Folding kinetics
description
Transcript of Folding kinetics
Folding kinetics
Folding equilibrium vs. folding kinetics
Transient intermediate!
Partially Folded States(Molten globule)
Molten globule state:An intermediate conformation assumed by many globular proteins at equilibrium under mildly denaturing conditions
R.H. Pain, Mechanisms of protein folding
Protein folding time scale
ns s ms
Randomcoils
-helix -sheetturns
Hydrophobic collapse
Annealing, Assembly
>S, min, hr..
“Proteins are characterized by:
a) High frequency small amplitude fluctuations (10-12 – 10-9 sec) of individual side chains (NOISE!)
b) Large amplitude collective fluctuations of protein domains (10-6 sec)
c) Slower conformational transitions”
Dr. Sunney Chan
How to detect folding intermediates?
Stopped-flow designed (1970s) Continuous-flow Quench-flow
Bieri and Kiefhber, Biol. Chem., 1999
N Uk1
k2
GNU= -RT lnKeq = -RT ln (k1/k2)
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A0
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A Bk
[A] = [A0] * exp (-kt)
Amplitude Rate constant
Burst Phase
•instrumental dead time•(burst phase)
Start
End
Kinetics of chemical reaction
k obs=kUN*exp (-m U-TS*[urea]/RT) + kNU*exp(-m N-TS*[urea]/RT)
GUN= -RT *ln (kUN / kNU)
m U-N = m U-TS + m N-TS
k obs=Refolding rate constant + unfolding rate constant
• G• m
Chevron Plot
Log
(R
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con
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Denaturant
Refolding(UN)
Unfolding(N U)
Equilibrium mid-point
• compactness of the transition state (TS)
‡
1
Stopped-flow
H1 H2 H3 H4
Most helical bundle
(CARD of RICK)
H1
H4
H3 H2
H6
H5
Stopped-flow Apparatus:Single Mixing Experiments
Stepping Motor
Stepping Motor
Photomultiplier
Computer Control
1 2 3 4
N (0 M urea) U (~4 M urea)UnfoldingRefolding N (~0 M urea)U (4 M urea)
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Equilibrium folding
Four Kinetic Phases are Found in Unfolding and Refolding (burst, fast, medium, slow)
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4 to 4 in %
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0 to 4 9.5.02 unfodling data in %
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U signal
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Unfolding NU
Refolding U N
3 exponential equations[A] = [A1] * exp (-k1t)+ [A2] * exp (-k2t)+ [A3] * exp (-k3t)
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3 exponential equations[A] = [A1] * exp (-k1t)+ [A2] * exp (-k2t)+ [A3] * exp (-k3t)
RefoldingU to N
UnfoldingN to U
[urea]
Sequential Mixing Experiments
Stepping Motor
Stepping Motor
Photomultiplier
Computer Control
1 2 3 4
t(details of fast forming unfolded species)
N U(fast) U(slow)
example:
U(cis)N(cis) U(trans)
%N
Delay time
100%
1. Double jump: N U t (details in unfolding)
N U N
2. Interrupted refolding:U N
t(N formation in refolding)
1. Double jump: N U t (details in unfolding)
Figure 6 (A) Fraction of native protein versus delay time in double jump experiments. The initial ( ) and final signals (·) of the signal traces were normalized to fraction folded and plotted versus delay time. (B) Fraction of native protein versus delay time in interrupted refolding experiments. The initial signal was normalized and plotted versus delay time.
Parallel folding model
Sequential folding pathway
Slow folding kinetics
cis-trans isomerization Large arrangement Subunit assembly Complex topology
Are the intermediate on-pathway?
Natively Unfolded Proteins
Natively unfolded proteins occupy a unique niche within the protein kingdom in that they lack ordered structure under conditions of neutral pH in vitro.
A lot of them became structured while binding to their binding partners
Natively unfolded proteins Anthony L Fink Department of Chemistry and Biochemistry, University of California, Santa Cruz, CA 95064, USA
It is now clear that a significant fraction of eukaryotic genomes encode proteins with substantial regi
ons of disordered structure. In spite of the lack of structure, these proteins nevertheless are functio
nal; many are involved in critical steps of the cell cycle and regulatory processes. In general, intrinsi
cally disordered proteins interact with a target ligand (often DNA) and undergo a structural transition
to a folded form when bound. Several features of intrinsically disordered proteins make them well su
ited to interacting with multiple targets and to cell regulation. New algorithms have been developed t
o identify disordered regions of proteins and have demonstrated their presence in cancer-associate
d proteins and proteins regulated by phosphorylation.
Current Opinion in Structural Biology Volume 15, Issue 1, February 2005, Pages 35-41
Ultra-fast foldingContinuous flow
Methods 34 (2004)15-27
Quench-flow microsecond time scale
Chem Rev. 2006 May;106(5):1769-84. LinksProbing protein folding and conformational transitions with fluorescence.Royer CA.Centre de Biochimie Structurale, 29, rue de Navacelles 34090 Montpellier Cedex France.
Figure 1 (a) Structure of the regulatory domains of an activated mutant of LicT (tryptophan residues are in purple); (b) intrinsic fluorescence emission spectra of the WT and activated mutant forms
Trp location
Figure 2 Intrinsic tryptophan emission of P13MTCP1 in buffer (full line) and 3 M guanidine hydrochloride (dotted line).
Figure 3 Intrinsic emission spectra of WT protein L (circles) and the Y43W mutant (squares) under native (closed symbols) and denaturing (open symbols) conditions.
Figure 4 Stereoview of ribbon model overlays of the WT* form (green) and the WT NMR-derived solution #1 model (orange). The proteins exhibit the same overall fold, although the main-chain rmsd is 1.46. Major differences occur in the loop regions as well as the N-terminus of -strand 4. The side chains for residues Y47/W47, Y34, and I60 are shown
Figure 5 Ribbon representation of the 3-D structure of nuclease.29 Note that the tryptophan residue (purple) is the last residue in the sequence that exhibits order.
Trp Repressor
Figure 7 Steady-state spectra as a function of urea concentration for (A) trpR W99F and (B) W19F mutants. Increasing numbers correspond to increasing urea concentration. Insets: Urea unfolding profiles based on the average emission wavelength.
tryptophan 19 are red, tryptophan 99 are blue.
Figure 8 DAS (decay associated spectra) as a function of urea concentration for (A) trpR W99F and (B) W19F mutants. Increasing numbers correspond to increasing urea concentration
ANS used to probe folding intermediates
Figure 9 Spectrum of ANS in the presence of beta-lactamase at (A) pH 1.7 and (B) pH 12: (1) no added salt; (2) 0.6 M salt; dotted lines correspond to the spectrum of ANS in the absence of protein.
Figure 10 Stopped-flow fluorescence of ANS and the trp repressor in a refolding experiment. IAA (indole acrylic acid)
Figure 11 Time-dependence of ANS fluorescence from stopped-flow refolding studies of DHFR as a function of increasing acrylamide concentration (higher acrylamide concentration corresponds to lower intensities).
Figure 12 Burst phase intermediate ANS intensity as a function of increasing denaturant.
FRET
Figure 14 Recovered energy transfer distance distributions from the global analysis of the time-resolved decays of D-PGK and D-PGK-A. The predicted dye-to-dye distances from MD simulations (diamonds) are shown for comparison.
Figure 15 Unfolding of PGK monitored by tryptophan emission from D-PGK and D-PGK-A (circles and squares), AF emission from D-PGK-A (inverted triangles), AEDANS emission from D-PGK (triangles), and FRET efficiency from D-PGK-A (diamonds) for all six FRET pairs.
Figure 16 Stopped-flow FRET distances for the six FRET pairs of PGK. (A, 202-412; B, 135-412; C, 412-75; D, 290-412; E, 135-290; F, 75-290).
Demonstrate unfolding of PGK is a multistep process and bending of the hinge between the two domain occurs.
Figure 17 Positions of the four inserted tryptophan residues and the seven inserted cysteine residues in the RNase A structure.
Figure 18 FRET distance distributions at different GuHCl concentrations for doubly labeled RNase A mutants in the reduced native (Rn) and (U) unfolded states. The distances were also determined in the unfolded, oxidized state (Oxi 6M GdnHCl).
How fast can folding be
Figure 19 Transient from the folding of horse apomyoglobin after a laser T-jump. The inset (B) shows the response of free tryptophan.
Figure 21 Ultrafast fluorescence intensity kinetic traces after laser T-jumps of (a) 265-275 K, (b) 273-293 K, and 303-319 K. Relaxation times obtained from fits of the data to single exponential decays were on the order of tens of nanoseconds.
Designed small helical peptide labeled with extrinsic probe
Figure 23 Autocorrelation function and residuals of the simple diffusion model and an additional dynamics model for V60Flu FABP (Fatty acid binding protein).
Single molecule study
FRETfluorescence resonant energy transfer
thio
Case in a helix bundle(B) Unmodified ACBP (sharp decrease due to FL quenching)(C) AEDANS-labeled ACBP.Ex the Trp, Em at AEDANSLarge increase at first 80usec (due to major decrease of distance) following by decay phase from 10-100msec
First 80us forms loosely packed and highly dynamic ensembles of statesThe dimension is similar to the native state
New Technology for protein folding
Direct Observation of the Three-State Folding of a Single Protein Molecule Ciro Cecconi,1,2* Elizabeth A. Shank,1* Carlos Bustamante,1,2,3 Susan Marqusee UC Berkeley
Science 23 September 2005:Vol. 309. no. 5743, pp. 2057 - 2060
We used force-measuring optical tweezers to induce complete mechanical unfolding and refolding of individual Escherichia coli ribonuclease H (RNase H) molecules. The protein unfolds in a two-state manner and refolds through an intermediate that correlates with the transient molten globule–like intermediate observed in bulk studies. This intermediate displays unusual mechanical compliance and unfolds at substantially lower forces than the native state. In
a narrow range of forces, the molecule hops between the unfolded and intermediate states in real time. Occasionally, hopping was observed to stop as the molecule crossed the folding barrier directly from the intermediate, demonstrating that the intermediate is on-pathway. These studies allow us to map the energy landsc
ape of RNase H.
Mechanical Unfolding
Membrane protein folding
Nature 438, 581-589 (1 December 2005) Solving the membrane protein folding problem
Membrane protein foldingStephen White
laboratory UC Irvine
General Principles of Membrane Protein
Folding and Stability
Constitutive Membrane Protein Assembly
Inconstitutive Membrane Protein Assembly
WHY IS PROTEIN FOLDING SO DIFFICULT TO UNDERSTAND?
It's amazing that not only do proteins self-assemble -- fold -- but they do so amazingly quickly: some as fast as a millionth of a second. While this time is very fast on a person's timescale, it's remarkably long for computers to simulate.
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!
Computation
Molecular Dynamicsab initio protocol: Ignore sequence homology and atte
mpts to predict the folded state from fundamental energetic or physicochemical properties associated with the constituents residues. (Finding a single structure of low energy)
secondary structure predictionbased on the preference of a.a. for certain conformational states (50%) In combination of homologous sequences (70%)
(computational prediction of folding is not yet reliable)
http://www.stanford.edu/group/pandegroup/folding/villin/index.html
Simulations of the villin headpiece The villin headpiece is a small, 36-residue alpha helical protein. It has been heavily studied experimentally and by simulation since is perhaps one of the smallest, fastest folding proteins. It has a hydrophobic core made of 3 phenylalanines, but also has two groups (a tryptophan and another phenylalanine) which are hydrophobic, but are solvent exposed (for functional reasons). Duan and Kollman simulated 1 microsecond of MD time, in a ground breaking simulation. However, since the folding time is on the order of 10 microseconds, it is not surprising that they did not see it fold. Our simulations contain hundred of microseconds of MD time, and we have seen 35 simulations which have folded.
http://folding.stanford.edu/