| Records |
| Author |
Chiba, K.; Ikai, A.; Kawamura-Konishi, Y.; Kihara, H. |
| Title |
Kinetic study on myoglobin refolding monitored by five optical probe stopped-flow methods |
Type |
Journal Article |
| Year |
1994 |
Publication |
Proteins |
Abbreviated Journal |
Proteins |
| Volume |
19 |
Issue |
2 |
Pages |
110-119 |
| Keywords |
Animals; Chromatography, Gel; Circular Dichroism; Horses; Kinetics; Metmyoglobin/analogs & derivatives/chemistry; Myoglobin/*chemistry; *Protein Folding; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet; Urea |
| Abstract |
The refolding kinetics of horse cyanometmyoglobin induced by concentration jump of urea was investigated by five optical probe stopped-flow methods: absorption at 422 nm, tryptophyl fluorescence at around 340 nm, circular dichroism (CD) at 222 nm, CD at 260 nm, and CD at 422 nm. In the refolding process, we detected three phases with rate constants of > 1 x 10(2) s-1, (4.5-9.3) s-1, and (2-5) x 10(-3) s-1. In the fastest phase, a substantial amount of secondary structure (approximately 40%) is formed within the dead time of the CD stopped-flow apparatus (10.7 ms). The kinetic intermediate populated in the fastest phase is shown to capture a hemindicyanide, suggesting that a “heme pocket precursor” recognized by hemindicyanide must be constructed within the dead time. In the middle phase, most of secondary and tertiary structures, especially around the captured hemindicyanide, have been constructed. In the slowest phase, we detected a minor structural rearrangement accompanying the ligand-exchange reaction in the fifth coordination of ferric iron. We present a possible model for the refolding process of myoglobin in the presence of the heme group. |
| Address |
Laboratory of Biodynamics, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Kanagawa, Japan |
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Thesis |
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Place of Publication |
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Editor |
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| Language |
English |
Summary Language |
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Original Title |
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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 |
0887-3585 |
ISBN |
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Medium |
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| Area |
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Expedition |
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Conference |
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| Notes |
PMID:8090705 |
Approved |
no |
| Call Number |
Equine Behaviour @ team @ |
Serial  |
3799 |
| Permanent link to this record |
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| Author |
Pierce, M.M.; Nall, B.T. |
| Title |
Coupled kinetic traps in cytochrome c folding: His-heme misligation and proline isomerization |
Type |
Journal Article |
| Year |
2000 |
Publication |
Journal of Molecular Biology |
Abbreviated Journal |
J Mol Biol |
| Volume |
298 |
Issue |
5 |
Pages |
955-969 |
| Keywords |
Amino Acid Sequence; Amino Acid Substitution/genetics; Binding Sites; Cytochrome c Group/*chemistry/genetics/*metabolism; *Cytochromes c; Enzyme Stability/drug effects; Fluorescence; Guanidine/pharmacology; Heme/*metabolism; Histidine/genetics/*metabolism; Hydrogen-Ion Concentration; Isomerism; Kinetics; Models, Molecular; Molecular Sequence Data; Mutation/genetics; Proline/*chemistry/metabolism; Protein Conformation/drug effects; Protein Denaturation/drug effects; *Protein Folding; Protein Renaturation; Saccharomyces cerevisiae/enzymology/genetics; Sequence Alignment; Thermodynamics |
| 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. |
| Address |
Center for Biomolecular Structure, Department of Biochemistry, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, USA |
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English |
Summary Language |
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Original Title |
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Series Title |
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Abbreviated Series Title |
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Edition |
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| ISSN |
0022-2836 |
ISBN |
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Expedition |
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Conference |
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| Notes |
PMID:10801361 |
Approved |
no |
| Call Number |
refbase @ user @ |
Serial |
3853 |
| Permanent link to this record |
