Directed denaturation: Room temperature and stoichiometric unfolding of cytochrome c by a metalloporphyrin dimer

Article abstract of DOI:10.1021/ja028574m

Using circular dichroism, UV-vis, and trypsin proteolysis, we have shown how a metalloporphyrin dimer induces the unfolding of a protein, cytochrome c, under physiologically relevant conditions and accelerates its rate of proteolytic degradation. Copyrigh

Full text of DOI:10.1021/ja028574m

Published on Web 03/21/2003
Directed Denaturation: Room Temperature and Stoichiometric Unfolding of
Cytochrome c by a Metalloporphyrin Dimer
Andrew J. Wilson, Kevin Groves, Rishi K. Jain, Hyung Soon Park, and Andrew D. Hamilton*
Department of Chemistry, P.O. Box 208107, 225 Prospect Street, Yale UniVersity,
New HaVen, Connecticut 06520-8107
Received September 17, 2002; E-mail: andrew.hamilton@yale.edu
Protein denaturation normally requires elevated temperatures,
high concentrations of denaturants such as guanidinium hydrochlo-
ride, urea, and various detergents or drastic changes in pH.1 Other
than the chaperones or related proteins,2 there are very few examples
of molecules that unfold target proteins at stoichiometric concentra-
tions under physiologically relevant conditions.3 In the present
paper, we report synthetic agents that selectively denature cyto-
chrome c at or near room temperature.
We have previously described molecules that bind to protein
exteriors by matching hydrophobic and charged domains over a
large surface area of the target protein.4 In the case of anionic
tetraphenylporphyrin derivatives 1a and 2a and cytochrome c, we
observed not only strong binding4b to the native state but also a
sizable decrease in the melting temperature (∆Tm ≈ 20 °C) of the
protein.5 These results opened the possibility that appropriate
modification of the porphyrins might lead to an agent able to fully
denature cytochrome c at room temperature and under physiological
conditions.
Figure 1. CD spectra of 2 µM cytochrome c in the presence and absence
of porphyrin receptors 1a and 1c at different pH’s.
To probe the effects of porphyrin aggregation on the unfolding
of cytochrome c, we prepared different metalloporphyrin deriva-
tives. Water-soluble zinc(II)-porphyrins have been shown to exhibit
minimal aggregation in aqueous buffer7 due to axial coordination
of water to the metal. Consistent with this, 1b showed no difference
in its Soret band at pH’s 7.4 and 5.8 or on addition of acetone. At
pH 7.4, zinc complex 1b behaves similarly to 1a or 2a, forming a
strong 1:1 complex with cytochrome c (Kd ) 28 nM) and lowering
the Tm by ∼20 °C. In contrast to 1a, however, 1b has no effect on
the CD of cytochrome c at room temperature even in large excess
at pH 5.8. These results suggest that for 1a at pH 7.4 or zinc
derivative 1b at both pH 5.8 and 7.4, strong binding but weakly
denaturing monomeric porphyrins are the dominant porphyrin
species interacting with the protein.
To enhance the aggregation of the functionalized porphyrins, we
prepared the Cu(II) derivatives 1c and 2b. Copper(II) porphyrins
are known to undergo extensive dimerization in water7a through
enhanced π-π stacking interactions.8 The UV-vis spectrum of
1c shows a broadened Soret band at pH 7.4, that sharpens upon
addition of acetone, suggesting the presence of aggregated por-
phyrins. An exciton coupling band in the CD spectrum of 1c that
decreases upon addition of acetone as well as an isosbestic point
in dilution studies indicate that a porphyrin dimer is the predominant
species in solution. Because the Cu(II)-porphyrins do not fluoresce,
UV-difference spectroscopy was used to show that 2b forms a 2:1
complex (confirmed by Job analysis) with cytochrome c with a Kd
value of 60 nM (at pH 7.4, 5 mM phosphate).9 The thermal
denaturation curve for cytochrome c in the presence of 2 equiv of
2b (Figure 2) shows a 50 °C decrease in Tm to 35 °C and confirms
the enhanced effect of the porphyrin dimer as compared to the
monomer. The effect is even more dramatic with 1c and required
an increase in ionic strength of the solution to derive accurate
binding data. Cu complex 1c bound cytochrome c with 2:1
stoichiometry (Job plot) and a Kd of 600 nM (5 mM phosphate, 50
mM NaCl, pH 7.4).9 Two equivalents of 1c at room temperature
(pH 7.4, 50 mM NaCl, 5 mM phosphate) led to a loss of the CD
A hint of the chemical features that would be necessary for room-
temperature denaturation came in our studies on the effect of pH
on the interaction of 1a with cytochrome c. At pH 7.4, 1a forms a
1:1 complex (Kd ) 20 nM) with the cationic heme edge region of
cytochrome c and causes a decrease in the Tm from 85 to 65 °C.4b,5
In contrast, at room temperature, 1a, even in excess, had little effect
on the native conformation of the protein (as determined by CD).
However, at pH 5.8 and room temperature, titration of 1a into a
solution of cytochrome c in 5 mM phosphate buffer led to a loss
of the CD signal at 222 nm that reached saturation at 4 equiv and
corresponded to an unfolding of the tertiary structure of the protein
(Figure 1). pH 5.8 buffer had no effect on the thermal stability of
cytochrome c alone, suggesting the denaturing properties of 1a at
low pH must be due not to a change in the protein but in the
chemical behavior of the porphyrin. Indeed, the Soret band of 1a
showed a significant broadening at pH 5.8 relative to 7.4 that could
be reversed by addition of acetone.6 This is consistent with a degree
of self-association at the lower pH and suggests that the denaturation
effect of 1a may be due to an aggregated species.7
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J. AM. CHEM. SOC. 2003, 125, 4420-4421
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C O M M U N I C A T I O N S
activity of trypsin and by the minimal acceleration in cleavage rate
of myoglobin and R-lactalbumin (data not shown) on addition of 4
equiv of 1c.
Figure 4. SDS-PAGE analysis of the trypsin proteolysis of 20 µM
cytochrome c (5 mM phosphate, 50 mM NaCl, pH 7.4) followed over 60
min at room temperature: (a) alone, (b) 80 µM 1c, (c) 80 µM 1a.
Figure 2. Thermal unfolding profile for cytochrome c as a function of
added porphyrin receptor (5 mM phosphate, pH 7.4). CD spectra recorded
at (a) 222 nm and (b) 410 nm.
At this point, the exact mechanism of cytochrome c unfolding by
1c and 2b has not been determined. However, the large denaturation
effect of the Cu porphyrin dimers suggests a preferential interaction
with the unfolded state of the protein relative to the folded form.
Presumably, the increased size and overall charge of the porphyrin
dimers, relative to monomeric 1a, 1b, and 2a, leads to stronger
interaction with the larger hydrophobic surface area of the unfolded
protein. Selectivity appears to derive from a charge matching of
surface residues on the protein with the porphyrin dimer. Some
degree of cooperativity between the two porphyrins bound to the
denatured state as a dimer must also be considered to explain why
2 equiv of monomeric porphyrin do not bind as well to the
denatured state as the dimer. We are currently investigating the
interaction of 1c and 2c with molten globule and unfolded forms
of cytochrome c formed by mutated or chemically modified
derivatives.
signal at 222 nm and formation of a spectrum (Figure 1) that closely
resembles that of thermally denatured (>90°) cytochrome c. This
effect reaches saturation between 1 and 2 equiv of dimer and is
not observed for either 1a or 1b. In the thermal denaturation
experiment, cytochrome c in the presence of 1c (2 equiv) is almost
fully unfolded over the whole temperature range (Figure 2).
Furthermore, these large denaturation effects are selective for
cytochrome c as compared to certain other proteins with a range
of pI and Tm values. Figure 3a-e shows the thermal unfolding
profiles of R-lactalbumin, Bcl-xL, cytochrome c551, myoglobin,
and RNase A in the presence and absence of 2 equiv of 1c. None
of these proteins shows a decrease in Tm of more than 15 °C, while
that of cytochrome c is >50 °C (Figure 3f) under equivalent
conditions. Notably, cytochrome c551, which has a similar structure
to cytochrome c but different charge distribution, shows almost no
change in Tm on addition of 1c.
Acknowledgment. We are grateful to the National Institutes
of Health for support of this research.
Supporting Information Available: Synthetic procedures and
analytical data, UV-vis spectra describing dimerization studies, titration
data, and Job plots (PDF). This material is available free of charge via
the Internet at http://pubs.acs.org.
References
(1) (a) Arakawa, T.; Prstrelski, S. J.; Kenney, W. C.; Carpenter, J. F. AdV.
Drug DeliVery ReV. 2001, 46, 307-326. (b) Yang, A.-S.; Honig, B. J.
Mol. Biol. 1993, 231, 459-474.
(2) (a) Walter, S.; Buchner, J. Angew. Chem., Int. Ed. 2002, 41, 1098-1113.
(b) Weber-Ban, E. U.; Reid, B. G.; Miranker, A. D.; Horwich, A. L. Nature
1999, 401, 90-93. For an example of a nonchaperone protein that performs
in a similar way, see: (c) Huntington, J. A.; Read, R. J.; Carrell, R. W.
Nature 2000, 407, 923-926.
Figure 3. Melting curves for 2 µM protein (5 mM phosphate, 50 mM
NaCl, pH 7.4) (in black alone, in red with 4 µM 1c): (a) R-lactalbumin,
(b) Bcl-xL, (c) cytochrome c551, (d) myoglobin, (e) RNase A, (f) cytochrome
c.
(3) (a) Fischer, N. O.; McIntosh, C. M.; Simard, J. M.; Rotello, V. M. Proc.
Natl. Acad. Sci. U.S.A. 2002, 99, 5018-5023. (b) Peterson, J. R.; Lokey,
R. S.; Mitchison, T. J.; Kirschner, M. W. Proc. Natl. Acad. Sci. U.S.A.
2001, 98, 10624-10629. (c) Leung, D. K.; Yang, Z.; Breslow, R. Proc.
Natl. Acad. Sci. U.S.A. 2000, 97, 5050-5053. (d) Foster, B. A.; Coffey,
H. A.; Morin, M. J.; Rastinejad, F. Science 1999, 286, 2507-2510.
(4) (a) Park, H. S.; Lin, Q.; Hamilton, A. D. Proc. Natl. Acad. Sci. U.S.A.
2002, 99, 5105-5109. (b) Jain, R. K.; Hamilton, A. D. Org. Lett. 2000,
2, 1721-1723. (c) Blaskovich, M. A.; Lin, Q.; Delarue, F. L.; Sun, J.;
Park, H. S.; Coppola, D.; Hamilton, A. D.; Sebti, S. M. Nat. Biotechnol.
2000, 18, 1065-1070.
Cytochrome c is known to undergo more rapid proteolysis in
the denatured state as compared to the compact native form.10 We
therefore assessed the full extent of the unraveling of cytochrome
c by 1c at room temperature by measuring the sensitivity of the
protein to proteolysis by trypsin. SDS-PAGE analysis of cytochrome
c cleavage by trypsin alone11 (Figure 4a) shows very little change
in the integrity of the folded protein after 60 min at room
temperature. However, in the presence of 4 equiv of 1c, tryptic
digestion of cytochrome c is advanced after 15 min and complete
after 60 min (Figure 4b). In contrast, 4 equiv of porphyrin 1a, which
under the same conditions is monomeric and thus weakly denatur-
ing, has little effect on the rate of trypsin cleavage of the protein
(Figure 4c). These results point to a specific denaturing effect for
dimeric 1c that leads not only to a disruption of the tertiary structure
of cytochrome c but also to an enhanced rate of proteolysis. Again,
selectivity of this effect is suggested both by the unaffected catalytic
(5) Jain, R. K.; Hamilton, A. D. Angew. Chem., Int. Ed. 2002, 41, 641-643.
(6) See Supporting Information.
(7) (a) Pasternack, R. F.; Francesconi, L.; Raff, D.; Spiro, E. Inorg. Chem.
1973, 12, 2606-2611. (b) Dolphin, D., Ed. The Porphyrins; Academic
Press: New York, 1978; Vol. 5, pp 303-339.
(8) Hunter, C. A.; Sanders, J. K. M. J. Am. Chem. Soc. 1990, 112, 5525-
5534 and references therein.
(9) The Cu(II) porphyrins were treated as a single dimeric entity in the
calculations.
(10) Wang, L.; Chen, R. X.; Kallenbach, N. R. Proteins: Struct., Funct., Genet.
1998, 30, 435-441.
(11) See Supporting Information.
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