Kinetics folding

The key question we want to answer is what are the intrinsic sequence dependent factors tliat not only detennine tire folding rates but also tire stability of tire native state It turns out tliat many of tire global aspects of tire folding kinetics of proteins can be understood in tenns of tire equilibrium transition temperatures. In particular, we will show tliat tire key factor tliat governs tire foldability of sequences is tire single parameter... 

The various folding mechanisms expected in foldable sequences may be classified in tenns of the (Sj,. We have already shown that sequences that fold extremely rapidly have very small values of Gj,. Based on our study of several model proteins as well as analysis of real proteins we classify the folding kinetics of proteins in the following [7]. 

R Zwanzig. Simple model of protein folding kinetics. Pi oc Natl Acad Sci USA 92 9801-9804, 1995. 

Berriz GF, Shakhnovich El. Characterization of the folding kinetics of a three-helix bundle protein via a minimalist Langevin model. J Mol Biol 2001 310 673-85. 

Shimada J, Shakhnovich El. The ensemble folding kinetics of protein G from an all-atom Monte Carlo simulation. Proc Natl Acad Sci USA 2002 99 11175-80. 

The burst-phase intermediate has been studied intensively in the folding of bovine o -lactalbumin (Arai and Kuwajima, 1996 Ikeguchi et al., 1986 Kuwajima et al., 1985). The CD spectrum of the intermediate has been determined by measuring the folding kinetics at various wavelengths and extrapolating the CD to zero time. The results are shown as open circles (holoprotein) and squares (apoprotein) in Figure 35. The... 

Levy, Y, Jortner, J., and Becker, O. M. (2001). Solvent effects on the energy landscapes and folding kinetics of polyalanine. Proc. Natl. Acad. Sci. USA 98, 2188-2193. 

Proteins in vivo protein-protein interactions, protein folding kinetics, protein subunit exchange, enzyme activity assay, etc. 

Bashford D Weaver D. L. and Karplus M. Diffusion-collision model for the folding kinetics of the lambda-repressor operatorbinding domain. J. Biomol. Str. Dyn. (1984) 1 1243-1255. 

Zwanzig R. Two-state models of protein folding kinetics. Proc. Natl. Acad. Sd., USA (1997) 94(1) 148-150. 

The structure of the ribosomal protein L9 from B. stearothermophilus is shown in Fig. 3. The folding kinetics and thermodynamics of its C-terminal domain have been studied as a function of pH by NMR and CD spectroscopies. The ionization state of the two histidines (Hisl06 and His 134) was found to be essential for the global stability and the folding rate of the protein. ... 

Fig. 3. (A) Ribbon diagram of ribosomal L9 protein from B. stearothermophilus. The N- and C-terminal domains are labelled. The C-terminal construct consisting of residues 58-149 is shaded. (B) Ribbon diagram of the C-terminal domain showing the location of the three histidine residues. Reprinted from J. Mol. Biol., Vol. 318, S. Sato and D. P. Raleigh, pH-dependent stability and folding kinetics of a protein with an unusual alpha-beta topology The C-terminal domain of the ribosomal protein L9 , pp. 571-582, Copyright 2002, with permission from Elsevier Science.

F NMR was used to monitor the folding kinetics of TmCsp labelled with 5-fluorotryptophan. It was found that the increased thermostability of TmCsp (rui = 87°C) compared to CspB (ri =52°C) was due to lower unfolding rate constants over a wide temperature range. This suggested that entropic factors play an important role in the thermostabilization of TmCsp. 

Since values of fccat he between 1 and 107 s-1, measurements must be made in a time range of 1 to 10 7 s. This requires either techniques for rapidly mixing and then observing the enzyme and substrate, or totally new methods. Also, since the events that are to be observed occur on the enzyme itself, the enzyme must be available in substrate quantities. The development of apparatus for measuring these rapid reactions and of techniques for isolating large quantities of pure proteins has revolutionized enzyme and protein folding kinetics. 

The structure of the transition state for the folding of CI2 has been mapped by kinetic measurements and d>-value analysis of more than 100 mutants under a variety of conditions.29,30 The properties were measured in water from folding kinetics and extrapolated to water from unfolding kinetics as a function of [GdmCl]. The results from both directions agree. 

The average properties of the transition state may be estimated from a variety of criteria. The Tanford j8t value, measured from the relative sensitivities of the folding kinetics and equilibrium constants to [GdmCl] (Chapter 18, section Bl, equation 18.9), is 0.6, indicating that about 60% of the surface area of the protein is buried in the transition state for folding, relative to that buried in the native structure. The change in heat capacity of the transition state relative to that... 

Chymotrypsinogen 480, 481 Chymotrypsin inhibitor 2 (CI2) folding kinetics 544-577, 577 GroEL binding 605 fragments 577, 578, 587, 588, 595 mechanism of folding 576-588 structure 576, 577 Circular dichroism (CD) 193-195 optimal absorbance for signal to noise 212-214... 

Cold-shock /3-barrel proteins, folding kinetics 551... 

Label-free optical detection 199 Lactate dehydrogenase 465 -469, 472 A-repressor folding kinetics 551 Lattice simulations 597 Leaving group 81, 86, 88-93 Levinthal s paradox 575,576, 598, 599,600... 

T4 lysozyme 33,497 helix stability of 528, 529 hydrophobic core stability of 533, 544 Tanford j8 value 544, 555, 578, 582-Temperature jump 137, 138, 541 protein folding 593 Terminal transferase 408,410 Ternary complex 120 Tertiary structure 22 Theorell-Chance mechanism 120 Thermodynamic cycles 125-131 acid denaturation 516,517 alchemical steps 129 double mutant cycles 129-131, 594 mutant cycles 129 specificity 381, 383 Thermolysin 22, 30,483-486 Thiamine pyrophosphate 62, 83 - 84 Thionesters 478 Thiol proteases 473,482 TNfn3 domain O-value analysis 594 folding kinetics 552 Torsion angle 16-18 Tbs-L-phenylalanine chloromethyl ketone (TPCK) 278, 475 Transaldolase 79 Tyransducin-o 315-317 Transit time 123-125 Transition state 47-49 definition 55... 

Chyraotrypsin inhibitor 2 (CI2) folds rapidly by simple two-state kinetics that is, D N, with a r1/2of 13 ms.18,19 CI2 is a small 64-residue protein that has all its peptidyl-proline bonds in the favorable trans conformation.20 (There are, of course, additional slow cis —> trans peptidyl-prolyl isomerization events, which account for about 20-30% of the refolding amplitudes.) The occurrence of two-state kinetics does not prove that there are no intermediates on the folding pathway there could be intermediates that are present at high energy and are kineti-cally undetectable (see section B4). Two-state behavior has subsequently been found for many other small proteins. The simplicity of two-state folding kinetics provides the ideal starting point for the analysis and illumination of the basic principles of folding. 

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