Protein metabolism

1
Protein metabolism
2
Birth of a protein
Predicted genes or genes of unknown function are
typically called open reading
frames (ORFs)
3
Complexity of protein synthesis
4
Aminoacyl-tRNA synthetases charge tRNAs with
amino acids

• Specificity is shown
• Here ?
• Note two non-standard
• amino acids have been
• shown to be incorporated
• into proteins in this way
• selenocysteine, and
• pyrrolysine

5
Initiation requires several auxiliary factors

• Bacterial translation is regulated
• by specific incorporation of fMet
• Note three sites for tRNA binding
• two encompass both subunits

6
Structural analysis reveals RNA binding sites on
50S subunit
7
Structures have been solved for both 50S and 30S
ribosomal subunits
8
(No Transcript)
9
RNA as a catalyst in peptide bond formation
10
Translation elongation
11
The ribosome is a molecular machine
12
Ribosomes are the site for various antibiotic
action
13
A Proteins Adolescence
14
Fate of proteins in cells
Prokaryote vs. Eukaryote
15
Additional codes are intrinsic to a
proteins primary sequence
Acquire secondary structure either coming off
ribosome (a), or by interactions with chaperones
(b), however,some proteins fold into tertiary
structures autonomously Post-translational
processing and modifications Insulin and
proteases Inteins Glycosylation, etc.
16
Protein targeting utilizes signal peptides
Various protein activities are regulated by
processing to yield a mature enzyme
17
The PCC functions in protein translocation
18
Introns versus Inteins
Intron Intein
DNA DNA RNA RNA RNA
Protein Protein Protein
Splicing event
19
Formation of disulfide bonds addition of
prosthetic groups
20
Glycosylation provides a ligand and targeting to
proteins
21
Buttering proteins
22
Some amino acids are modified post-translationally
23
Some nascent polypeptides can fold spontaneously
24
while others need help
25
DnaKJ and GrpE are chaperones
26
GroESL is a chaperonin
27
Other important enzymes in protein folding

• Protein disulfide isomerase (PDI) assists
  protein in forming proper disulfide bridges
• Peptide prolyl cis-trans isomerase (PPI)
  interconverts cis-trans isomers of proline

28
The state of proteins are monitored by the cell
20 of new polypeptides are immediately degraded
because of abnormalities, incomplete assembly
others inactivated by heat or chemical stress,
while some are unstable
Quality control for proteins
Signal for quality control is surface-exposed
hydrophobic regions
29
Why do some proteins need help folding, while
others do not?

• What are the molecular mechanisms of protein
  folding?
• How important is protein folding?
• Why should I care?

30
Two-state folding of small proteins

• Modeled after Anfinsen experiments
• A large number of proteins fewer than 150 amino
  acids can efficiently refold upon dilution
• During refolding, only observe two distinct
  states folded and unfolded on a time scale of
  seconds or less

31
A non-random process

• The unfolded protein undergoes specific
  kinetically preferred steps on way to the native
  state

32
Long-range interactions lead to non-randomness

• In non-native proteins residual structure appears
  as hydrophobic clusters, in which tryptophan or
  histidine residues are surrounded by hydrophobic
  residues
• Do these act as nucleation sites for protein
  folding?

33
Lysozyme folding
34
H-exchange and lysozyme folding

• Refolding experiments indicated both domains
  achieve native structure in folding intermediates
  prior to tertiary interactions spanning the two
  domains
• Amide hydrogens become protected in the alpha
  domain much faster than beta

35
Hydrogen exchange measures solvent accessibility
of amide hydrogens
36
Trp-63 is an exception

• The amide hydrogen of Trp-63 becomes protected as
  rapidly as the alpha domain residues, despite
  its location in the beta domain
• This result suggests Trp-63 may be involved in
  alpha domain folding

37
Other Trps are important too

• Replacement of Trp-62 and Trp-108 with Tyr lead
  to increased rate of refolding
• Chemical modification of Trp-62 leads to
  increased misfolding
• Trp-62 is also important for correct formation of
  disulfide binds in peptide fragments

38
Folding interactions

• Native state Trp62 is solvent accessible and side
  chain disordered in crystal structure
• Denatured state, this Trp and others are
  inaccessible
• Trp62 and 63 (in a non-native b domain state)
  associate with native hydrophobic cluster in a
  (Trp108-111)
• These non-native interactions stabilize a native
  core (W62G destabilizes core and causes
  misfolding)

39
Take home

• Although a residue such as tryptophan may be
  exposed in the native state for functional
  reasons, it could be buried in early stages of
  folding to reduce the tendency of transiently
  populated species to aggregate
• Protein sequence codes for structural
  characteristics other than those of the native
  fold!

40
Larger proteins have a more difficult time
becoming native
41
Kinetic barriers

• Folding intermediates can become trapped in
  energy minima
• Some may be necessary intermediates, which can
  accumulate to significant populations
• This may lead to aggregation

42
Tryptophanase folding

• 8 M urea ? Dilution ? aggregates and native
  protein
• 3 M urea ? Dilution ? only aggregates
• Suggests a folding intermediate whose population
  is favored under this urea
• Occurs in the presence of other denatured
  proteins generating a homogenous population

43
A competition

• Kinetics of aggregate formation indicates a
  competition between unimolecular, intrachain
  reaction and multimolecular, interchain reaction

44
Competition evident in domain swapping between
monomers

• Three domains in diptheria toxin
• In dimer, one domain loses contact with other two
  (rotates 180 degrees and translates 65 angstroms)
  and forms similar contacts with other chain.
• Observed in several proteins

45
(No Transcript)
46
Chaperonin function

• Open rings of chaperonin provide a hydrophobic
  binding surface to bind hydrophobic clusters of
  non-native structures (compete with multimer
  formation, no access in cavity)
• GroES and ATP binding cause conformational
  changes in GroEL ? leading to alternation of
  surface from hydrophobic to hydrophilic
  (encourages burial of hydrophobic surfaces of
  folding protein)
• Oligomeric structures form in bulk solution

47
GroESL cycle
48
Why should I care?

• Amyloid Protein deposit in b-pleated sheets
• Associated with numerous disease states
• Alzheimers ? Ab peptides
• Creutzfeldt-Jacob ? Prion
• Elderly cardiac ? Transthyretin

49
The amyloid state

• Various proteins share NO primary sequence
  identity
• Amyloid state must be accessible to any protein
  as a very stable alternative state (may be more
  thermodynamically stable than native)
• Expect sequences that readily form amyloids to be
  selected against (very few form in vivo)

50
Amyloid formation

• Often results from destabilization of already
  folded native protein
• Is structural disruption thermodynamic or
  kinetic?
• Thermodynamic ? free energy difference between
  native and monomeric intermediate leading to
  amyloid formation
• Kinetic ? refers to free energy barrier between
  native state and transition state for amyloid,
  reflected in rate constant for conversion

51
Mutations affect kinetics and thermodynamics

• Mutations increase free energy of native state or
  decrease free energy of amyloid monomer
• Mutations affect kinetics by increasing free
  energy of transition state or decreasing the
  transition state
• Both can contribute

52
Variant proteins vs. wild type lysozyme

• Crystal structures look the same, although subtle
  changes indicated destabilization in domain
  interface
• Variants more sensitive to temperature in vitro
• Deuterium exchange show all hydrogens
  exchangeable in variant, while 55 are
  inaccessible in wild type

53
(No Transcript)
54
Polymerization of amyloid intermediates

• Like microtubule assembly, nucleation is a
  critical step
• Indicates that a critical concentration of
  monomers is needed for amyloids to form
• Prion diseases can be seeded
• Nucleation behavior might account for long term
  development of amyloidic disease

55
Clinical treatments

• Suggestions??