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Uzawa, T., Akiyama, S., Kimura, T., Takahashi, S., Ishimori, K., Morishima, I., et al. (2004). Collapse and search dynamics of apomyoglobin folding revealed by submillisecond observations of alpha-helical content and compactness. Proc. Natl. Acad. Sci. U.S.A., 101(5), 1171–1176.
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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Gulotta, M., Gilmanshin, R., Buscher, T. C., Callender, R. H., & Dyer, R. B. (2001). Core formation in apomyoglobin: probing the upper reaches of the folding energy landscape. Biochemistry, 40(17), 5137–5143.
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.
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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Pierce, M. M., & Nall, B. T. (2000). Coupled kinetic traps in cytochrome c folding: His-heme misligation and proline isomerization. J Mol Biol, 298(5), 955–969.
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.
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
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Ballew, R. M., Sabelko, J., & Gruebele, M. (1996). Direct observation of fast protein folding: the initial collapse of apomyoglobin. Proc. Natl. Acad. Sci. U.S.A., 93(12), 5759–5764.
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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Abbruzzetti, S., Crema, E., Masino, L., Vecli, A., Viappiani, C., Small, J. R., et al. (2000). Fast events in protein folding: structural volume changes accompanying the early events in the N-->I transition of apomyoglobin induced by ultrafast pH jump. Biophys J, 78(1), 405–415.
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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Hoang, L., Maity, H., Krishna, M. M. G., Lin, Y., & Englander, S. W. (2003). Folding units govern the cytochrome c alkaline transition. J Mol Biol, 331(1), 37–43.
Abstract: 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.
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Chiba, K., Ikai, A., Kawamura-Konishi, Y., & Kihara, H. (1994). Kinetic study on myoglobin refolding monitored by five optical probe stopped-flow methods. Proteins, 19(2), 110–119.
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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Miksovska, J., & Larsen, R. W. (2003). Photothermal studies of pH induced unfolding of apomyoglobin. J Protein Chem, 22(4), 387–394.
Abstract: 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.
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Gulotta, M., Rogatsky, E., Callender, R. H., & Dyer, R. B. (2003). Primary folding dynamics of sperm whale apomyoglobin: core formation. Biophys J, 84(3), 1909–1918.
Abstract: The structure, thermodynamics, and kinetics of heat-induced unfolding of sperm whale apomyoglobin core formation have been studied. The most rudimentary core is formed at pH(*) 3.0 and up to 60 mM NaCl. Steady state for ultraviolet circular dichroism and fluorescence melting studies indicate that the core in this acid-destabilized state consists of a heterogeneous composition of structures of approximately 26 residues, two-thirds of the number involved for horse heart apomyoglobin under these conditions. Fluorescence temperature-jump relaxation studies show that there is only one process involved in Trp burial. This occurs in 20 micro s for a 7 degrees jump to 52 degrees C, which is close to the limits placed by diffusion on folding reactions. However, infrared temperature jump studies monitoring native helix burial are biexponential with times of 5 micro s and 56 micro s for a similar temperature jump. Both fluorescence and infrared fast phases are energetically favorable but the slow infrared absorbance phase is highly temperature-dependent, indicating a substantial enthalpic barrier for this process. The kinetics are best understood by a multiple-pathway kinetics model. The rapid phases likely represent direct burial of one or both of the Trp residues and parts of the G- and H-helices. We attribute the slow phase to burial and subsequent rearrangement of a misformed core or to a collapse having a high energy barrier wherein both Trps are solvent-exposed.
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Gilmanshin, R., Callender, R. H., & Dyer, R. B. (1998). The core of apomyoglobin E-form folds at the diffusion limit. Nat Struct Biol, 5(5), 363–365.
Abstract: 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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