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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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Abstract |
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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Call Number |
Equine Behaviour @ team @ |
Serial |
3792 |
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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 |
Type |
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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Call Number |
Equine Behaviour @ team @ |
Serial |
3789 |
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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 |
Uzawa, T.; Akiyama, S.; Kimura, T.; Takahashi, S.; Ishimori, K.; Morishima, I.; Fujisawa, T. |
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Title |
Collapse and search dynamics of apomyoglobin folding revealed by submillisecond observations of alpha-helical content and compactness |
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Journal Article |
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Year |
2004 |
Publication |
Proceedings of the National Academy of Sciences of the United States of America |
Abbreviated Journal |
Proc. Natl. Acad. Sci. U.S.A. |
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Volume |
101 |
Issue |
5 |
Pages |
1171-1176 |
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Keywords |
Animals; Apoproteins/*chemistry; Circular Dichroism; Cytochromes c/chemistry; Horses; Myoglobin/*chemistry; *Protein Folding; *Protein Structure, Secondary; Scattering, Radiation |
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Abstract |
The characterization of protein folding dynamics in terms of secondary and tertiary structures is important in elucidating the features of intraprotein interactions that lead to specific folded structures. Apomyoglobin (apoMb), possessing seven helices termed A-E, G, and H in the native state, has a folding intermediate composed of the A, G, and H helices, whose formation in the submillisecond time domain has not been clearly characterized. In this study, we used a rapid-mixing device combined with circular dichroism and small-angle x-ray scattering to observe the submillisecond folding dynamics of apoMb in terms of helical content (f(H)) and radius of gyration (R(g)), respectively. The folding of apoMb from the acid-unfolded state at pH 2.2 was initiated by a pH jump to 6.0. A significant collapse, corresponding to approximately 50% of the overall change in R(g) from the unfolded to native conformation, was observed within 300 micros after the pH jump. The collapsed intermediate has a f(H) of 33% and a globular shape that involves >80% of all its atoms. Subsequently, a stepwise helix formation was detected, which was interpreted to be associated with a conformational search for the correct tertiary contacts. The characterized folding dynamics of apoMb indicates the importance of the initial collapse event, which is suggested to facilitate the subsequent conformational search and the helix formation leading to the native structure. |
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Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Nishikyo, Kyoto 615-8510, Japan |
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0027-8424 |
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PMID:14711991 |
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Equine Behaviour @ team @ |
Serial |
3779 |
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