Transcript Protein folding

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Proteins: structure, translation, etc.
Structure of proteins
- amino acids, peptide bond, primary-quaternary structures, disulfide bond
Protein synthesis
-protein translation, co-translational folding, stalling, etc.
Protein folding and unfolding
- Levinthal paradox, acquisition of native structure, loss of structure
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Amino acid structures
methionine (M)
isoleucine (I)
phenylalanine (F)
valine (V)
tyrosine (Y)
leucine (L)
tryptophan (W)
aspartic
acid (D)
glutamic
acid (E)
aspargine (N)
glutamine (Q)
serine (S)
lysine (K)
arginine (R)
histidine (H)
glycine (G)
alanine (A)
+
+
3
2
threonine (T)
cysteine
proline
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Amino acid relationships
hydrophobic
MILV
FYW
C
P
Suggested amino acid substitutions
Solvent exposed
(SEA>30 Å2 ,
)
Interior
(SEA<10 Å2,
)
SEA, solvent
exposed area
small neutral
G(A*)ST
hydrophilic
EDNQ
KRH
*A is also
fairly hydrophobic
aromatic
Amino acids connected by a line can be substituted with
95% confidence
Adapted from D. Bordo and P. Argos (1991) J. Mol. Biol. 217, 721-729.
Peptide bond formation
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©Alberts et al. (1998)
 You should know the structure of a polypeptide chain (protein)!
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The peptide bond
Ri+1
Ri
R=side chain
O=C-N-H is planar
(double-bond character)
Phi (Φ) and Psi (ψ) angles can vary;
their rotation allows polypeptides
to adopt their various structures
(alpha-helices, beta-sheets, etc.)
cis conformation is rare except for proline
potential for steric hindrance
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Protein structure: overview
Structural element
Description
primary structure
amino acid sequence of protein
secondary structure
helices, sheets, turns/loops
super-secondary structure
association of secondary structures
domain
self-contained structural unit
tertiary structure
folded structure of whole protein
• includes disulfide bonds
quaternary structure
assembled complex (oligomer)
• homo-oligomeric (1 protein type)
• hetero-oligomeric (>1 type)
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Protein structure: helices
alpha
3.10
pi
- alpha helices are about
10 residues on average
- side chains are well
staggered, preventing
steric hindrance
- helices can form
bundles, coiled coils, etc.
H-bonding
amino acids
per turn:
3.6
frequency
~97% ~3%
3.0
4.4
rare
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Protein structure: sheets
parallel
‘twisted’
anti-parallel
- the basic unit of a
beta-sheet is called a
beta-strand
- unlike alpha-helix, sheets
can be formed from
discontinuous regions of a
polypeptide chain
- beta-sheets can form
various higher-level
structures, such as a
beta-barrel
Green
Fluorescent
Protein
(GFP)
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Protein structure: sheets (detail)
- notice the difference
in H-bonding pattern
between parallel and
anti-parallel beta-sheets
- also notice orientation
of side chains relative
to the sheets
‘twisted’
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Protein structure: turns/loops
alpha-helix
beta-sheet
- there are various types of
turns, differing in the
number of residues and
H-bonding pattern
- loops are typically longer;
they are often called coils
and do not have a
‘regular’,
or repeating, structure
ribonuclease A
loop
(usually exposed on surface)
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Ramachandran plot
Psi (ψ)
no steric
clashes
Phi (Φ)
- Phi (Φ) and Psi (ψ) rotate,
allowing the polypeptide to assume
its various conformations
permitted
if atoms are
more closely
spaced
- some conformations of the
polypeptide backbone result in
steric hindrance and are disallowed
- glycine has no side chain and is
therefore conformationally highly
flexible (it is often found in turns)
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Types of non-covalent interactions
interaction
nature
ionic
electrostatic
(salt bridge)
bond
length
“bond”
strength
1.8-4.0 Å 1-6
(3.0-10 Å kcal/mol
for like
charges)
example
positive: K, R, H,
N-terminus
negative: D, E,
C-terminus
hydrophobic entropy
-
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hydrophobic side chains
(M,I,L,V,F,W,Y,A,C,P)
H-bond
H-bonding
2.6-3.5
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H donor, O acceptor
van der
Waals
attraction/
repulsion
2.8-4.0
<1
closely-spaced atoms;
if too close, repulsion
aromaticaromatic
p-p
4.5-7.0
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F,W,Y (stacked)
2.9-3.6
2.7-4.9
N-H donor to F,W,Y
aromaticH-bonding
amino group
these all contribute to some extent to protein structure & stability;
- important to understand extremophilic (or any other) proteins
Protein-solvent interactions
hydrophilic amino acids (D, E, K, R, H, N, Q)
- these amino acids tend to interact extensively with solvent in
context of the folded protein; the interaction is mostly ionic and Hbonding
- there are instances of hydrophilic residues being buried in the
interior of the protein; often, pairs of these residues form salt
bridges
hydrophobic amino acids (M, I, L, V, F, W, Y, A*, C, P)
- these tend to form the ‘core’ of the protein, i.e., are buried
within the folded protein; some hydrophobic residues can be
entirely (or partially) exposed
small neutral amino acids (G, A*, S, T)
- less preference for being solvent-exposed or not
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The disulfide bond
protein
+
protein
oxidation
protein
protein
+ 2 H+ +
2 e-
reduction
• disulfide bond formation is a covalent modification; the
oxidation reaction can either be intramolecular (within the same
protein) or inter-molecular (within different proteins, e.g.,
antibody light and heavy chains). The reaction is reversible.
- most disulfide-bonded proteins are extracellular
(e.g. lysozyme contains four disulfide bonds);
the conditions inside the cytosol are reducing,
meaning that the cysteines are usually in reduced form
- cellular enzymes (protein disulfide isomerases) assist
many proteins in forming proper disulfide bond(s)
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Protein folding
“arguably the single most important process in biology”
in the test tube
~40 years
versus
in the cell
~20 years
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Folding of RNAse A in the test tube
denaturation
renaturation
Incubate protein
100-fold
in guanidine
dilution of protein
hydrochloride
into physiological
(GuHCl)
buffer
(aggregation)
or urea
- 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.
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Levinthal paradox
in vitro
in vivo
denatured
protein:
random coil
106 possible
conformations
folding
folding
Native protein
1 stable
conformation
t = seconds or much less
t = seconds
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Protein folding theory
• limited number of
secondary structure
elements: helices,
sheets and turns
• folding can be thought
to occur along
“energy surfaces or
landscapes”
Dobson, CM (2001)
Phil Trans R Soc Lond
356, 133-145
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Folding of lysozyme
hydrophobic collapse
- upon dilution of unfolded
protein in buffer, the protein
will ‘collapse’ onto itself,
trying to bury as many
hydrophobic surfaces as
possible
- in doing so, the protein
may fold properly, or:
- misfold and aggregate
- go through a ‘trapped
intermediate’ stage
• hen lysozyme has 129 residues,
consists of 2 domains (α and β)
Protein synthesis: the ribosome
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Yusupov et al. (2001) Science 292, 883.
- whole 70S ribosome from Thermus
thermophilus at 5.5Å
- small (30S) subunit: 16S RNA, ~20
proteins
- large (50S) subunit: 23S RNA, 5S RNA,
>30 proteins
- high concentration in the cell (~ 50 μM)
Protein synthesis cycle
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interface view of 50S subunit
E-, P-, A-site
tRNAs and mRNA
1. acylation of tRNAs with respective amino acids
2. binding of tRNA charged with methionine to P-site
on the AUG start codon (present on the mRNA)
3. next tRNA charged with appropriate amino acid
binds A-site
4. transpeptidation (peptide bond formation) between
P-site (N-terminal) amino acid and A-site amino acid
leads to the growth of the polypeptide chain. The
catalysis is by the peptidyltransferase, which consists
only of RNA. The ribosome is thus a ribozyme.
5. the E-site represents the ‘exit’ site for the
uncharged tRNA
6. release from tRNA and disassembly then occurs
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Elongation of the polypeptide chain
- PT = peptidyltransferase site
- rRNAs are in grey
- proteins are in green
- polypeptide chain model is
shown to traverse the
ribosome channel from the PT
site to the polypeptide exit site
adapted from Selmer et al. (1999) Science 286: 2349-2352
- the channel/tunnel and exit site are quite narrow, meaning that
there is likely to be little if any co-translational protein folding
in the channel
- possibility of an alpha-helix forming? (“yes”)
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Co-translational protein folding
Fact:
- first ~30 amino acids of the polypeptide chain
present within the ribosome is constrained
(the N-terminus emerges first)
folding
assembly
Assumption:
as soon as the nascent chain is extruded, it will start
to fold co-translationally (i.e., acquire secondary
structures, super-secondary structures, domains)
until the complete polypeptide is produced and
extruded
Observing co-translational folding
Experiment:
1. translate firefly luciferase RNA in vitro in the
presence of 35S-methionine for 2 min
2. Prevent re-initiation of translation with
aurintricarboxylic acid (ATCA): ‘synchronizing’
3. at set timepoints, quench translation, incubate with
proteinase K (digests unstructured/non-compact
regions in proteins, but not folded domains/proteins)
4. add denaturing (SDS) buffer, then perform SDSPAGE (polyacrylamide gel electrophoresis)
5. dry gel, observe by autoradiography
Firefly
Luciferase
(62 kDa)
Result:
2 3
4
5
6
7
8
10
12 min
60 kDa
40 kDa
20 kDa
no
ProK
N-terminal
domain
(~22 kDa)
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C-terminal
domain
(~40 kDa)
2
with
ProK
3
4
5
6
7
8
10
12 min
60 kDa
40 kDa
20 kDa
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Antibiotics & protein synthesis
antibiotics can be useful tools for manipulating translation, folding
antibiotic
effect
cyclohexamide
inhibits the eukaryotic peptidyltransferase;
prevents release of the polypeptide chain. Can
be used to isolate ribosome-nascent chain
complexes
chloramphenicol
inhibits the prokaryotic peptidyltransferase
puromycin
causes premature chain termination and release
from ribosome. Puromycin is similar to a
tyrosyl-tRNA and acts as a substrate during
elongation. Once added to the carboxyl end of
the nascent chain, protein synthesis is aborted
tetracycline
inhibits aminoacyl tRNA binding to the A-site
kanamycin
causes misreading of the mRNA
streptomycin
causes misreading of the mRNA
ssrA RNA in bacteria
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Problem:
- turnover (degradation) of mRNA occurs
very quickly in bacteria, and the 3’ end of
the mRNA has a higher probability of being
degraded first
- if the stop codon is removed, there are no
signals for mRNA release from the
ribosome, and the mRNA will stall
Solution:
- SsrA, or 10SA RNA is a small RNA (363 nt)
that resembles a tRNA and can be charged
with alanine. It is placed into the
peptidyltransferase site by the protein SsrB
- SsrA can be used as a template, and codes
a peptide, ANDENYALAA
- the fusion protein containing this sequence
is recognized and degraded by the ClpAP or
ClpPX proteases
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Nascent chain stalling in eukaryotes
- can make proteins that are of a defined length by translating
an RNA that is truncated at the 3’ end (i.e., has no stop codon)
Steps:
1. linearize a vector encoding a gene of interest using a restriction
enzyme, such that the cut is precisely where you want the
polypeptide to end (before the stop codon)
2. make RNA using nucleotides and polymerase enzyme
3. add to an in vitro translation system (rabbit reticulocyte lysate),
which has all of the required components to translate the RNA
4. if the RNA is not truncated, the full-length protein will be made
and released; if the RNA is truncated, it will remain bound to the
ribosome
Note: the protein can be labeled this way with
co-translational folding still takes place
35S-methionine;
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Chain stalling: in practice
Fact: only full-length firefly luciferase is functional
Goal: show that firefly luciferase can adopt a folded, functional
conformation co-translationally
Experiment:
1. prepare DNA construct that encodes firefly luciferase and an extra 35
amino acids at its C-terminus
2. digest construct such that the last 2 amino acids and the stop codon are
removed
3. prepare RNA using polymerase and nucleotides
4. in vitro translate the RNA in rabbit reticulocyte lysate
5. assay for firefly luciferase activity (light emission at 560 nm occurs when
luciferin substrate is oxidatively decarboxylated)
Problem? Hint: does this experiment show physiological relevance?
Protein folding:
in 3 different environments
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• ex vivo refolding rabbit reticulocyte lysate
- rabbit reticulocyte lysate is an abundant source of molecular
chaperones, many of which are ATP-dependent
• in vitro folding environments
- protein folding (from denaturant), when possible, requires the
proper environment:
proper pH, salts, concentration of protein, temperature,
stabilizing agents (e.g., other proteins, glycerol, etc.)
• in vivo folding
- molecular chaperones, protein folding catalysts, proper redox
environment, availability of binding partners
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Following the acquisition
of (native) structure
denaturation
• regain of 2º, 3º and 4º structures
- by circular dichroism and
fluorescence measurements
- by other criteria (e.g., native gel
electrophoresis, SEC,
protease sensitivity assays, etc.)
• regain of activity
- activity not necessarily enzymatic
renaturation
native
structure?
refolding
unfolding
Circular
dichroism
Acquisition of native structure:
examples
• actin
- chemically denatured actin can be refolded by incubating it in
rabbit reticulocyte lysate; native gel electrophoresis, and
binding to DNAse I is used to assess folding
• various small proteins (RNAse A, lysozyme, etc.)
- can be denatured chemically and refolded simply by dilution
of the denaturing agent; activity assays are available, but
folding can be monitored using spectroscopic techniques
• other
- small-angle light x-ray scattering (SAXS), NMR are some
other techniques used to monitor protein folding
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Protein denaturants
• high temperatures
- cause protein unfolding, aggregation
• low temperatures
- some proteins are sensitive to cold denaturation
• heavy metals (e.g., lead, cadmium, etc.)
- highly toxic; efficiently induce the ‘stress response’
• proteotoxic agents (e.g., alcohols, cross-linking agents, etc.)
• oxygen radicals, ionizing radiation
- cause permanent protein damage
• chaotropes (urea, guanidine hydrochloride, etc.)
- highly potent at denaturing proteins;
often used in protein folding studies
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Following the loss of structure
• loss of secondary structure
- the far-UV circular dichroism spectrum of a protein changes
at the so-called ‘melting temperature’ or Tm
- fluorescence characteristics will likely also change
• loss of tertiary structure
- the far- and near-UV circular dichroism spectra of a protein
change, but the Tm of both spectra may be different
- fluorescence characteristics will likely also change
• loss of activity
- the activity of a protein can be monitored over time
• aggregation
- can measure light scattering (e.g., at 320 nm) spectrophotometrically, or by detecting the protein in a precipitate
Loss of structure: example
intermediate
Urea (M)
0 0
1 2
chymotrypsin no Yes Yes Yes
folded
unfolded
Far-UV
spectrum
native
unfolded
Fluorescence
spectrum
2M
urea
Bacterial luciferase (α subunit)
Noland et al. (1999) Biochemistry 38, 16136.
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