Probing Stability and Dynamics of Proteins by Protease Digestion I: Comparison of Protease Susceptibility and Thermal Stability of Cytochromes c

Endo, Shigeru; Nagayama, Kuniaki; Wada, Akira

doi:10.1080/07391102.1985.10508426

Cited by 13 publications

(13 citation statements)

References 22 publications

(23 reference statements)

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“…2 for the cytochromes in D 2 O, and the T m values follow the order: bovine (74 7C) 1 horse Similarly, plots of the 1615-cm -1 aggregation band or a-helical amide Ib (1640-1660 cm -1 ) intensities versus temperature (data not shown) yielded the same T m values, confirming that aggregation accompanies loss of secondary structure as reported for other proteins [20]. The order of the T m values observed here for the three cytochromes agrees well with those reported by calorimetry [12] and CD [7], but the values are over 10 7C lower than those obtained in the absence of 0.2 M KCl [12,17], which was added to the samples in the present study because the limited-proteolysis studies were performed in 0.2 M KCl [7,8]. The perturbing effects of KCl and other salts on cytochrome c conformation [31] would explain the 1 10 7C reduction in our T m values, which cannot be ascribed to the high protein concentrations (4 mM) used in the FTIR studies since a T m of 82 7C was previously reported for the FTIR-monitored thermal denaturation of 5 mM horse c in the absence of KCl [17].…”

Section: Resultssupporting

confidence: 80%

“…Bovine cytochrome c is assumed to be essentially identical in structure to horse c since the two proteins share high sequence identity ( 1 97%). Despite remarkably similar main-chain structures [1,6], cytochromes c from different species exhibit different local stabilities as probed by tryptic digestion ( [7,8], A. Filosa, A.M. English, unpublished results) and 695-nm absorption ( [4,5,9], A. Filosa, A.M. English, unpublished results). The latter measures the integrity of the Fe III -S(Met80) bond since disruption or weakening of this bond results in quenching of the 695-nm absorption and opening of the heme crevice [10].…”

Section: Introductionmentioning

confidence: 99%

“…The lower thermal stability of the heme crevice of tuna c relative to the horse and bovine cytochromes is somewhat curious given that the three proteins possess similar global thermal stabilities as monitored by circular dichroism (CD) [7] and calorimetry [12]. Hence, in the present study, Fourier transform infrared (FTIR) spectroscopy is used to compare the conformational changes occurring during the thermal titration of tuna cytochrome c with those in the horse and bovine cytochromes.…”

Section: Introductionmentioning

confidence: 99%

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FTIR-monitored thermal titration reveals different mechanisms for the alkaline isomerization of tuna compared to horse and bovine cytochromes c

1999

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Fourier transform infrared (FTIR) spectroscopy is used to compare the thermally induced conformational changes in horse, bovine and tuna ferricytochromes c in 50 mM phosphate/0.2 M KCl. Thermal titration in D2O at pD 7.0 of the amide II intensity of the buried peptide NH protons reveals tertiary structural transitions at 54 degrees C in horse and at 57 degrees C in bovine c. These transitions, which occur well before loss of secondary structure, are associated with the alkaline isomerization involving Met80 heme-ligand exchange. In tuna c, the amide-II-monitored alkaline isomerization occurs at 35 degrees C, followed by a second amide II transition at 50 degrees C revealing a hitherto unreported conformational change in this cytochrome. Amide II transitions at 50 degrees C (tuna) and 54 degrees C (horse) are also observed during the thermal titration of the CN(-)-ligated cytochromes (where CN- displaces the Met80 ligand), but a well-defined 35 degrees C amide II transition is absent from the titration curve of the CN- adduct of tuna c. The different mechanisms suggested by the FTIR data for the alkaline isomerization of tuna and the mammalian cytochromes c are discussed. After the alkaline isomerization, loss of secondary structure and protein aggregation occur within a 5 degrees C range with Tm values at 74 degrees C (bovine c), 70 degrees C (horse c) and 65 degrees C (tuna c), as monitored by changes in the amide I' bands. The FTIR spectra were also used to compare the secondary structures of the ferricytochromes c at 25 degrees C. Curve fitting of the amide I (H2O) and amide I' (D2O) bands reveals essentially identical secondary structure in horse and bovine c, whereas splitting of the alpha-helical absorption of tuna c indicates the presence of less-stable helical structures. CN- adduct formation results in no FTIR-detectable changes in the secondary structures of either tuna or horse c, indicating that Met80 ligation does not influence the secondary structural elements in these cytochromes. The data provided here demonstrate for the first time that the selective thermal titration of the amide II intensity of buried peptide NH protons in D2O is a powerful tool in protein conformational analysis.

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Section: Resultssupporting

confidence: 80%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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FTIR-monitored thermal titration reveals different mechanisms for the alkaline isomerization of tuna compared to horse and bovine cytochromes c

1999

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“…Third, PCH only detects one species after protease digestion of TMR-cytochrome c with trypsin. With 19 potential sites for trypsin cleavage (30), this digest should completely destroy the tertiary structure of cytochrome c and reduce the number of fluorescent species to one, consistent with our observation. Additionally, the brightness of this single species is similar to TMRcysteine, consistent with the notion that the fluorophore is no longer quenched.…”

supporting

confidence: 79%

Cytochrome c conformations resolved by the photon counting histogram: Watching the alkaline transition with single-molecule sensitivity

Perroud

Bokoch

Zare

2005

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

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We apply the photon counting histogram (PCH) model, a fluorescence technique with single-molecule sensitivity, to study pHinduced conformational changes of cytochrome c. PCH is able to distinguish different protein conformations based on the brightness of a fluorophore sensitive to its local environment. We label cytochrome c through its single free cysteine with tetramethylrhodamine-5-maleimide (TMR), a fluorophore with specific brightnesses that we associate with specific protein conformations. Ensemble measurements demonstrate two different fluorescence responses with increasing pH: (i) a decrease in fluorescence intensity caused by the alkaline transition of cytochrome c (pH 7.0 -9.5), and (ii) an increase in intensity when the protein unfolds (pH 9.5-10.8). The magnitudes of these two responses depend strongly on the molar ratio of TMR used to label cytochrome c. Using PCH we determine that this effect arises from the proportion of a nonfunctional conformation in the sample, which can be differentiated from the functional conformation. We further determine the causes of each ensemble fluorescence response: (i) during the alkaline transition, the fluorophore enters a dark state and discrete conformations are observed, and (ii) as cytochrome c unfolds, the fluorophore incrementally brightens, but discrete conformations are no longer resolved. Moreover, we also show that functional TMR-cytochrome c undergoes a response of identical magnitude regardless of the proportion of nonfunctional protein in the sample. As expected for a technique with single-molecule sensitivity, we demonstrate that PCH can directly observe the most relevant conformation, unlike ensemble fluorometry.single-molecule fluorescence spectroscopy ͉ protein conformations ͉ protein labeling ͉ confocal microscopy ͉ metalloprotein I n cellular signaling networks, rare conformations of proteins are believed to be crucial for determining outcomes of the entire system (1, 2). Traditional biophysical methods rely on ensemble averages, that is, measurements from a large number of molecules simultaneously. As a consequence, rare, but important, populations can be obscured and go undetected. Recent advances in singlemolecule methodology have allowed researchers to break the ensemble average and more thoroughly characterize the heterogeneity of biological samples (3). This report describes the application of the photon counting histogram (PCH) model (4-7), a fluorescence spectroscopy technique with single-molecule sensitivity, to study the conformational heterogeneity of cytochrome c as a function of pH.Fluorescence spectroscopy has long been an important methodology for studying the conformational states of proteins, which is commonly done either by measuring the intrinsic fluorescence of the protein (for example, tryptophan residues) or covalently attaching an external fluorophore to specific residues (8). The latter approach assumes that the fluorophore does not significantly perturb the protein under investigation and that the photophysical propertie...

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“…For example, cytochromes c from yeast and horse have amino acid sequences that are >60% identical and their tertiary structures are remarkably similar [1,2]. However, protease susceptibility and thermal denaturation studies have shown that the yeast iso-1-cytochrome c (iso-1-cyt c) is much less stable than the horse cytochrome c (h-cyt c) [3,4] To identify the residues responsible for this large difference in protein stability, site-specific mutagenesis, followed by protein expression and purification from Escherichia coli cells, has been used to obtain yeast iso-1-and h-cyt c variants. We engineered residue substitutions within -helices with the goal of assessing the importance of charge distribution for the stabilization of -helix dipole and for the global stability of cyt c. In particular, we chose to introduce mutations into the N-terminal end of two -helices (the -helix encompassing residues 60 to 70 and the C-terminal helix) (Fig.…”

Section: Introductionmentioning

confidence: 99%

Probing the Effect of Mutations on Cytochrome c Stability

Agueci¹,

Polticelli²,

Sinibaldi³

et al. 2007

PPL

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Although the tertiary structures of mitochondrial cytochromes c (cyts c) seem to be remarkably similar, there are variations in their amino acid sequences, stability and functional properties. GdnHCl-induced unfolding experiments on engineered yeast and horse cyt c were carried out with the aim to to clarify, at molecular level, some aspects concerning the stability of this class of proteins. The results obtained are discussed in the light of the three-dimensional structures of the two proteins.

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Probing Stability and Dynamics of Proteins by Protease Digestion I: Comparison of Protease Susceptibility and Thermal Stability of Cytochromes c

Cited by 13 publications

References 22 publications

FTIR-monitored thermal titration reveals different mechanisms for the alkaline isomerization of tuna compared to horse and bovine cytochromes c

FTIR-monitored thermal titration reveals different mechanisms for the alkaline isomerization of tuna compared to horse and bovine cytochromes c

Cytochrome c conformations resolved by the photon counting histogram: Watching the alkaline transition with single-molecule sensitivity

Probing the Effect of Mutations on Cytochrome c Stability

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