Pierce, M. M., & Nall, B. T. (2000). Coupled kinetic traps in cytochrome c folding: His-heme misligation and proline isomerization. J Mol Biol, 298(5), 955–969.
Abstract: The effect of His-heme misligation on folding has been investigated for a triple mutant of yeast iso-2 cytochrome c (N26H,H33N,H39K iso-2). The variant contains a single misligating His residue at position 26, a location at which His residues are found in several cytochrome c homologues, including horse, tuna, and yeast iso-1. The amplitude for fast phase folding exhibits a strong initial pH dependence. For GdnHCl unfolded protein at an initial pH<5, the observed refolding at final pH 6 is dominated by a fast phase (tau(2f)=20 ms, alpha(2f)=90 %) that represents folding in the absence of misligation. For unfolded protein at initial pH 6, folding at final pH 6 occurs in a fast phase of reduced amplitude (alpha(2f) approximately 20 %) but the same rate (tau(2f)=20 ms), and in two slower phases (tau(m)=6-8 seconds, alpha(m) approximately 45 %; and tau(1b)=16-20 seconds, alpha(1b) approximately 35 %). Double jump experiments show that the initial pH dependence of the folding amplitudes results from a slow pH-dependent equilibrium between fast and slow folding species present in the unfolded protein. The slow equilibrium arises from coupling of the His protonation equilibrium to His-heme misligation and proline isomerization. Specifically, Pro25 is predominantly in trans in the unligated low-pH unfolded protein, but is constrained in a non-native cis isomerization state by His26-heme misligation near neutral pH. Refolding from the misligated unfolded form proceeds slowly due to the large energetic barrier required for proline isomerization and displacement of the misligated His26-heme ligand.
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Hagen, S. J., & Eaton, W. A. (2000). Two-state expansion and collapse of a polypeptide. J Mol Biol, 301(4), 1019–1027.
Abstract: The initial phase of folding for many proteins is presumed to be the collapse of the polypeptide chain from expanded to compact, but still denatured, conformations. Theory and simulations suggest that this collapse may be a two-state transition, characterized by barrier-crossing kinetics, while the collapse of homopolymers is continuous and multi-phasic. We have used a laser temperature-jump with fluorescence spectroscopy to measure the complete time-course of the collapse of denatured cytochrome c with nanosecond time resolution. We find the process to be exponential in time and thermally activated, with an apparent activation energy approximately 9 k(B)T (after correction for solvent viscosity). These results indicate that polypeptide collapse is kinetically a two-state transition. Because of the observed free energy barrier, the time scale of polypeptide collapse is dramatically slower than is predicted by Langevin models for homopolymer collapse.
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Kihara, H., Nakatani, H., Hiromi, K., & Hon-Nami, K. (1977). Kinetic studies on redox reactions of hemoproteins. I. Reduction of thermoresistant cytochrome c-552 and horse heart cytochrome c by ferrocyanide. Biochim Biophys Acta, 460(3), 480–489.
Abstract: The oxidation-reduction reaction of horse heart cytochrome c and cytochrome c (552, Thermus thermophilus), which is highly thermoresistant, was studied by temperature-jump method. Ferrohexacyanide was used as reductant. (Formula: see text.) Thermodynamic and activation parameters of the reaction obtained for both cytochromes were compared with each other. The results of this showed that (1) the redox potential of cytochrome c-552, + 0.19 V, is markedly less than that of horse heart cytochrome c. (2) deltaHox of cytochrome c-552 is considerably lower than that of horse heart cytochrome c. (3) deltaSox and deltaSred of cytochrome c-552 are more negative than those of horse heart cytochrome c. (4) kred of cytochrome c-552 is much lower than that of horse heart cytochrome c at room temperature.
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Steinhoff, H. J. (1988). A continuous wave laser T-jump apparatus and its application to chemical reactions in hemoglobin single crystals. J Biochem Biophys Methods, 15(6), 319–330.
Abstract: A laser temperature jump apparatus is constructed where the T-jump is achieved by means of the direct absorption of continuous laser radiation of low intensity by a solid sample. The final temperature in the irradiated volume element is reached when the absorbed radiation power equals the dissipation of heat by heat conduction. The time range from the beginning of irradiation to the stationary state depends on the geometry of the irradiated volume element and is less than 10 ms. The heating laser beam is simultaneously used to detect the relaxation to the new chemical equilibrium in the sample. Relaxation processes with relaxation rates between 10(2) s-1 and less than 10(-3) s-1 on samples with volumes less than 10(-3) mm3 may be investigated using this T-jump method. One application of this method is the determination of reaction rates of ligand reactions in hemoglobin single crystals. Rate constants obtained for the reaction of thiocyanate with crystallized horse methemoglobin are presented.
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Steinhoff, H. J., Lieutenant, K., & Redhardt, A. (1989). Conformational transition of aquomethemoglobin: intramolecular histidine E7 binding reaction to the heme iron in the temperature range between 220 K and 295 K as seen by EPR and temperature-jump measurements. Biochim Biophys Acta, 996(1-2), 49–56.
Abstract: Temperature-dependent EPR and temperature-jump measurements have been carried out, in order to examine the high-spin to low-spin transition of aquomethemogobin (pH 6.0). Relaxation rates and equilibrium constants could be determined as a function of temperature. As a reaction mechanism for the high-spin to low-spin transition, the binding of N epsilon of His E7 to the heme iron had been proposed; the same mechanism had been suggested for the ms-effect, found in temperature-jump experiments on aquomethemoglobin. A comparison of the thermodynamic quantities, deduced form the measurements in this paper, gives evidence that indeed the same reaction is investigated in both cases. Our results and most of the findings of earlier studies on the spin-state transitions of aquomethemoglobin, using susceptibility, optical, or EPR measurements, can be explained by the transition of methemoglobin with H2O as ligand (with high-spin state at all temperatures) and methemoglobin with ligand N epsilon of His E7 (with a low-spin ground state). Thermal fluctuations of large amplitude have to be postulated for the reaction to take place, so this reaction may be understood as a probe for the study of protein dynamics.
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