Records |
Author |
Uzawa, T.; Akiyama, S.; Kimura, T.; Takahashi, S.; Ishimori, K.; Morishima, I.; Fujisawa, T. |
Title |
Collapse and search dynamics of apomyoglobin folding revealed by submillisecond observations of alpha-helical content and compactness |
Type |
Journal Article |
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. |
Volume |
101 |
Issue |
5 |
Pages |
1171-1176 |
Keywords |
Animals; Apoproteins/*chemistry; Circular Dichroism; Cytochromes c/chemistry; Horses; Myoglobin/*chemistry; *Protein Folding; *Protein Structure, Secondary; Scattering, Radiation |
Abstract ![sorted by Abstract field, descending order (down)](img/sort_desc.gif) |
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. |
Address |
Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Nishikyo, Kyoto 615-8510, Japan |
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English |
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Series Volume |
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Series Issue |
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Edition |
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ISSN |
0027-8424 |
ISBN |
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Medium |
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Conference |
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Notes |
PMID:14711991 |
Approved |
no |
Call Number |
Equine Behaviour @ team @ |
Serial |
3779 |
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Author |
Hoang, L.; Maity, H.; Krishna, M.M.G.; Lin, Y.; Englander, S.W. |
Title |
Folding units govern the cytochrome c alkaline transition |
Type |
Journal Article |
Year |
2003 |
Publication |
Journal of Molecular Biology |
Abbreviated Journal |
J Mol Biol |
Volume |
331 |
Issue |
1 |
Pages |
37-43 |
Keywords |
Animals; Cytochrome c Group/*chemistry; Horses; Hydrogen/chemistry; Hydrogen-Ion Concentration; Kinetics; Models, Molecular; *Protein Folding; Protein Structure, Tertiary; Spectrum Analysis; Titrimetry |
Abstract ![sorted by Abstract field, descending order (down)](img/sort_desc.gif) |
The alkaline transition of cytochrome c is a model for protein structural switching in which the normal heme ligand is replaced by another group. Stopped flow data following a jump to high pH detect two slow kinetic phases, suggesting two rate-limiting structure changes. Results described here indicate that these events are controlled by the same structural unfolding reactions that account for the first two steps in the reversible unfolding pathway of cytochrome c. These and other results show that the cooperative folding-unfolding behavior of protein foldons can account for a variety of functional activities in addition to determining folding pathways. |
Address |
Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6059, USA. lhoang@mail.upenn.edu |
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Language |
English |
Summary Language |
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Series Issue |
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Edition |
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ISSN |
0022-2836 |
ISBN |
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Medium |
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Conference |
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Notes |
PMID:12875834 |
Approved |
no |
Call Number |
Equine Behaviour @ team @ |
Serial |
3781 |
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Author |
Haruta, N.; Kitagawa, T. |
Title |
Time-resolved UV resonance Raman investigation of protein folding using a rapid mixer: characterization of kinetic folding intermediates of apomyoglobin |
Type |
Journal Article |
Year |
2002 |
Publication |
Biochemistry |
Abbreviated Journal |
Biochemistry |
Volume |
41 |
Issue |
21 |
Pages |
6595-6604 |
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 |
Abstract ![sorted by Abstract field, descending order (down)](img/sort_desc.gif) |
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. |
Address |
School of Mathematical and Physical Sciences, The Graduate University for Advanced Studies, Myodaiji, Okazaki 444-8585, Japan |
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English |
Summary Language |
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Series Editor |
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Series Title |
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Abbreviated Series Title |
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Series Volume |
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Series Issue |
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Edition |
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ISSN |
0006-2960 |
ISBN |
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Medium |
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Area |
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Conference |
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Notes |
PMID:12022863 |
Approved |
no |
Call Number |
Equine Behaviour @ team @ |
Serial |
3785 |
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Author |
Hirota, S.; Suzuki, M.; Watanabe, Y. |
Title |
Hydrophobic effect of trityrosine on heme ligand exchange during folding of cytochrome c |
Type |
Journal Article |
Year |
2004 |
Publication |
Biochemical and Biophysical Research Communications |
Abbreviated Journal |
Biochem Biophys Res Commun |
Volume |
314 |
Issue |
2 |
Pages |
452-458 |
Keywords |
Amino Acids/chemistry; Animals; Cytochromes c/*chemistry; Heme/*chemistry; Histidine/chemistry; Horses; Hydrogen-Ion Concentration; Kinetics; Ligands; Myocardium/chemistry; Peptides/chemistry; Protein Folding; Spectrophotometry; Spectrum Analysis, Raman; Tyrosine/*analogs & derivatives/*chemistry |
Abstract ![sorted by Abstract field, descending order (down)](img/sort_desc.gif) |
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. |
Address |
Department of Physical Chemistry, Kyoto Pharmaceutical University, Yamashina-ku, 607-8414 Kyoto, Japan. hirota@mb.kyoto-phu.ac.jp |
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Place of Publication |
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English |
Summary Language |
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Abbreviated Series Title |
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Edition |
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ISSN |
0006-291X |
ISBN |
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Conference |
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Notes |
PMID:14733927 |
Approved |
no |
Call Number |
Equine Behaviour @ team @ |
Serial |
3777 |
Permanent link to this record |
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Author |
Miksovska, J.; Larsen, R.W. |
Title |
Photothermal studies of pH induced unfolding of apomyoglobin |
Type |
Journal Article |
Year |
2003 |
Publication |
Journal of Protein Chemistry |
Abbreviated Journal |
J Protein Chem |
Volume |
22 |
Issue |
4 |
Pages |
387-394 |
Keywords |
Acoustics; Animals; Apoproteins/*chemistry/metabolism; Circular Dichroism; Horses; Myocardium/chemistry; Myoglobin/*chemistry/metabolism; Photolysis; Protein Conformation/radiation effects; Protein Denaturation/radiation effects; *Protein Folding; Temperature; Thermodynamics |
Abstract ![sorted by Abstract field, descending order (down)](img/sort_desc.gif) |
Conformational dynamic and enthalpy changes associated with pH induced unfolding of apomyoglobin were studied using photoacoustic calorimetry and photothermal beam deflection methods. The transition between the native state and the I intermediate was induced by a nanosecond pH jump from o-nitrobenzaldehyde photolysis. Deconvolution of photoacoustic waves indicates two kinetic processes. The fast phase (T < 50 ns) is characterized by a volume expansion of 8.8 ml mol(-1). This process is followed by a volume contraction of about -22 ml mol(-1) (tau approximately 500 ns). Photothermal beam deflection measurements do not reveal any volume changes on the time scale between approximately 100 micros and 5 ms. We associate the volume contraction with structural changes occurring during the transition between the native state and the I intermediate. The lack of any processes on the ms time scale may indicate the absence of structural events involving larger conformational changes of apomyoglobin after the pH jump. |
Address |
Department of Chemistry, University of South Florida, Tampa, Florida 33620, USA |
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English |
Summary Language |
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Series Editor |
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Abbreviated Series Title |
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Edition |
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ISSN |
0277-8033 |
ISBN |
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Medium |
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Area |
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Conference |
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Notes |
PMID:13678303 |
Approved |
no |
Call Number |
Equine Behaviour @ team @ |
Serial |
3780 |
Permanent link to this record |
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Author |
Gulotta, M.; Gilmanshin, R.; Buscher, T.C.; Callender, R.H.; Dyer, R.B. |
Title |
Core formation in apomyoglobin: probing the upper reaches of the folding energy landscape |
Type |
Journal Article |
Year |
2001 |
Publication |
Biochemistry |
Abbreviated Journal |
Biochemistry |
Volume |
40 |
Issue |
17 |
Pages |
5137-5143 |
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 |
Abstract ![sorted by Abstract field, descending order (down)](img/sort_desc.gif) |
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. |
Address |
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, USA |
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English |
Summary Language |
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Series Editor |
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Edition |
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ISSN |
0006-2960 |
ISBN |
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Notes |
PMID:11318635 |
Approved |
no |
Call Number |
Equine Behaviour @ team @ |
Serial |
3789 |
Permanent link to this record |