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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 |
Type |
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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no |
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Call Number |
Equine Behaviour @ team @ |
Serial |
3792 |
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Author |
Ballew, R.M.; Sabelko, J.; Gruebele, M. |
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Title |
Direct observation of fast protein folding: the initial collapse of apomyoglobin |
Type |
Journal Article |
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Year |
1996 |
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 |
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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Abstract |
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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Notes |
PMID:8650166 |
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no |
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Call Number |
Equine Behaviour @ team @ |
Serial |
3798 |
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Author |
Chiba, K.; Ikai, A.; Kawamura-Konishi, Y.; Kihara, H. |
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Title |
Kinetic study on myoglobin refolding monitored by five optical probe stopped-flow methods |
Type |
Journal Article |
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Year |
1994 |
Publication |
Proteins |
Abbreviated Journal |
Proteins |
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Volume |
19 |
Issue |
2 |
Pages |
110-119 |
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Keywords |
Animals; Chromatography, Gel; Circular Dichroism; Horses; Kinetics; Metmyoglobin/analogs & derivatives/chemistry; Myoglobin/*chemistry; *Protein Folding; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet; Urea |
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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. |
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Laboratory of Biodynamics, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Kanagawa, Japan |
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English |
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ISSN |
0887-3585 |
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Notes |
PMID:8090705 |
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no |
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Call Number |
Equine Behaviour @ team @ |
Serial |
3799 |
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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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Address |
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, USA |
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English |
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ISSN |
0006-2960 |
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Notes |
PMID:11318635 |
Approved |
no |
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Call Number |
Equine Behaviour @ team @ |
Serial |
3789 |
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Author |
Hagen, S.J.; Eaton, W.A. |
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Title |
Two-state expansion and collapse of a polypeptide |
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Journal Article |
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Year |
2000 |
Publication |
Journal of Molecular Biology |
Abbreviated Journal |
J Mol Biol |
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Volume |
301 |
Issue |
4 |
Pages |
1019-1027 |
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Keywords |
Animals; Computer Simulation; Cytochrome c Group/*chemistry/*metabolism; Horses; Kinetics; Lasers; Models, Chemical; Peptides/*chemistry/*metabolism; Protein Conformation; Protein Denaturation; *Protein Folding; Spectrometry, Fluorescence; Temperature; Thermodynamics |
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Abstract |
The initial phase of folding for many proteins is presumed to be the collapse of the polypeptide chain from expanded to compact, but still denatured, conformations. Theory and simulations suggest that this collapse may be a two-state transition, characterized by barrier-crossing kinetics, while the collapse of homopolymers is continuous and multi-phasic. We have used a laser temperature-jump with fluorescence spectroscopy to measure the complete time-course of the collapse of denatured cytochrome c with nanosecond time resolution. We find the process to be exponential in time and thermally activated, with an apparent activation energy approximately 9 k(B)T (after correction for solvent viscosity). These results indicate that polypeptide collapse is kinetically a two-state transition. Because of the observed free energy barrier, the time scale of polypeptide collapse is dramatically slower than is predicted by Langevin models for homopolymer collapse. |
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Address |
Laboratory of Chemical Physics, NIDDK, National Institutes of Health, Building 5, Bethesda, MD, 20892-0520, USA |
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English |
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ISSN |
0022-2836 |
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Notes |
PMID:10966803 |
Approved |
no |
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Call Number |
Equine Behaviour @ team @ |
Serial |
3790 |
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Author |
Jallon, J.M.; Risler, Y.; Iwatsubo, M. |
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Title |
Beef liver L-Glutamate dehydrogenase mechanism: presteady state study of the catalytic reduction of 2.oxoglutarate by NADPH |
Type |
Journal Article |
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Year |
1975 |
Publication |
Biochemical and biophysical research communications |
Abbreviated Journal |
Biochem Biophys Res Commun |
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Volume |
67 |
Issue |
4 |
Pages |
1527-1536 |
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Keywords |
Animals; Cattle; Glutamate Dehydrogenase/*metabolism; Ketoglutaric Acids; Kinetics; Liver/*enzymology; Nadp; Oxidation-Reduction; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet |
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English |
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0006-291X |
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Notes |
PMID:1038 |
Approved |
no |
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Call Number |
Admin @ knut @ |
Serial |
21 |
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Author |
Permyakov, S.E.; Khokhlova, T.I.; Nazipova, A.A.; Zhadan, A.P.; Morozova-Roche, L.A.; Permyakov, E.A. |
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Title |
Calcium-binding and temperature induced transitions in equine lysozyme: new insights from the pCa-temperature “phase diagrams” |
Type |
Journal Article |
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Year |
2006 |
Publication |
Proteins |
Abbreviated Journal |
Proteins |
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Volume |
65 |
Issue |
4 |
Pages |
984-998 |
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Keywords |
Animals; Apoproteins/chemistry/metabolism; Binding Sites; Calcium/chemistry/*metabolism; Cattle; Edetic Acid/metabolism; Horses/metabolism; Hydrogen-Ion Concentration; Lactalbumin/chemistry/metabolism; Muramidase/*chemistry/*metabolism; Protein Denaturation; Spectrometry, Fluorescence; *Temperature; Thermodynamics; Tryptophan/chemistry/metabolism |
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Abstract |
The most universal approach to the studies of metal binding properties of single-site metal binding proteins, i.e., construction of a “phase diagram” in coordinates of free metal ion concentration-temperature, has been applied to equine lysozyme (EQL). EQL has one relatively strong calcium binding site and shows two thermal transitions, but only one of them is Ca(2+)-dependent. It has been found that the Ca(2+)-dependent behavior of the low temperature thermal transition (I) of EQL can be adequately described based upon the simplest four-states scheme of metal- and temperature-induced structural changes in a protein. All thermodynamic parameters of this scheme were determined experimentally and used for construction of the EQL phase diagram in the pCa-temperature space. Comparison of the phase diagram with that for alpha-lactalbumin (alpha-LA), a close homologue of lysozyme, allows visualization of the differences in thermodynamic behavior of the two proteins. The thermal stability of apo-EQL (transition I) closely resembles that for apo-alpha-LA (mid-temperature 25 degrees C), while the thermal stabilities of their Ca(2+)-bound forms are almost indistinguishable. The native state of EQL has three orders of magnitude lower affinity for Ca(2+) in comparison with alpha-LA while its thermally unfolded state (after the I transition) has about one order lower (K = 15M(-1)) affinity for calcium. Circular dichroism studies of the apo-lysozyme state after the first thermal transition show that it shares common features with the molten globule state of alpha-LA. |
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Address |
Institute for Biological Instrumentation of the Russian Academy of Sciences, Pushchino, Moscow region 142290, Russia |
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ISSN |
1097-0134 |
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Notes |
PMID:17022083 |
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no |
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Call Number |
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Serial |
1858 |
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Author |
Rodier, F. |
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Title |
[Spectral properties of porcine plasminogen: study of the acidic transition (author's transl)] |
Type |
Journal Article |
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Year |
1976 |
Publication |
European journal of biochemistry / FEBS |
Abbreviated Journal |
Eur J Biochem |
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Volume |
63 |
Issue |
2 |
Pages |
553-562 |
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Keywords |
Animals; Binding Sites; Guanidines; Hydrogen-Ion Concentration; *Plasminogen; Protein Binding; Protein Conformation; Spectrometry, Fluorescence; Spectrophotometry; Spectrophotometry, Ultraviolet; Swine; Temperature |
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Abstract |
The acidic transition of porcine plasminogen, prepared by affinity chromatography, was studied by non-destructive methods. These methods are based on the analysis of the behaviour of the tryptophyls under various conditions. The perturbation of the absorption and emission spectra by pH or temperature and the dynamic quenching of the intrinsic fluorescence are used to obtain information on structural changes which affect the environment of these residues. It is shown that by decreasing pH the fluorescence emission spectra are shifted toward the long wavelengths, with a broadening of the fluorescence band. The same effect can be obtained at constant pH by heating the protein solution. In order to analyze these phenomena, it is assumed that the fluorescence intensities at 355 nm and 328 nm reflect the proportion of the tryptophans which are exposed to the solvent, and buried, respectively. The plot of the ratio of the fluorescence intensities at these wavelengths versus pH or temperature leads to a titration curve showing an unmasking of tryptophans. The proportion of exposed tryptophans is measured by the dynamic fluorescence quenching technique and the data analyzed according to Lehrer. The plot of the fraction of exposed tryptophyls versus pH also shows the unmasking of these chromophores. Thermal perturbation of a solution of plaminogen at neutral pH induces a difference absorption spectrum whose amplitudes at the maxima are proportional to the number of exposed aromatic residues. The comparison with a solution of fully denatured plasminogen in 6 M guanidium chloride, where all the tryptophyls are exposed, shows that the percentage of exposure is equal to 59%. This number is significantly higher than the percentage found by the fluorescence quenching technique (20%), indicating that some tryptophyls are located in crevices, exposed to the solvent but not to the iodide. At acidic pH the absorption difference spectra induced by thermal perturbation are not classical, since they show an inversion and a new band between 300 nm and 305 nm. This band is mentioned in the literature as a minor band of tryptophan which appears when this chromophore is located in an asymmetric environment. On plotting the maximum amplitude of these spectra obtained at acidic pH versus temperature, we obtain a curve indicating that two types of antagonistic interactions are involved in the perturbation of the chromophores spectra. The spectrophotometric titration of plasminogen gives classical absorption difference spectra. By plotting the maximum amplitude at 292 nm versus pH, we obtain a titration curve with an apparent pK of 2.9 units. This pK is acidic which respect to the pK value of a normal carboxyl. This low value can be due to a positively charged group in the neighbourhood of a carboxyl, which interacts with one or more chromophores. When the carboxyl becomes protonated, this positively charged group is free and available to perturb the environment of some chromophores... |
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French |
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Original Title |
Proprietes spectrales du plasminogene porcin. Etude de la transition acide |
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0014-2956 |
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Notes |
PMID:4326 |
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Call Number |
Admin @ knut @ |
Serial |
22 |
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Author |
Tsong, T.Y. |
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Title |
Conformational relaxations of urea- and guanidine hydrochloride-unfolded ferricytochrome c |
Type |
Journal Article |
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Year |
1977 |
Publication |
The Journal of Biological Chemistry |
Abbreviated Journal |
J Biol Chem |
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Volume |
252 |
Issue |
24 |
Pages |
8778-8780 |
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Keywords |
*Cytochrome c Group; Guanidines/*pharmacology; Protein Conformation/drug effects; Spectrometry, Fluorescence; Urea/*pharmacology |
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Abstract |
Several recent studies of protein the unfolded proteins. In urea- and guanidine HCl-unfolded ferricytochrome c (horse heart), an acid-induced spin state transformation of the heme group has been detected by the heme absorptions, Trp-59 fluorescence, and the intrinsic viscosity of protein. Kinetics of this second conformational transition, by the temperature jump and stopped flow methods, are complex. One rapid reaction (tau1), pH-independent, occurs in a 50-mus range; the second reaction (tau2), in a 1-ms range, depends linearly upon pH and is faster at the alkaline side; a third reaction (tau3), in a 1-s range, shows a sigmoidal transition at pH 5.1 and is faster at the acidic side. The results are consistent with a kinetic scheme which involves protein conformational changes in the transformation of the heme coordination state. The kinetics, along with previous equilibrium studies, indicate that ligand or charge interactions within a protein molecule are not completely prohibited even in strongly denaturing conditions, such as in high concentrations of urea and guanidine HCl. Thus, local structures of peptide chain associated with these interactions can exist in the unfolded protein. |
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English |
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0021-9258 |
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Notes |
PMID:200618 |
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Call Number |
refbase @ user @ |
Serial |
3882 |
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