globular domains may be a complete small protein or a number of these in larger proteins can interact more slowly to fold into the final folded structure.
    polypeptide folded protein secondary structures domains
  • 10. Co-translational protein folding folding assembly - initially, the first ~30 amino acids of the polypeptide chain present within the ribosome are constrained and cannot fold until they exit from the ribosome (the amino, or N-terminus emerges first, and the C-terminus emerges last). - as soon as the first part of the nascent chain is extruded, it will start to fold co-translationally (i.e., acquire secondary structures, domains etc.); as the complete polypeptide is produced and extruded, it will fold in a similar fashion and then the final tertiary structure will be established, followed (in some cases) by assembly of subunits to form the quaternary structure. NH 3 + definition: co-translational is a process which occurs during the translation ( synthesis ) of a protein on the ribosome
  • 11. Molecular chaperones - the great majority of proteins can fold without assistance, in a co-translational manner - some proteins, which may have ‘difficulties’ reaching their native states, must be stabilized by molecular chaperones (or chaperonins) by assisted folding - these bind to nascent (emerging) polypeptides and stabilize them (usually by associating hydrophobic residues). - otherwise these hydrophobic residues tend to associate with other hydrophobic residues, leading to intra- or inter-molecular associations with other proteins that prevent proper folding - there are dozens of different types of molecular chaperones, and some accomplish functions different from helping protein folding - e.g. , some help protein assembly, some help to transport proteins to various parts of the cell, some help damaged proteins from refolding
  • 12. Most extensively-studied of all chaperonins: the GroEL-GroES complex of E . coli EL
  • 13. Protein misfolding can cause serious human diseases e.g. the prion-based, Creutzfeld-Jacob disease (CJD)-basic mechanism for many neurodegenerative diseases is similar the formation of protein aggregates that kill nerve cells. Prion diseases are self-infectious: misfolded version of the prion protein, PrP*, can induce the normal PrP protein to misfold into a more  strand-based structure, resulting in damaging aggregation via formation of cross-  filaments. these filaments are visualized cytologically as amyloid stacks (see pp362-363 of Alberts et al.)
  • 14. depiction of cross  -filament structure resulting from extensive stacking of misformed  sheets; this type of structure is resistant to proteases Model for conversion of PrP tp PrP*: shows the change of two  -helices into four  strands
  • 15. Protein quaternary structure
    • Association of multiple polypeptides into a functional unit
      • - many, although not all proteins engage in this
      • - individual proteins in the quaternary structure are called‘subunits’
    • Example: prefoldin (a so-called molecular chaperone
    • that assists the co-translational stabilization
    • of proteins during their folding in vivo (in the cell)
  • 16. Prefoldin quaternary structure - structure of prefoldin hexamer - oligomerization (assembly) domain is a double beta-barrel structure composed of beta-strands - coiled coils consist of two helices winding around each other two types of proteins (subunits) assemble into a hexamer (6 subunits)
  • 17. Symmetries of Protein Quaternary Structure Figure 6.30 Figure 6.32 there are also ways that units can associate into higher order structures without symmetry
  • 18. Protein modifications: requirements for activity - cleavage and covalent modifications of proteins (often after synthesis) but may also be co-translational
    • INSULIN
    • Synthesized as PRE PRO INSULIN
    • - 1 st cleavage removes signal sequence ( PRE )
    • - 2 nd and 3 rd cleavages remove joining ( PRO )
    • peptide sequences
      • - di-sulphide bonds hold the two peptides
      • together
    H 2 N- H 2 N- A zymogen is a catalytically inactive protein precursor that must be cleaved proteolytically to be activated disulfide bonds I. CLEAVAGE some proteins require sections of the polypeptide chain to be removed for correct maturation. H 2 N- H 2 N-
  • 19. Zymogens in action - the pancreatic proteases (such as trypsin, chymotrypsin, elastase , and carboxypeptidase )  covalent enzyme activation by proteolytic cleavage - synthesized in the pancreas in an inactive form because if they were active in the pancreas, they would digest the pancreatic tissue. Rather, they are made as slightly longer, catalytically inactive molecules called zymogens ( trypsinogen, chymotrypsinogen, proelastase , and procarboxypeptidase , respectively) Figure 11.39 * * * * * * represents active enzyme cleavage event
  • 20. Chymotrypsin activation Figure 11.40 Activation of Chymotrypsinogen (No Enzymatic Activity) Chymotrypsin – Serine Protease One of the most complex examples of proteolytic activation 1st cut is stabilized by S-S bond  chymotrypsin Series of modifications. Each triggering the next Final state  chymotrypsin (by Trypsin) (Ile)
  • 21. II. Covalent Modifications (p. 403-408)
    • Sometimes proteins are covalently modified after synthesis
    • These modifications can be:
      • Required to obtain the active conformation (e.g.. collagen)
      • Used to control the activity of a protein
        • (e.g. histones, signal transducing proteins, etc.)
    • Examples:
    • Collagen: Proline  Hydroxyproline (hydroxylation)
      • This requires Vitamin C ; No Vitamin C  No Hydroxyproline  Scurvy
      • Due to weakening of collagen fibres- hydroxylation of prolines
      • somehow stabilizes structure
    OH
  • 22. Prothrombin, histones Prothrombin: Glutamate  gamma- Carboxy Glutamate ( carboxylation ) This requires Vitamin K ; No vitamin K  No Blood Clotting Histones: Histones are proteins involved in the folding/compacting of nuclear DNA. They are often modified in regions of active transcription. Acetylation of Lysine is the MOST common- (decreases net positive charge of histones).
  • 23. Signal Transduction Proteins: Phosphorylation of Hydroxyls (-OH) - these proteins become transiently phosphorylated which either activates or inhibits their activity - phosphorylation can be on one of a 3 different amino acids - a particular protein will only have specific modification sites Serine  phosphoserine Threonine  phosphothreonine Tyrosine  phosphotyrosine kinases are the cellular proteins that phosphorylate these residues phosphatases are the cellular proteins that remove the phosphate groups Phosphorylation- an important kind of protein modification. together these modulate protein activity
  • 24. More protein modifications protein activity Asn, Ser, Thr Glycosylation various Lys C-terminus Tyr Lys, N-terminus N-terminus Cys Cys Tyr Pro, Lys, Asn, Asp Arg, Lys, His, Glu Ser, Thr, Tyr target site activation degradation/other bioactive peptides protein-protein intera’n gene expression membrane association membrane association signalling, oncogenesis protein-protein intera’n collagen structure prot. repair, chemotaxis signalling, activation cellular process Ubiquitylation Truncation Amidation Sulfation Acetylation Myristoylation Palmitoylation Prenylation Sulfation Hydroxylation Methylation Phosphorylation modification