Transcript Co-translational Folding

Cotranslational Protein Folding
National Seminar on Bioinformatics and Functional Genomics
February 15-17, 2006
madhav kulkarni
The challenges of ‘Self Assembly’
• Challenges in general
– living organisms put themselves together, all by themselves.
– getting into the right shape can't happen just by chance. So where are
the directions? And how do living things follow them?
• Questions that echo through all of biology:
–
–
–
–
transformation of embryo to infant
complexity of organs
design of a single cell
the building materials of life -- the proteins
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Proteins
•
Building blocks of life.
•
Assembled in biological organisms to form cell structures, enzymes
and other chemicals necessary for life.
•
Each amino acid (20) has unique properties including size, 3D
shape and polarity
Institut fur Cheme. (1998) Amino Acid Dictionary. Amino Acid Dictionary
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Protein Biosynthesis
•
Protein Biosynthesis
– Transcription
– Translation
– Events following Protein
Biosynthesis
DNA
Gene
mRNA
Ribosome & tRNA
Protein
http://www.stanford.edu/group/pandegroup/folding/education/protfold.html
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Translation
•
•
The message of mRNA is decoded to make proteins.
Initiation and elongation
– the ribosome recognizes the starting codon on the mRNA strand
and binds to it.
– tRNA, has an anticodon that matched with the codon on the
mRNA. tRNA also has a single unit of amino acid attached to it.
– As the ribosome travels down the mRNA one codon at a time,
another tRNA is attached to the mRNA at one of the ribosome
site.
– The first tRNA is released, but the amino acid that is attached to
the first tRNA is now moved to the second tRNA, and binds to its
amino acid. This translocation continues on, and a long chain of
amino acid (protein), is formed.
•
As the entire unit reaches the end codon on the mRNA, it falls apart
and a newly formed protein is released.
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Events followed by Biosynthesis
•
Protein folding (complete)
– protein takes its functional shape or conformation
– There are hydrophilic and hydrophobic amino acids in protein, wherein
the main driving force for folding is from the hydrophobic portions of the
protein chain to fold away from the outside water environment (typical of
globular protein)
– Folding process creates cavity containing amino acids which can make
non-covalent bonds (hydrogen bond and/or ionic interactions) only with
certain ligands
•
Post-translational modifications
– formation of disulfide bridges and attachment of any of a number of
biochemical functional groups, such as acetate, phosphate, various
lipids and carbohydrates.
– Removal of one or more amino acids from the amino end of the
polypeptide chain, or cutting the polypeptide in the middle of the chain
– two or more polypeptide chains that are synthesized separately may
associate to become subunits of a protein with quaternary structure
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Protein Folding – The Problem
•
•
The mechanism by which it happens
The short time span in which it happens
– How an amino acid sequence folds into unique 3-D shape?
– How can native conformation be found and recognized?
– The entire duration of the folding process varies dramatically
depending on the protein of interest
– Slowest folding proteins - many minutes or hours to fold
– Small proteins, with lengths of a hundred or so amino acids,
typically fold on time scales of milliseconds
– Very fastest known protein folding reactions are complete
within a few microseconds
– Possible intermediates have a very short lifetime
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Objective of protein folding studies
•
To learn the details of the pathways involved (in misfolding)
– The kinetics of the folding process, the partitioning of
polypeptides among alternative forms, and the yield of
correctly folded protein are consequences of kinetic partitioning
between alternative pathways.
– When proteins do not fold correctly (i.e. "misfold"), there can be
serious consequences, including many well known diseases,
such as Alzheimer's, Mad Cow (BSE), CJD, ALS, Huntington's,
Parkinson's disease, and many Cancers and cancer-related
syndromes.
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Implications
•
Would greatly enhance the ability to utilize the enormous amount of
data being generated by genome sequencing project.
•
No/less need to rely on resource-intensive experimental methods for
determining protein structures but could determine them
computationally.
•
Drug discovery could be accelerated, saving significant resources.
•
Genetic engineering experiments to improve the function of
particular proteins would be possible.
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Current Studies
Protein folding
Folding process
Theoretical
Practical
Final Fold
Theoretical
Practical
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Theoretical approach – Folding Process
•
Leading strategy - To find an amino acid chain's state of
minimum energy
– The shape that yields the lowest energy state must be a
protein's natural shape, or, as chemists call it, its "native
conformation." (http://wsrv.clas.virginia.edu/~rjh9u/protfold.html)
•
Calculation of protein energy landscapes.
•
Folding funnel
– proposed that natural proteins have evolved such that this
complicated energy surface has a funneled shape which leads
towards the native state, which is the lowest-energy
conformation available to the protein.
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Practical Approach – Folding Process
•
•
•
Studying folding process by techniques like
– Renaturation
– Permuted version of a protein/mutant protein
– Incorporation of probes (fluorescent) in the protein
Analytical methods
– photochemical methods
– laser temperature jump spectroscopy
– SDS/page with conformation-dependent antigenicity
– ultrafast mixing of solutions
Denaturation and renaturation to study the unfolding and/or
refolding process (kinetics and pathway)
– Many proteins refolded from a fully denatured state to the native
biologically active structure.
– The same principle have been assumed to govern the folding of
protein during biosynthesis.
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Theoretical approach – Final Fold
Bioinformatics (Protein Structure Prediction)
– prediction of native structure from amino-acid sequences alone
•
Comparative modeling algorithms (homology)
– To build a model based on a previously determined structure of
related sequence
•
Threading algorithms
– To identify proteins that are structurally similar to one another,
although sequence similarity is negligible
•
Ab initio folding algorithms
– To fold the proteins according to basic structural template.
•
The native fold can often be predicted on the basis of homology or
threading.
•
Only around 2000 distinct protein folds in nature!
(Fetrow J.S. et. al., Current Pharmaceutical Biotechnology, 3, 329-347 (2002)
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Practical Approach – Final Fold
•
•
X-ray crystallography and
NMR
– determination of the folded
structure of a protein
• lengthy and complicated
process
Source: www.rcsb.org updated: 14 February 2006
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Amino acid sequence to 3D structure
The primary sequence of a protein contains all information needed for a
protein to attain active conformation
Anfinsen, C. B., et al., Proc. Natl. Acad. Sci. USA, 47, 1309-1314 (1961) in Fetrow J.S. et. al., Current
Pharmaceutical Biotechnology, 3, 329-347 (2002)
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Levinthal Paradox
•
It would be impossible for a protein to fold at observed rates by
randomly searching all possible conformations of the polypeptide chain.
(Levinthal, C., Chem. Phys., 65, 44-45 (1968) in Fetrow J.S. et. al., Current Pharmaceutical Biotechnology, 3,
329-347 (2002))
•
•
Three possible conformations: a, b, and L (Ramachandran Plot)
If each residue of a 100 residue polypeptide had only three
conformations, the total number of conformations would be 3100 = 5 x
1047. Since conformational changes occur on the timescale of 10-13
seconds, the time required by the 100 residue protein to search all
conformations would be 5x1047x10-13 » 1037 years. Nevertheless,
proteins are observed to fold in 10-1 - 103 seconds both in-vivo and invitro
•
Thus, proteins might be going through a sequence of progressively more
structured intermediate states that limit the conformational search and
direct the polypeptide chain along a preferred route toward the native
conformation.
(Roder H. and Colon W., Current Opinion in Structural Biology, 7, 15-28 (1997))
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Protein Folding
•
Is it really spontaneous? How spontaneous?
•
Does it happen only after the polypeptide chain is completely
synthesized?
•
Does it overlap with the translation process (Cotranslational
protein)?
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Cotranslational folding
•
Does it occur?
– If yes, is the folding pathway the same as when starting from an
unfolded protein?
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Cotranslational folding
Cartoon depiction of Cotranslational
folding of a polypeptide.
Schematic representation of a
section through a protein folding
landscape in which the basic funnel
concept for refolding polypeptides
has been adapted to include
the processes of Cotranslational
folding.
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Difficulties in study of Cotranslational folding
•
Low concentration of nascent polypeptide
•
Heterogeneity of the translation mixture
(Fedorov A.N., et. al., Journal of Molecular Biology, 228, 2, 351-358 (1992))
•
Aggregation of the intermediates through exposed hydrophobic
groups
•
Formation of incorrect disulfide bonds
•
Isomerization of proline residues
(Branden C. and Tooze J., in Introduction to Protein Structure 2 nd edition, pg. 91, 1999))
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Cotranslational folding
•
The process of protein folding is concomitant with synthesis was
articulated, and experimental testing was begun in the early 1960s
(Kiho, Y., and Rich, A. (1964) J. Mol. Biol. 51, 111–118)
•
Today there is substantial experimental support for the
Cotranslational folding hypothesis.
(Fedorov A.N. and Baldwin T.O., JMB, 294, 579-586 (1999)
(Fedorov A. N. and Baldwin T. O., The Journal of Biological chemistry, 272, 52, 32715-32718 (1997)
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Cotranslational folding
Evidences
•
Escherichia coli tryptophan synthase b chains begin to fold during
translation, even before appearance of the entire N-terminal domain
showing conformation dependent antigenicity (Fedorov, A. N., Friguet, B.,
Djavadi-Ohaniance, L., Alakhov, Yu. B., and Goldberg, M. E. (1992) J. Mol. Biol. 228, 351–358;
Friguet, B., Fedorov, A. N., Serganov, A., Navon, A., and Goldberg, M. E.(1993) Anal. Biochem. 210,
344–350)
•
No lag was detected between synthesis of the nascent chains and
appearance of immunoreactivity
(Tokatlidis, K., Friguet, B., Deville-Bonne, D., Baleux, F., Fedorov, A. N., Navon, A., Djavadi-haniance,
L. & Goldberg, M. E. (1995) Philos. Trans. R. Soc. Lond. B Biol. Sci. 348, 89–95 in Fedorov A. N. and
Baldwin T. O., The Journal of Biological chemistry, 272, 52, 32715-32718 (1997))
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Cotranslational folding
Evidences
•
Ribosome-bound bovine rhodanese form protease-resistant Nterminal domains.
(Reid, B. G., and Flynn, G. C. (1996) J. Biol. Chem. 271, 7212–7217)
•
Enzymatically active forms of rhodanese and firefly luciferase still
bound to the ribosomes when these polypeptides are expressed with
extended C-terminal segments so that each enzyme was in the bulk
solution.
(Kudlicki, W., Chirgwin, J., Kramer, G., and Hardesty, B. (1995) Biochemistry 34, 14284–14287 23;
Makeyev, E. V., Kolb, V. A., and Spirin, A. S. (1996) FEBS Lett. 378, 166–170)
•
Important to note that the full length Luciferase is virtually inactive
in the ribosome bound sate, although acquisition of the activity
occurs immediately upon release from the ribosome.
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Cotranslational folding
Evidences
•
Rat serum albumin - a secretory protein with 17 disulfide bonds in
the native structure - spread throughout the polypeptide chain.
– In the nascent polypeptides, about one half of the cysteinyl
residues exist in disulfide bonds, indicating completion of a
substantial part of the overall folding process
(Peters, T., and Davidson, L. K. (1982) J. Biol. Chem. 257, 8847–8853)
(Fedorov A. N. and Baldwin T. O., The Journal of Biological chemistry, 272, 52, 32715-32718 (1997)
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Cotranslational folding
Role of Molecular Chaperones and Folding Catalysts
• Involved in proper folding and assembly as well as preventing
premature folding.
• While being elongated, nascent polypeptide portions reaching the
funnel opening interact with ribosome-associated chaperons
assisting the folding process. (Baram D, and Yonath A., FEBS Letters 579, 948-954
(2005))
•
SecB can bind nascent polypeptides of E. coli secretory proteins,
apparently preventing premature folding in the cytoplasm.
(Randall, L. L., et. al. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 802–807)
•
•
Protein disulfide isomerase (PDI)1 - affects folding of disulfidecontaining proteins, both in vivo and in vitro.
PDI is essential for efficient cotranslational formation of disulfide
bonds in a coupled translation/translocation system.
(Bulleid, N. J., and Freedman, R. B. (1988) Nature 335, 649–651)
•
Eucaryotic peptidylprolyl isomerase (PPI).
(Fedorov A. N. and Baldwin T. O., The Journal of Biological chemistry, 272, 52, 32715-32718 (1997)
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Cotranslational folding
Ribosomes as general protein folding modulators
•
•
•
General protein folding activity observed in the large subunit of the
ribosome
– located in the peptidyl transferase domain of the large RNA of
this subunit.
In contrast to the protein folding activity of the molecular
chaperones, this activity is
– (a) present in the RNA and is
– (b) universal, not selective for any protein.
– The overlap of this active site with the peptidyl transferase centre
on the ribosomal RNA suggests a functional overlap between
protein synthesis and folding by ribosome in the cell.
Ribosomes from both prokaryotic and eukaryotic sources could
refold a large number of proteins from their denatured states to
active form.
(Fedorov A. N. and Baldwin T. O., The Journal of Biological chemistry, 272, 52, 32715-32718 (1997)
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Cotranslational folding
Ribosomes as general protein folding modulators
•
A large number of proteins
like bacterial alkaline phosphatase, glucose 6phosphate dehydrogenase, glucose oxidase, lactate dehydrogenase, horse radish
peroxidase, malate dehydrogenase, b lactamase, restriction endonucleases like EcoR1,
BamH1, HindIII, PstI, b -galactosidase, carbonic anhydrase, etc. could be folded by
the ribosomes.
(Das, B., Chattopadhyay, S. and Das Gupta, C., Biochem.; iophys. Res. Commun., 1992, 183, 774–
780.; Chattopadhyay, S., Das, B., Bera, A. K., Dais Gupta, D. and Das Gupta, C., Biochem. J., 1994,
300, 717–721; Bera, A. K., Das, B., Chattopadhyay, S. and Das Gupa, C., Biochem. Mol. Biol. Int.,
1994, 32 215–223; Das, B., Chattopadhyay, C., Bera, A. K. and Das Gupta, C., Eur. J. Biochem.,
1996, 235 613–621.)
•
Renaturation of some proteins is improved by the presence of
ribosomes
– attributed to the large ribosomal subunit, specifically to its RNA,
the 23 S and 28 S RNA of prokaryotic and eukaryotic ribosomes,
respectively
(Chattopadhyay, S., Das, B., and Dasgupta, C. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 8284–8287;
Kudlicki, W., Coffman, A., Kramer, G., and Hardesty, B. (1997) Fold. Des. 2, 101–108)
http://www.ias.ac.in/currsci/aug25/articles27.htm by DasGupta Chanchal
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Cotranslational folding
Is ribosome-mediated protein folding co-translation or post-translational?
•
•
•
•
•
Growing polypeptide chain fairly flexible
Cross linking of growing polypeptide chain with the 50S particle
showed many contacts, especially with the nucleotides in the
domain V
Two major activities, polypeptide synthesis and its folding into active
form
Folding intermediates having large part of its secondary structures
formed and even with tertiary structure formation
The final level of folding - outside the ribosome - ‘post translational’.
But the released polypeptide chain received the instructions for
folding from the ribosome
(Fedorov, A. N., Friguet, B., Djavadi-Ohaniance, L., Alakhov, Yu, B. and Goldberg, M. E., J. Mol. Biol.,
1992, 228, 351–358 in http://www.ias.ac.in/currsci/aug25/articles27.htm by DasGupta
Chanchal)
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Cotranslational folding
•
Statistical analysis of more than 200 protein structures has revealed
the tendency that, within the length of polypeptide typical for a
domain, residues tend to interact with the N-terminal portion of the
polypeptide and that the N-terminal region is, on average, more
compact than the C-terminal region. This observation is consistent
with vectorial folding of nascent polypeptides beginning from the
N terminus and proceeding to the C terminus.
(Alexandrov,
N. (1993) Protein Sci. 2, 1989–1991)
(Fedorov A. N. and Baldwin T. O., The Journal of Biological chemistry, 272, 52, 32715-32718 (1997))
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Cotranslational folding
•
•
•
•
Biosynthetic folding, proceeding through a series of intermediate structures (I1, I2, I3),
avoids certain kinetic traps, such as Mi in the Figure, which are encountered during
refolding of denatured protein.
In the absence of cotranslational folding (Iu1 , Iu2 , Iu3 ), the fully synthesized
polypeptide would begin folding from an unfolded ensemble, Mu, similar to the refolding
reaction and unavoidably proceeds through the slow-folding Mi intermediate.
The rate of either reaction is limited by the highest activation barrier.
In Cotranslational folding, the protein released from the ribosome is close to the
transition state, TS, and therefore rapidly assumes the native structure Mn.
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Cotranslational folding
•
Quick
•
Secondary structure formation and compaction require much less
than 1 s (Roder, H., and Colo´n, W. (1997) Curr. Opin. Struct. Biol. 7, 15–28)
•
Formation of compact globular intermediates usually requires no
more then a few seconds (Ptitsyn, O. B. (1995) Adv. Protein Chem. 47, 83–229)
•
Polypeptide synthesis requires many seconds (50–300 residues/min
for cell-free systems and somewhat faster in vivo; compact
intermediates must be formed in the process of synthesis. (Fedorov, A. N.,
and Baldwin, T. O. (1998) Methods Enzymol. 290)
•
Stereochemical analysis suggests that the nascent polypeptide
emerges from the peptidyltransferase center in an a-helical
configuration (Lim, V. I., and Spirin, A. S. (1986) J. Mol. Biol. 188, 565–574)
(Fedorov A. N. and Baldwin T. O., The Journal of Biological chemistry, 272, 52, 32715-32718 (1997)
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Cotranslational folding
Kinetics and Pathway
•
An upper limit of the rate of Cotranslational folding is imposed by
the rate of polypeptide synthesis.
•
For many proteins, as mentioned above, the C-terminal segment of
20–30 amino acid residues, which is sheltered by the ribosome prior
to the release of the full-length polypeptide into the bulk solution, is
essential for formation of the native, biologically active structure.
Consequently, folding cannot be completed before release of the
nascent polypeptide from the ribosome.
•
Kinetics of folding would be a function of the rates of polypeptide
synthesis, folding of the full-length monomer, and for oligomeric
proteins, subunit assembly.
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Cotranslational folding
Kinetics and Pathway
•
Cotranslational folding of the bacterial luciferase ‘b’ subunit is ratelimiting in the formation of the native ‘ab’ heterodimer when
prefolded ‘a’ subunit is available at a sufficiently high concentration
•
Coexpression of both subunits leads to much slower formation of
the native enzyme, apparently because association becomes the
rate-limiting step
•
Biosynthetic folding seems to be much faster and more efficient than
renaturation for several proteins.
(Fedorov A. N. and Baldwin T. O., The Journal of Biological chemistry, 272, 52, 32715-32718 (1997))
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Cotranslational folding
Kinetics and Pathway
•
Formation of secondary structural elements like alpha-helices, betasheets or beta turns which act as nucleation sites for the further
collapse of the native structure.
•
Secondary structure formation -timescale– nanoseconds to
microseconds
http://svr.ssci.liv.ac.uk/~volk/folding/Fasteventinprotein folding.htm
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Biosynthetic folding & Renaturation
Stages of protein folding
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Biosynthetic folding & Renaturation
•
One of the basic differences between biosynthetic protein folding
and protein renaturation is Cotranslational folding, folding that
occurs during synthesis.
•
The same conformations are achieved by polypeptides folded in
cells as a consequence of biosynthetic processes and as a result of
refolding of the full-length polypeptide from the denatured state.
•
However, identification of the final protein structures does not
necessarily mean identity of the pathways leading to their
formation
(Baldwin, R. L. (1975) Annu. Rev. Biochem. 44, 454–477)
•
How the pattern observed for refolding in vitro relate to protein
folding within the living system?
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Biosynthetic folding & Renaturation
•
Protein folding in the cell significantly faster than refolding of the
denatured protein in vitro
– bacterial luciferase
• contains no disulfide bonds.
• association of ‘a’ with ‘b’ chain determines the overall rate of
enzyme formation.
• the ‘b’ subunit released from the ribosome associates with
the ‘a’ subunit much faster than does ‘bi’, which
predominates in refolding experiments,
• suggesting that the structure of the ‘b’-subunit when it is
released from the ribosome (partially folded) is different from
bi (predominant intermediate in renaturation).
•
The ‘b’ subunit produced by biosynthetic folding is a folding
intermediate which is beyond a rate-limiting step encountered
during refolding of the subunit.
(Fedorov, A. N., and Baldwin, T. O. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 1227–1231)
(Fedorov A. N. and Baldwin T. O., The Journal of Biological chemistry, 272, 52, 32715-32718 (1997))
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Biosynthetic folding & Renaturation
•
The evolutionary pressure for fast folding operates in the context of
biosynthetic folding, including biosynthesis and concomitant folding
of the nascent polypeptide chain, obviously not on refolding of the
full-length polypeptide.
•
In this case, unlike the biosynthetic folding, all residues are initially
present to influence the folding pathway.
•
However, in renaturation experiments, especially for large,
multidomain and multisubunit proteins under conditions
approximating physiological conditions, low final yields, slow rates
and even an inability to achieve the native structure from the
denatured state are often experienced.
•
Many proteins fail to fold to their native state.
(Fedorov A. N. and Baldwin T. O., The Journal of Biological chemistry, 272, 52, 32715-32718 (1997))
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Cotranslational folding & Computational methods
Focus of theoretical studies
•
•
•
•
What are the sequence requirements for proteins to fold rapidly and
be stable in their native conformations?
What are the thermodynamic mechanism(s) of protein stabilization
and the kinetic mechanism(s) of folding?
Are there special native structures (motifs) that are more likely to
corresponds to the native structures of foldable proteins?
What is the best approximation for protein folding energetic
(potentials)?
(Shakhnovich E. I., Current Opinion in Structureal Bology, 7, 29-40 (1997))
•
Challenges
– What are good models for the potential energy surface?
– How can native conformation be found and recognized?
(Fetrow J.S. et. al., Current Pharmaceutical Biotechnology, 3, 329-347 (2002)
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Theoretical Studies
•
Homology modeling or threading could result in the final folded
structure without giving insights into the folding process.
•
Ab initio with complete sequence could probably reach the nativelike structure but the probability that it would follow the natural
pathway is remote.
•
How to know that the pathways are similar or not?
•
In fact, it takes about a day to simulate a nanosecond
(1/1,000,000,000 of a second). Unfortunately, proteins fold on the
tens of microsecond timescale (10,000 nanoseconds). Thus, it would
take 10,000 CPU days to simulate folding -- i.e. it would take 30
CPU years! That's a long time to wait for one result!
(http://folding.stanford.edu/science.html)
•
Classical molecular dynamics may miss many features of the folding
process as the process involves ensemble of transition states.
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The folding funnel
•
energy landscape perspectives, describe the in vitro progression of
an isolated polypeptide chain from an ensemble of denatured,
random conformations to the native structure at the global energy
minimum
•
do not account for the behavior of newly synthesized polypeptide
chains released from ribosomes in cells.
•
cannot describe the behavior of most polypeptide chains under
physiological conditions.
•
describes the folding behavior of only a single polypeptide chain at
infinite dilution. They do not consider populations or incorporate
realistic intermolecular collision frequencies.
(Clark P., TRENDS in Biochemical Sciences, 29 (10) 527-534 (2004)
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The folding funnel
•
an intrinsic feature of actual folding processes – namely, collisions
between partially folded chains that lead to self-association – is
excluded from consideration
•
misfolding associated with self-association, polymerization or
aggregation is not considered
•
the cotranslational appearance of the polypeptide chain outside the
ribosome therefore corresponds to a specific portion of the folding
funnel, and the chain presumably folds reasonably quickly and
efficiently to this available local energy minimum
(Clark P., TRENDS in Biochemical Sciences, 29 (10) 527-534 (2004)
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The folding funnel -
Open questions
– Is the observed folded conformation the one with lowest free
energy? Or
– Is it the most stable of the kinetically accessible conformations?
(kinetically trapped in local minima)
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SUMMARY
How nascent protein can fold correctly?
•
Protein folding, no matter how it worked, has to be pretty
simple and fast.
•
Reasonable approach
– Folding is hierarchical process with primary structure
preceding secondary structure which is then followed by
tertiary structure (and finally quaternary structure).
(Johnson A. E., FEBS Letters 579,916-920 (2005)) Figure from http://folding.stanford.edu/science.html
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Other methods
•
Renaturation of denatured protein may not give correct
insights into the folding kinetic and/or pathways.
•
Computational techniques like homology modeling, threading
techniques and ab initio algorithms also may not give correct
insights into the folding kinetic and/or pathways.
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The problem remains unsolved
“Despite all the efforts…
understanding of protein folding mechanism remains elusive”.
•
We are not very close to realizing this goal, and so the
Protein Folding problem remains
one of the most basic unsolved problems in biology
.
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Thank you
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Additional Slides Ahead…
•
Nascent chain of Influenza Hemagglutinin (HA) can be
simultaneously engaged in translation, translocation, glycosylation,
glycal trimming, folding and association with calnexin. Disulfide
bonds begin to form after both cysteins entered the ER lumen.
(Chen W. et. al., PNAS, 92, 6229-6233 (1995))
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Cotranslational folding
Evidences
•
•
•
•
Immunoglobin light chains - two domain polypeptides - two
intramolecular disulfide bonds, one in the N-terminal domain and
the other in the C-terminal domain.
Nascent light chain polypeptides fold in the lumen of the
endoplasmic reticulum.
The disulfide bond between Cys-35 and Cys-100 of the N-terminal
domain starts to form when the nascent chains achieve 15.5 kDa
length.
Formation of this bond is almost quantitative when the nascent
polypeptide has achieved a length of 18 kDa; formation of the
disulfide requires ~3 s.
(Bergman, L. W., and Kuehl, W. M. J. Biol. Chem. 254, 8869–8876 (1979) in Fedorov A. N. and
Baldwin T. O., The Journal of Biological chemistry, 272, 52, 32715-32718 (1997))
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Cotranslational folding
Evidences
Oligomers
•
Formation of enzymatically active b-galactosidase oligomer from
nascent polypeptides on ribosomes. (Kiho, Y., and Rich, A. (1964) J. Mol. Biol. 51,
111–118)
•
The modular organization of the monomer and independent folding
of each domain provides an explanation for how this large tetrameric
complex could be formed with one monomer not yet completely
synthesized.
(Jacobson, R. H., Zhang, X. J., DuBose, R. F., and Matthews, B. W. (1994) Nature 369, 761–766)
(Fedorov A. N. and Baldwin T. O., The Journal of Biological chemistry, 272, 52, 32715-32718 (1997)
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Cotranslational folding
Kinetics and Pathway
•
Firefly luciferase fold much more efficiently during synthesis than
during renaturation under the same conditions. N-terminal domain
folds cotranslationally, avoiding intramolecular misfolding, which
may be critical in the folding of multidomain proteins. Sequential
domain formation observed contranslationally but not in vitro.
(Frydman J. et.al., Nature Structural Biology, 6, 7, 697-705))
•
Firefly luciferase also folds efficiently upon translocation into
proteoliposomes depleted of chaperones.
(Tyedmers, J., Brunke, M., Lechte, M., Sandholzer, U., Dierks, T., Schlotterhose, P., Schmidt, B., and
Zimmermann, R. (1996) J. Biol. Chem. 271, 19509–19513)
•
These observations imply a crucial role for biosynthetic folding of
nascent chains.
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Cotranslational folding
•
•
•
•
•
•
•
The nascent polypeptide may start to fold from the N-terminus to
the C-terminus to form the native protein structure in vivo. during
biosynthesis on the ribosome;
Isomerizations within the partially folded N-terminal segment of a
polypeptide occurs concomitantly;
Restricted diffusion and attachment of the nascent chain to the
large ribosomal particle reduces the aggregation potential of the
nascent polypeptides;
Cell regulates the rate of protein folding for both structural and
trafficking purposes (translocation, premature folding, posttranslational modification)
Multiple cellular components (e.g. peptidopropyl isomerase, protein
disulfide isomerase or heat shock proteins) catalyze or assist the
folding of newly synthesized polypeptides.
Can help in preventing nonproductive side reactions
May allow folding to progress in an orderly domain-by-domain
sequence
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Cotranslational folding
Ribosomes as general protein folding modulators
•
Ribosomes not only synthesize the polypeptide chains, but also work
on it to see that the chains fold to meaningful proteins to carry on
cellular activities.
(Moazed, D. and Noller, H. F., Biochimie, 1987, 69, 879–884. )
•
The central loop of domain V of 23S rRNA (active core: nucleotides
2000-2624 ) where aminoacyl tRNA and a number of antibiotics
bind and the peptidyl transferase reaction takes place helps in
renaturing denatured proteins.
(Chattopadhyay S at. al., PNAS, 93, 8284-8287 (1996))
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Cotranslational folding
•
Evidence are available for cotranslatational folding in both
eukaryotic proteins and prokaryotic proteins.
•
Both experimental and theoretical studies of protein refolding
suggest that there is evolutionary pressure for proteins to fold fast
and folding of larger proteins generally involves smaller independent
folding units.
(Shakhnovich, E. I. (1997) Curr. Opin. Struct. Biol. 7, 29–40)
•
Even inclusion body aggregates are typically formed from partially
folded conformations, rather than native states or fully denatured
polypeptide chains.
(Clark P.L., Trends in Biochemical Sciences, 29, 10, 527-534 (2000))
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