Acid-Denatured States of Proteins

Fink, Anthony L.; Calciano, Linda J.; Goto, Yuji; Palleros, Daniel R.

doi:10.1016/b978-0-12-721955-4.50044-0

Cited by 13 publications

(8 citation statements)

References 7 publications

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“…1B) an additional exchange envelope that is also in the 17+ for 113 ± 3 Hs exchanged (state I), corresponding to a more ordered structure. Possibly analogous, cytochrome c in solution forms a more compact A-state from the denatured state with decreasing pH (4,5 (4,5).…”

Section: Resultsmentioning

confidence: 99%

“…Water is thought to be a key factor in the spontaneous folding of a protein into its bioactive conformation (1)(2)(3)(4)(5); "water interactions are of the essence in the function of real-life protein molecules" (6). However, a preliminary report (7) of the research described here gave hydrogen/deuterium (H/D) exchange evidence of at least three conformations of waterfree cytochrome c cations in the gas phase.…”

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confidence: 95%

“…Three distinct exchange levels were dominant, with some charge values exhibiting two of three levels, but the level of highest exchange occurred at an intermediate charge value. Although in solution nonionized cytochrome c mimics this behavior, with the native structure first denatured and then reorganized into the molten globule state with increasing HI concentration (4,5,22), the importance of water for in vivo folding appears well established: "the likelihood of a protein refolding in the gas phase is exceedingly small" (23). We report here more definitive high-resolution MS data showing that multiply protonated equine cytochrome c ions undergo H/D exchange with D20 at six distinct levels, with conversion between these levels (presumably unfolding and folding) effected by infrared laser heating and high-velocity collisions or by reducing the number of charges on the ion.…”

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Gas-phase folding and unfolding of cytochrome c cations.

Wood

Chorush

Wampler

et al. 1995

Proc. Natl. Acad. Sci. U.S.A.

251

267

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Water is thought to play a dominant role in protein folding, yet gaseous multiply protonated proteins from which the water has been completely removed show hydrogen/deuterium (H/D) exchange behavior similar to that used to identify conformations in solution. Indicative of the gas-phase accessibility to D20, multiply-charged (6+ to 17+) cytochrome c cations exchange at six (or more) distinct levels of 64 to 173 out of 198 exchangeable H atoms, with the 132 H level found at charge values 8+ to 17+. Infrared laser heating and fast collisions can apparently induce ions to unfold to exchange at a higher distinct level, while chargestripping ions to lower charge values yields apparent folding as well as unfolding.Water is thought to be a key factor in the spontaneous folding of a protein into its bioactive conformation (1-5); "water interactions are of the essence in the function of real-life protein molecules" (6). However, a preliminary report (7) of the research described here gave hydrogen/deuterium (H/D) exchange evidence of at least three conformations of waterfree cytochrome c cations in the gas phase. These multiply protonated species were formed by electrospray ionization, introduced into the high-vacuum region of the Fouriertransform mass spectrometer, and allowed to exchange with D20 at 10-7 Torr (1 Torr = 133.3 Pa). In solution, the degree and sites of H/D exchange are well-established indicators of steric inaccessibility (8) and, thus, of protein conformation. To measure H/D exchange rates, these solution studies have used nuclear magnetic resonance (3, 9, 10), neutron diffraction (11), and mass spectrometry (MS), with MS employed to measure solution-phase H/D exchange rates (12-16), transient intermediates (17), and gaseous noncovalent complexes (18-21).For multiprotonated cytochrome c in the gas phase (7), H/D exchange exhibited pseudo-first-order kinetics for 7+ to 15+ ions. Three distinct exchange levels were dominant, with some charge values exhibiting two of three levels, but the level of highest exchange occurred at an intermediate charge value. Although in solution nonionized cytochrome c mimics this behavior, with the native structure first denatured and then reorganized into the molten globule state with increasing HI concentration (4, 5, 22), the importance of water for in vivo folding appears well established: "the likelihood of a protein refolding in the gas phase is exceedingly small" (23). We report here more definitive high-resolution MS data showing that multiply protonated equine cytochrome c ions undergo H/D exchange with D20 at six distinct levels, with conversion between these levels (presumably unfolding and folding) effected by infrared laser heating and high-velocity collisions or by reducing the number of charges on the ion. MATERIALS AND METHODSSolutions of 20 ,uM equine cytochrome c (Sigma) were electrosprayed [6+ to 9+ ions in pure aqueous solution and 10+ to 17+ ions in methanol/water/acetic acid, 76:22:2 (vol/vol)], and the resulting ions were transported by three rf-...

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 95%

mentioning

confidence: 99%

See 1 more Smart Citation

Gas-phase folding and unfolding of cytochrome c cations.

Wood

Chorush

Wampler

et al. 1995

Proc. Natl. Acad. Sci. U.S.A.

251

267

View full text Add to dashboard Cite

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“…Cyt c can be unfolded at pH 2 in the absence of salt (U-state). However, the protein can also form a partially folded state, called the A-state, under very acidic conditions (pH < 2) or at pH 2 in the presence of 0.5-1.5 M NaCl or KC1 (Fink et al, 1990;Goto et al, 1990a;Jeng et al, 1990;Jeng & Englander, 1991). The A-state structure is stabilized because the salt ions screen the repulsive electrostatic interactions that occur upon protonation of carboxylate side chains (Goto et al, 1990b;Goto & Nishikiori, 1991;Stigter et al, 1991).…”

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confidence: 99%

Secondary and tertiary structure of the A‐state of cytochrome c from resonance Raman spectroscopy

1995

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Ferricytochrome c can be converted to the partially folded A-state at pH 2.2 in the presence of 1.5 M NaCl. The structure of the A-state has been studied in comparison with the native and unfolded states, using resonance Raman spectroscopy with visible and ultraviolet excitation wavelengths. Spectra obtained with 200 nm excitation show a decrease in amide II intensity consistent with loss of structure for the 50s and 70s helices. The 230-nm spectra contain information on vibrational modes of the single Trp 59 side chain and the four tyrosine side chains (Tyr 48, 67, 74, and 97). The Trp 59 modes indicate that the side chain remains in a hydrophobic environment but loses its tertiary hydrogen bond and is rotationally disordered. The tyrosine modes Y8b and Y9a show disruption of tertiary hydrogen bonding for the Tyr 48, 67, and 74 side chains. The high-wavenumber region of the 406.7-nm resonance Raman spectrum reveals a mixed spin heme iron atom, which arises from axial coordination to His 18 and a water molecule. The low-frequency spectral region reports on heme distortions and indicates a reduced degree of interaction between the heme and the polypeptide chain. A structural model for the A-state is proposed in which a folded protein subdomain, consisting of the heme and the N-terminal, C-terminal, and 60s helices, is stabilized through nonbonding interactions between helices and with the heme.

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“…It is likely that these interactions, which are influenced by temperature or low pH, determine the stability of proteins. Lowering of pH can induce conformational changes in protein molecules, influencing their a-helix content (Fink et al, 1990) which leads to the gelation of fish myosin (Fretheim et al, 1985) and shark myofibrillar proteins (Venugopal et al., 1994). The heatinduced changes of the unacidified dispersions are comparable to the modori phenomenon of surimi gel, in which the homogeneous dispersion of a protein network might undergo intense shrinking at high temperatures.…”

Section: Resultsmentioning

confidence: 99%