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Author |
Haruta, N.; Kitagawa, T. |
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Title |
Time-resolved UV resonance Raman investigation of protein folding using a rapid mixer: characterization of kinetic folding intermediates of apomyoglobin |
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Journal Article |
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Year |
2002 |
Publication |
Biochemistry |
Abbreviated Journal |
Biochemistry |
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Volume |
41 |
Issue |
21 |
Pages |
6595-6604 |
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Keywords |
Animals; Apoproteins/*chemistry; Circular Dichroism; Holoenzymes/chemistry; Horses; Hydrochloric Acid/chemistry; Hydrogen-Ion Concentration; Imidazoles/chemistry; Kinetics; Models, Molecular; Myoglobin/*chemistry; Peptide Fragments/chemistry; *Protein Folding; Protein Structure, Secondary; Spectrum Analysis, Raman/*methods; Tryptophan/*chemistry; Ultraviolet Rays; Whales |
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Abstract |
The 244-nm excited transient UV resonance Raman spectra are observed for the refolding intermediates of horse apomyoglobin (h-apoMb) with a newly constructed mixed flow cell system, and the results are interpreted on the basis of the spectra observed for the equilibrium acid unfolding of the same protein. The dead time of mixing, which was determined with the appearance of UV Raman bands of imidazolium upon mixing of imidazole with acid, was 150 micros under the flow rate that was adopted. The pH-jump experiments of h-apoMb from pH 2.2 to 5.6 conducted with this device demonstrated the presence of three folding intermediates. On the basis of the analysis of W3 and W7 bands of Trp7 and Trp14, the first intermediate, formed before 250 micros, involved incorporation of Trp14 into the alpha-helix from a random coil. The frequency shift of the W3 band of Trp14 observed for this process was reproduced with a model peptide of the A helix when it forms the alpha-helix. In the second intermediate, formed around 1 ms after the start of refolding, the surroundings of both Trp7 and Trp14 were significantly hydrophobic, suggesting the formation of the hydrophobic core. In the third intermediate appearing around 3 ms, the hydrophobicity was relaxed to the same level as that of the pH 4 equilibrium intermediate, which was investigated in detail with the stationary state technique. The change from the third intermediate to the native state needs more time than 40 ms, while the appearance of the native spectrum after the mixing of the same solutions was confirmed separately. |
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School of Mathematical and Physical Sciences, The Graduate University for Advanced Studies, Myodaiji, Okazaki 444-8585, Japan |
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0006-2960 |
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PMID:12022863 |
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Call Number |
Equine Behaviour @ team @ |
Serial |
3785 |
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Author |
Abbruzzetti, S.; Viappiani, C.; Small, J.R.; Libertini, L.J.; Small, E.W. |
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Title |
Kinetics of histidine deligation from the heme in GuHCl-unfolded Fe(III) cytochrome C studied by a laser-induced pH-jump technique |
Type |
Journal Article |
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Year |
2001 |
Publication |
Journal of the American Chemical Society |
Abbreviated Journal |
J Am Chem Soc |
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Volume |
123 |
Issue |
27 |
Pages |
6649-6653 |
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Keywords |
Animals; *Bacterial Proteins; Cytochrome c Group/*chemistry; Guanidine/*chemistry; Heme/*chemistry; Histidine/*chemistry; Horses; Hydrogen-Ion Concentration; Kinetics; *Lasers; Ligands; Protein Folding |
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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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Dipartimento di Fisica, Universita di Parma, Istituto Nazionale per la Fisica della Materia, 43100 Parma, Italy |
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0002-7863 |
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PMID:11439052 |
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Call Number |
Equine Behaviour @ team @ |
Serial |
3788 |
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Author |
Gulotta, M.; Gilmanshin, R.; Buscher, T.C.; Callender, R.H.; Dyer, R.B. |
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Title |
Core formation in apomyoglobin: probing the upper reaches of the folding energy landscape |
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Journal Article |
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Year |
2001 |
Publication |
Biochemistry |
Abbreviated Journal |
Biochemistry |
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Volume |
40 |
Issue |
17 |
Pages |
5137-5143 |
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Keywords |
Animals; Apoproteins/*chemistry; Computer Simulation; Horses; Hydrogen-Ion Concentration; Kinetics; Models, Molecular; Myoglobin/*chemistry; *Protein Folding; Protein Structure, Secondary; Protein Structure, Tertiary; Spectrometry, Fluorescence/instrumentation/methods; Thermodynamics; Tryptophan/chemistry |
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Abstract |
An acid-destabilized form of apomyoglobin, the so-called E state, consists of a set of heterogeneous structures that are all characterized by a stable hydrophobic core composed of 30-40 residues at the intersection of the A, G, and H helices of the protein, with little other secondary structure and no other tertiary structure. Relaxation kinetics studies were carried out to characterize the dynamics of core melting and formation in this protein. The unfolding and/or refolding response is induced by a laser-induced temperature jump between the folded and unfolded forms of E, and structural changes are monitored using the infrared amide I' absorbance at 1648-1651 cm(-1) that reports on the formation of solvent-protected, native-like helix in the core and by fluorescence emission changes from apomyoglobin's Trp14, a measure of burial of the indole group of this residue. The fluorescence kinetics data are monoexponential with a relaxation time of 14 micros. However, infrared kinetics data are best fit to a biexponential function with relaxation times of 14 and 59 micros. These relaxation times are very fast, close to the limits placed on folding reactions by diffusion. The 14 micros relaxation time is weakly temperature dependent and thus represents a pathway that is energetically downhill. The appearance of this relaxation time in both the fluorescence and infrared measurements indicates that this folding event proceeds by a concomitant formation of compact secondary and tertiary structures. The 59 micros relaxation time is much more strongly temperature dependent and has no fluorescence counterpart, indicating an activated process with a large energy barrier wherein nonspecific hydrophobic interactions between helix A and the G and H helices cause some helix burial but Trp14 remains solvent exposed. These results are best fit by a multiple-pathway kinetic model when U collapses to form the various folded core structures of E. Thus, the results suggest very robust dynamics for core formation involving multiple folding pathways and provide significant insight into the primary processes of protein folding. |
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Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, USA |
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0006-2960 |
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PMID:11318635 |
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Equine Behaviour @ team @ |
Serial |
3789 |
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Author |
Abbruzzetti, S.; Crema, E.; Masino, L.; Vecli, A.; Viappiani, C.; Small, J.R.; Libertini, L.J.; Small, E.W. |
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Title |
Fast events in protein folding: structural volume changes accompanying the early events in the N-->I transition of apomyoglobin induced by ultrafast pH jump |
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Journal Article |
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Year |
2000 |
Publication |
Biophysical Journal |
Abbreviated Journal |
Biophys J |
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Volume |
78 |
Issue |
1 |
Pages |
405-415 |
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Keywords |
Animals; Apoproteins/*chemistry; Horses; *Hydrogen-Ion Concentration; Kinetics; Models, Molecular; Myoglobin/*chemistry; Protein Conformation; *Protein Folding; Protein Structure, Secondary; Spectrometry, Fluorescence |
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Ultrafast, laser-induced pH jump with time-resolved photoacoustic detection has been used to investigate the early protonation steps leading to the formation of the compact acid intermediate (I) of apomyoglobin (ApoMb). When ApoMb is in its native state (N) at pH 7.0, rapid acidification induced by a laser pulse leads to two parallel protonation processes. One reaction can be attributed to the binding of protons to the imidazole rings of His24 and His119. Reaction with imidazole leads to an unusually large contraction of -82 +/- 3 ml/mol, an enthalpy change of 8 +/- 1 kcal/mol, and an apparent bimolecular rate constant of (0.77 +/- 0.03) x 10(10) M(-1) s(-1). Our experiments evidence a rate-limiting step for this process at high ApoMb concentrations, characterized by a value of (0. 60 +/- 0.07) x 10(6) s(-1). The second protonation reaction at pH 7. 0 can be attributed to neutralization of carboxylate groups and is accompanied by an apparent expansion of 3.4 +/- 0.2 ml/mol, occurring with an apparent bimolecular rate constant of (1.25 +/- 0.02) x 10(11) M(-1) s(-1), and a reaction enthalpy of about 2 kcal/mol. The activation energy for the processes associated with the protonation of His24 and His119 is 16.2 +/- 0.9 kcal/mol, whereas that for the neutralization of carboxylates is 9.2 +/- 0.9 kcal/mol. At pH 4.5 ApoMb is in a partially unfolded state (I) and rapid acidification experiments evidence only the process assigned to carboxylate protonation. The unusually large contraction and the high energetic barrier observed at pH 7.0 for the protonation of the His residues suggests that the formation of the compact acid intermediate involves a rate-limiting step after protonation. |
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Dipartimento di Fisica, Universita di Parma, 43100 Parma, Italia |
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0006-3495 |
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PMID:10620304 |
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Equine Behaviour @ team @ |
Serial |
3792 |
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Author |
Gilmanshin, R.; Callender, R.H.; Dyer, R.B. |
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Title |
The core of apomyoglobin E-form folds at the diffusion limit |
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Journal Article |
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Year |
1998 |
Publication |
Nature Structural Biology |
Abbreviated Journal |
Nat Struct Biol |
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Volume |
5 |
Issue |
5 |
Pages |
363-365 |
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Keywords |
Animals; Apoproteins/*chemistry; Diffusion; Horses; Myoglobin/*chemistry; *Protein Folding; Spectroscopy, Fourier Transform Infrared; Temperature |
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The E-form of apomyoglobin has been characterized using infrared and fluorescence spectroscopies, revealing a compact core with native like contacts, most probably consisting of 15-20 residues of the A, G and H helices of apomyoglobin. Fast temperature-jump, time-resolved infrared measurements reveal that the core is formed within 96 micros at 46 degrees C, close to the diffusion limit for loop formation. Remarkably, the folding pathway of the E-form is such that the formation of a limited number of native-like contacts is not rate limiting, or that the contacts form on the same time scale expected for diffusion controlled loop formation. |
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1072-8368 |
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PMID:9586997 |
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Equine Behaviour @ team @ |
Serial |
3795 |
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Author |
Ballew, R.M.; Sabelko, J.; Gruebele, M. |
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Direct observation of fast protein folding: the initial collapse of apomyoglobin |
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Journal Article |
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1996 |
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Proceedings of the National Academy of Sciences of the United States of America |
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Proc. Natl. Acad. Sci. U.S.A. |
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Volume |
93 |
Issue |
12 |
Pages |
5759-5764 |
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Keywords |
Animals; Apoproteins/*chemistry; Circular Dichroism; Horses; Kinetics; Muscle, Skeletal/chemistry; Myoglobin/*chemistry; *Protein Folding; Spectrometry, Fluorescence; Spectrophotometry, Infrared; Temperature |
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The rapid refolding dynamics of apomyoglobin are followed by a new temperature-jump fluorescence technique on a 15-ns to 0.5-ms time scale in vitro. The apparatus measures the protein-folding history in a single sweep in standard aqueous buffers. The earliest steps during folding to a compact state are observed and are complete in under 20 micros. Experiments on mutants and consideration of steady-state CD and fluorescence spectra indicate that the observed microsecond phase monitors assembly of an A x (H x G) helix subunit. Measurements at different viscosities indicate diffusive behavior even at low viscosities, in agreement with motions of a solvent-exposed protein during the initial collapse. |
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School of Chemical Sciences and Beckman Institute for Advanced Science and Technology, University of Illinois, Urbana, 61801, USA |
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0027-8424 |
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PMID:8650166 |
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Call Number |
Equine Behaviour @ team @ |
Serial |
3798 |
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