Making connection between ultra-fast folding kinetics and molecular - PowerPoint PPT Presentation

Making connection between ultra-fast folding kinetics and molecular dynamics simulation Group 4 Tae-yong Kim Donghwan Kim Jinah Lee

Concept pts of pr protein in fold ldin ing  Protein folding is essential for understanding biological processes and developing therapeutic approaches to misfolding-related diseases.  Protein folding can be followed in equilibrium experiments, which monitor protein states as a function of temperature or denaturant concentration, and kinetics

Un Unfold ldin ing/fold ldin ing ex exper erimen ents Image of mass spectroscopy  Mass spectrometry : effective tool for supporting both thermodynamic and kinetic protein-folding studies: in microseconds or even more  Fast photochemical oxidation of proteins(FPOP): combined rapid mixing with a chemical approach, FPOP, to afford potentially a considerably higher resolution probe of folding.

Tem emper erature-jumped method  Temperature jump is a technique used in the study of chemical kinetics. TJ measurement Example of TJ mehtod Biochem. J. (2001) 358, 165-173

Ult Ultrafast fold ldin ing  Proteins that fold on a microsecond timescale.  Small single domain proteins, containing less than 100 amino acid residues.  Simple folding mechanism. PNAS, 2003, Vol. 100, no.26

villin illin hea eadpiec ece e subdom omain, HP35( 35(His27) 27)  HP35 is one of the smallest naturally occurring protein domains.  Sequence : LSDED FKAVF GMTRS AFANL PLWKQ QNLKK EKGLF  Trp23 is the fluorescence probe; His27 replaces Asn27 of the wild-type sequence, and when protonated.  reduces the fluorescence of Trp23 upon folding; Lys24 and Lys29 make repulsive electrostatic interactions with protonated His27 and Arg14, respectively.

TJ TJ wit ith tryptophan f fluorescence det etection  Tryptophan quantum yield ( ϕ ) as a function of time at 10 ° C after a 5 K laser- induced T-jump for 300 μM solutions of Cys-HP35(Nle24,His27,Nle29) containing 20 mM sodium acetate, 1 mM TCEP, and either  (A) 2.25 M GdmCl (blue)  (B) 4 M GdmCl (green)  (C) 6 M GdmCl (red)  The circles are the experimental data and the lines are fits with a single exponential function.

Kin inetic ics: T Tryptophan T Trip iple let Lif ifetim ime  The population of the tryptophan triplet state was monitored by triplet-triplet absorption at 440 nm  Normalized tryptophan triplet–triplet absorbance at 440 nm as a function of time on a log scale at 10 ° C for 100 μM solutions of Cys- HP35(Nle24,Nle29) containing 20 mM sodium acetate, 1 mM TCEP, and  2.25 M GdmCl (blue)  4.5 M GdmCl (green)  6 M GdmCl (red)  The absorbance is proportional to the sum of the populations of the triplet state in the folded and unfolded states.  The circles are the experimental data and the lines are the fits with the kinetic model.

Kin inetic ic m model f l for t trip iple let-lif lifetim ime Triplet-triplet absorbances, A(t)/A(0), with the initial conditions (t=0) * k * k = = f u p ( 0 ) , p ( 0 ) + + F * U * * * * * k k k k f u f u is + p ( t ) p ( t ) = = − λ + − − λ F * U * ( ) / ( 0 ) exp( ) ( 1 ) exp( ) A t A a t a t + − + p ( 0 ) p ( 0 ) F * U * where 1 λ = ± − 2 ( k k 4 Q ) ± 2 = + + + * * k k k 2 k k u f s q = + + + ( ) * * dp Q ( k k )( k k ) k k = − + + * * u s s q f s F * k k p k p s U F * f U * dt − λ + + − λ * * ( k )( k k k ) − + = s u f s ( ) a dp + λ − λ = − + + * * * * ( k k )( ) U * k p k k k p + − u f u F * s q f U * dt

Com omparison on o of fol olding rate  Folding times obtained in a two-state analysis of the results from the two methods at denaturant concentrations varying from 1.5–6.0 M guanidinium chloride are in excellent agreement, with an average difference of only 20%.  Polynomial extrapolation of all the data to zero denaturant yields a folding time of 220 (+100, − 70) ns at 283 K, suggesting that under these conditions the barrier Comparison of folding times measured between folded and unfolded states has by temperature jump and tryptophan effectively disappeared—the so-called triplet lifetime at 283 K. “downhill scenario.”

Conclu lusio ion & D Dis iscussio ion  In this study, we confirmed ultra-fast folding protein time using triplet lifetime experiment.  For the future, we would better think about protein that bigger and having more complex mechanism than ultra-fast folding protein to folding rate.