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Dubois, C., Manfredi, E., & Ricard, A. (2008). Optimization of breeding schemes for sport horses. Livestock Science, 118(1-2), 99–112.
Abstract: A selection scheme for jumping sport horses is modelled with four stages of selection for males and one stage for females. The selection objective included three traits: conformation and gaits (CG, weighted 20%), competition jumping (CJ, weighted 60%) and a third trait (TT, weighted 20%) such as sperm quality or orthopaedic status. The first selection stage is based on knowledge of the pedigree with the aim of selecting horses suitable for CG test (at 3Â years old) and CJ test (at 5Â years old). The second stage includes the horse's own performance with respect to CG and CJ with the aim of selecting horses suitable for the TT test. The third stage is the selection of a limited number of males who are allowed to reproduce. The fourth stage (at 12Â years old) takes into account the results of the horse's progeny. Females are selected in one step, whatever the number of performances measured at 5Â years old. The annual genetic response was 9.4% genetic standard deviation of the objective, 2.6% for CG, 9.0% for CJ and 1.5% for TT. Results showed that selection by progeny testing did not contribute much to genetic response (12% of progeny issued from proven sires), the female pathway represented 26% of genetic response, TT was difficult to improve when the genetic correlation was unfavourable (-Â 0.6% genetic standard deviation for -Â 0.20 genetic correlation), and should consequently be directed towards the use of molecular markers. When compared with a selection scheme involving a station test, genetic response was the same if the breeding values used for selection before entering the station test took into account the results of the relatives for CJ and CG. This revealed the importance of an extensive performance test (like for competition performance) when designing breeding schemes for sport horses.
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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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Hirota, S., Suzuki, M., & Watanabe, Y. (2004). Hydrophobic effect of trityrosine on heme ligand exchange during folding of cytochrome c. Biochem Biophys Res Commun, 314(2), 452–458.
Abstract: Effect of a hydrophobic peptide on folding of oxidized cytochrome c (cyt c) is studied with trityrosine. Folding of cyt c was initiated by pH jump from 2.3 (acid-unfolded) to 4.2 (folded). The Soret band of the 2-ms transient absorption spectrum during folding decreased its intensity and red-shifted from 397 to 400 nm by interaction with trityrosine, whereas tyrosinol caused no significant effect. The change in the transient absorption spectrum by interaction with trityrosine was similar to that obtained with 100 mM imidazole, which showed that the population of the intermediate His/His coordinated species increased during folding of cyt c by interaction with trityrosine. The absorption change was biphasic, the fast phase (82+/-9s(-1)) corresponding to the transition from the His/H(2)O to the His/Met coordinated species, whereas the slow phase (24+/-3s(-1)) from His/His to His/Met. By addition of trityrosine, the relative ratio of the slow phase increased, due to increase of the His/His species at the initial stage of folding. According to the resonance Raman spectra of cyt c, the high-spin 6-coordinate and low-spin 6-coordinate species were dominated at pH 2.3 and 4.2, respectively, and these species were not affected by addition of trityrosine. These results demonstrated that the His/His species increased by interaction with trityrosine at the initial stage of cyt c folding, whereas the heme coordination structure was not affected by trityrosine when the protein was completely unfolded or folded. Hydrophobic peptides thus may be useful to study the effects of hydrophobic interactions on protein folding.
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Abbruzzetti, S., Viappiani, C., Small, J. R., Libertini, L. J., & Small, E. W. (2001). Kinetics of histidine deligation from the heme in GuHCl-unfolded Fe(III) cytochrome C studied by a laser-induced pH-jump technique. J Am Chem Soc, 123(27), 6649–6653.
Abstract: We have developed an instrumental setup that uses transient absorption to monitor protein folding/unfolding processes following a laser-induced, ultrafast release of protons from o-nitrobenzaldehyde. The resulting increase in [H(+)], which can be more than 100 microM, is complete within a few nanoseconds. The increase in [H(+)] lowers the pH of the solution from neutrality to approximately 4 at the highest laser pulse energy used. Protein structural rearrangements can be followed by transient absorption, with kinetic monitoring over a broad time range (approximately 10 ns to 500 ms). Using this pH-jump/transient absorption technique, we have examined the dissociation kinetics of non-native axial heme ligands (either histidine His26 or His33) in GuHCl-unfolded Fe(III) cytochrome c (cyt c). Deligation of the non-native ligands following the acidic pH-jump occurs as a biexponential process with different pre-exponential factors. The pre-exponential factors markedly depend on the extent of the pH-jump, as expected from differences in the pK(a) values of His26 and His33. The two lifetimes were found to depend on temperature but were not functions of either the magnitude of the pH-jump or the pre-pulse pH of the solution. The activation energies of the deligation processes support the suggestion that GuHCl-unfolded cyt c structures with non-native histidine axial ligands represent kinetic traps in unfolding.
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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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Dyson, H. J., & Beattie, J. K. (1982). Spin state and unfolding equilibria of ferricytochrome c in acidic solutions. J Biol Chem, 257(5), 2267–2273.
Abstract: Equilibrium, stopped flow, and temperature-jump spectrophotometry have been used to identify processes in the unfolding of ferricytochrome c in acidic aqueous solutions. A relaxation occurring in approximately 100 microseconds involves perturbation of a spin-equilibrium between two folded conformers of the protein with methionine-80 coordinated or dissociated from the heme iron. The protein unfolds more slowly, in milliseconds, with dissociation and protonation of histidine-18. These two transitions appear cooperative in equilibrium measurements at low (0.01 M) ionic strength, but are separated at higher (0.10 M) ionic strength. They are resolved under both conditions in the dynamic measurements. The spin-equilibrium description permits a unified explanation of a number of properties of ferricytochrome c in acidic aqueous solutions.
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