Irreversible thermal denaturation of cytochrome c studied by electrospray mass spectrometry

Abstract

This work uses electrospray ionization mass spectrometry (ESI-MS) in conjunction with hydrogen/deuterium exchange (HDX) and optical spectroscopy for characterizing the solutionphase properties of cytochrome c (cyt c) after heat exposure. Previous work demonstrated that heating results in irreversible denaturation for a subpopulation of proteins in the sample. However, that study did not investigate the physical reasons underlying this interesting effect. Here we report that the formation of oxidative modifications at elevated temperature plays a key role for the observed behavior. Tryptic digestion followed by tandem mass spectrometry is used to identify individual oxidation sites. Trp59 and Met80 are among the modified amino acids. In native cyt c both of these residues are buried deep within the protein structure, such that covalent modifications would be expected to be particularly disruptive. ESI-MS analysis after heat exposure results in a bimodal charge-state distribution. Oxidized protein appears predominantly in charge states around 11ϩ, whereas a considerably lower degree of oxidation is observed for the 7ϩ and 8ϩ peaks. This finding confirms that different oxidation levels are associated with different solution-phase conformations. HDX measurements for different charge states are complicated by peak distortions arising from oxygen adduction. Nonetheless, comparison with simulated peak shapes clearly shows that the HDX properties are different for high-and low-charge states, confirming that interconversion between unfolded and folded conformers is blocked in solution. In addition to oxidation, partial aggregation upon heat exposure likely contributes to the formation of irreversibly denatured protein. A number of well-established tools are available for experiments of this kind, including X-ray crystallography, nuclear magnetic resonance, calorimetry, and various spectroscopic techniques. Electrospray ionization mass spectrometry (ESI-MS) has become another widely used approach for exploring the properties of proteins in solution, providing information complementary to that obtained by traditional methods [1]. The ESI process generates intact gas-phase ions from proteins in solution. In the commonly used positive-ion mode these [M ϩ nH] nϩ species are multiply charged because of proton attachment.ESI of natively folded proteins results in protonation states n similar to those predicted for water droplets of the same size that are charged to the Rayleigh limit. This finding supports the idea that ionization follows the charged-residue model [2-6], although alternative scenarios have been proposed as well [7][8][9]. Much higher protonation states and wider charge-state distributions are generally seen for proteins that are unfolded in solution. Based on this empirical relationship, ESI-MS is now routinely being used for monitoring structural transitions in response to changes in pH, temperature, organic co-solvents, or covalent modifications [1, 10 -16]. The physical basis for the striking dependence of...

Mapping the Structure of an Integral Membrane Protein under Semi-Denaturing Conditions by Laser-Induced Oxidative Labeling and Mass Spectrometry

“…6h), consistent with earlier studies on other proteins that have demonstrated spontaneous MetO formation at elevated temperature. 61,69 As a side aspect, we note that these covalent modifications may contribute to the irreversible nature of BR thermal denaturation. 69 Equation (5) allows the calculation of backgroundcorrected F u corr values from the F u app and F u bgr measurements.…”

“…61,69 As a side aspect, we note that these covalent modifications may contribute to the irreversible nature of BR thermal denaturation. 69 Equation (5) allows the calculation of backgroundcorrected F u corr values from the F u app and F u bgr measurements. The F u corr progressions obtained in this way exclusively reflect oxidation events that are caused by laser-induced labeling (Fig.…”

Joule Heating and Thermal Denaturation of Proteins in Nano-ESI Theta Tips

“…It is generally accepted, for example, the magnitude of charges is relatively high for unfolded conformations, which are described as high charge state distributions, while more folded conformations usually display relatively low charge state distributions. [3, 4, 5] For this reason, charge state distributions have been used in a biophysical context to monitor protein conformations using mass spectrometry. The magnitude of protein ion charge also has analytical implications.…”

“…[11,12] These interactions can be affected by a variety of factors including temperature, [4, 13, 14, 15] pH, [16, 17, 18] ionic strength, [19] solvent, [20, 21, 22] surface effects, [23] as well as instrumental parameters[24]. Most methods intended to change protein conformation involve bulk solution manipulations, such as the addition of acid, base, organic solvent, supercharging reagents[14] or other additives, as well as heating.…”

Distribution and Transition of Native and Completely Unfolded Conformations in the Unfolding of Bovine Heart Cytochrome c Induced by Urea and Guanidine Hydrochloride

“…For example, Harrington studied the unfolding of horse ferricytochrome c in the presence of several inorganic salts under a variety of denaturing conditions by means of viscosity measurements and absorbance changes in the Soret and visible regions; 5 Nieman et al studied the 13 C NMR spectroscopy of acetyltyrosyl-guanidinated horse heart cytochrome c; 6 Hagihara et al compared the stability of the native and acidic molten globule states of horse ferricytochrome c against heat, urea and guanidine hydrochloride using the intact species and species modified by various degrees of acetylation of the lysyl ε-amino groups; 7 Zhu et al investigated the conformational transition of horse heart cytochrome c induced by bromopyrogal red in very low concentration by dynamic spectro-electrochemical technique; 8 Zhang et al studied the denaturation of horse heart cytochrome c induced by bromopyrogal red by scanning tunnelling microscopy on the electrochemically pretreated highly oriented pyrolytic graphite surface; 9 Yang et al studied conformational changes of horse heart cytochrome c determined by matrix-assisted laser desorption mass spectrometry; 10 Moosavi-Movahedi et al studied the differential scanning calorimetry of the molten globule state of horse heart cytochrome c induced by sodium n-dodecyl sulfate; 11 Krylov et al studied the denaturation and renaturation of horse heart cytochrome c immobilized on gold electrodes in DMSO-containing buffers; 12 Kumar et al studied the alkali molten globule state of horse ferricytochrome c in "cold denaturation"; 13 Latypov et al studied the equilibrium and kinetic properties of reduced horse heart cytochrome c in the presence of carbon monoxide; 14 Baker and Heller studied the characteristics of equine heart cytochrome c in aqueous solutions of the fully water-miscible IL 1-butyl-3-methyl-imidazolium chloride by small-angle neutron and X-ray scattering; 15 Liu and Konermann characterized the solution-phase properties of horse heart cytochrome c after heat exposure by electrospray ionization mass spectrometry in conjunction with hydrogen/deuterium exchange and optical spectroscopy. 16 Like horse heart cytochrome c, cytochrome c from bovine heart is also a globular protein molecule. The difference between them is that the three amino acid residues, Thr-47, Lys-60 and Thr-89, in horse heart cytochrome c are separately replaced by Ser, Gly and Gly in bovine heart cytochrome c. 17 In earlier works, Richards et al studied the retention behavior of bovine heart cytochrome c and a series of closely related cytochrome c by reversed-phase chromatography and found that the specific amino acid substitutions were directly involved in the chromatographic contact region of the cytochrome c molecules; 18,19 and lately, Mozal et al carried out the equilibrium studies of the effect of the amino acid residue difference in horse and bovine cytochrome c on their unfolding processes by c...…”