Spectroscopic Studies of Ferrocytochrome c Folding - ACS Publications

Chapter 10

Spectroscopic Studies of Ferrocytochrome c Folding

Downloaded by EAST CAROLINA UNIV on January 14, 2018 | http://pubs.acs.org Publication Date: June 9, 1998 | doi: 10.1021/bk-1998-0692.ch010

Gary A. Mines, Jay R. Winkler, and Harry B. Gray Beckman Institute 139-74, California Institute of Technology, Pasadena, CA 91125

Electron-transfer triggering has been employed in a comparison of the folding energetics and kinetics of cytochrome c from horse and Saccharomyces cerevisiae. These two proteins, with just 60% sequence identity but very similar backbone structures, fold at very different rates at a given denaturant concentration, but at nearly the same rate when their foldingfreeenergies are the same. Differences in the amino-acid sequences shift the position of the folding/unfolding equilibrium, but do not appear to alter the location of the transition state along the folding coordinate.

Determining the process by which a polypeptide attains its physiologically relevant conformation is of great current interest. One experimental approach involves measuring the folding kinetics of proteins by stopped-flow mixing: solutions of unfolded protein in high concentrations of a chemical dénaturant (typically guanidine hydrochloride (GuHCl) or urea) are rapidly mixed with buffer solutions in order to dilute the dénaturant concentration to a level at which the folded form is favored. Although this methodology is very useful, it can only be employed to observe folding processes that occur on timescales greater than -1 ms, the minimum time required for solutions to mix. Secondary structure fluctuations in small polypeptides can occur on timescales as short as nanoseconds (7,2); these motions may play a role in the early events in folding reactions (3). It also has been suggested that the so-called "burst phase" in folding, referring to the formation of secondary structure and/or a hydrophobic core, can be a submillisecond process (4). Indeed, experimental evidence for a burst phase (^5 ms) has been reported for at least seven proteins or protein fragments: ubiquitin (5), hen lysozyme (6), ferricytochrome c (7,8),ribonucleaseA (9), trp aporepressor (JO), barstar (77, 12), and the IgG binding domain of protein G (73). If measurements made by timeresolved circular dichroism are included (dead time of about 10-20 ms), several others can be added to the list (14).

198

©1998 American Chemical Society Solomon and Hodgson; Spectroscopic Methods in Bioinorganic Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 1998.


199

Methods have been developed that allow observation of submillisecond events in protein folding: photodissociation of CO from reduced cytochrome c has been employed to investigate folding processes occurring in the 10-ns to 1-ms time range (75); conventional temperature-jump methodology has been used to study a 300 μβ folding event in barstar (72); and laser-initiated temperature jump experiments have been employed to probe early events in the folding of apomyoglobin (76). In addition, a continuous-flow mixing apparatus that can achieve time resolution down to 100 μβ has been used to study fast folding events in ferricytochrome c (8). These methods are promising, but more techniques are needed to examine the submillisecond kinetics of protein folding. We have developed an electron-transfer (ET) method of triggering protein folding that can be used to study events in the ns to s time range (17). We have employed this method to investigate the relationship between folding rate and freeenergy change in ferrocytochromes cfromhorse (h-cyt c) and yeast (y-cyt c). These proteins have similar folds, but distinct stabilities due to differences in amino-acid sequences. We find that the rates of thefinalfolding phase in the two proteins differ significantly at a given dénaturant concentration, but are comparable when the free energies of folding are matched. Materials and Methods Guanidine hydrochloride (GuHCl, ultrapure grade) was used as receivedfromUnited States Biochemical. The concentration of GuHCl in all solutions used in equilibrium or kinetics experiments was determined using the empirical relationship between the concentration of GuHCl and the refractive index of GuHCl (18). K [Co(C 0 )3] (19) was obtainedfromT. Pascher. Horse heart cytochrome c (Sigma) was used without further purification. The methods used to determine equilibrium unfolding curves and to measure folding kinetics have been described previously (20). 3

2

4

Yeast iso-l-cytochrome c (Cysl02Ser). The protein was a mutant of Saccharomyces cerevisiae iso-l-cytochrome c containing serine at position 102 (h-cyt c numbering system) in place of cysteine in order to prevent interprotein disulfide formation (27). Protein was isolated from 10-L cultures of a previously prepared GM-3C-2 cell line containing a plasmid with the mutant cyt c sequence (22,23); purification followed the procedure of Smith and coworkers (24), with the following modification: after dialysis, the protein was loaded onto a cation exchange column (SP Sepharose, 4 cm χ 2.5 cm i.d.) and eluted with a linear salt gradient (0 to 1 M NaCl, pH 7.0), rather than subjected to a batch adsorption procedure. Final purification was achieved using cation-exchange FPLC (Mono S; salt gradient, 0 to 1 M NaCl, pH 7.0); y-cyt c eluted at -0.30-0.33 M NaCl. Results and Discussion Our method is based on the observation that the formal potentials of redox-active metal centers in proteins are "tuned" by the folding of the polypeptide chain around them; that is, metals in the hydrophobic interior of a folded protein often exhibit redox potentials

Solomon and Hodgson; Spectroscopic Methods in Bioinorganic Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 1998.

200 that are significantly differentfromthose they would possess in an aqueous environment (25). A simple thermodynamic cycle (Figure 1) (26) indicates that such a shift in redox potential (ΔΕ _ =Ε - E^) reflects a difference in conformational stability between the two redox forms of the protein (-nFLE^^ = àAG _ = AG - AG^ ). If this stability difference is sufficiently large, it should be possible tofindconditions at which one redox form of the protein is unfolded and the other form is folded. For example, if the reduced form of the protein (P j) is more stable than the oxidized protein (P ), it is expected that a higher concentration of a chemical dénaturant will be required to unfold the reduced protein compared to the oxidized protein. In solutions with dénaturant concentrations above the unfolding transition for the oxidized protein and below that for reduced protein, electron injection into the oxidized protein (unfolded) will lead to the formation of folded reduced protein. If the reduction is rapid enough, the folding of the reduced protein can be observed. Owing to the many well established techniques for reducing or oxidizing proteins on microsecond timescales or shorter (27,28), this method offers promise for studying very early events in protein folding. Cytochrome c is a good candidate for ET-triggered folding studies. First of all, the heme group is linked to the polypeptide through covalent (thioether) bonds to Cysl4 and Cysl7. This linkage ensures that heme binding is not the rate-limiting step in the folding process. Secondly, it is known that the redox potential of folded cyt c (260 mV vs. NHE) (29) is -400 mV higher than the potential for an exposed heme in aqueous solution (ca. -150 mV) (30), indicating that the reduced form of the protein is substantially more stable toward unfolding than the oxidized form (26,31). Thus, there should be a wide dénaturant-concentration window in which folding can be triggered by rapid reduction of the heme. ¥

υ

ρ

lnd

m

ired

ox


re(

ox

Unfolding. The degree of folding of cyt c has been determined by monitoring the intensity offluorescencefrom tryptophan-59 (32). Thefluorescenceis nearly completely quenched in the folded state, presumably via energy transfer to the nearby heme group (33). As the protein is unfolded by dénaturant, the distance between the two chromophores increases, and thefluorescencesignal intensifies dramatically (34). Unfolding curves generatedfromfluorescencedata for h-cyt c and y-cyt c in both oxidation states at 22.5 and 40.0 °C are well described as two-state transitions in which thefreeenergy of folding is a linear function of dénaturant concentration (slope = m ; Figure 2). It is evident from the unfolding curves (at both temperatures for both species of cyt c) that the reduced form is considerably more stable than the oxidized form (as predictedfromelectrochemical data). Indeed, the extrapolated values of A A G . to zero dénaturant concentration (AAG° _ = -30 to -35 kJ/mol; Table I) are close to those estimated from the difference in redox potentials (-ΔΕ _ - -400 mV = -39 U/mol). It also is seen that, in both oxidation states at both temperatures, y-cyt c is considerably less stable than the corresponding h-cyt c protein: the A G values extrapolated to zero GuHCl concentration (AG °) are -15 kJ/mol more positive for y-cyt c than for h-cyt c, and the unfolding midpoints ([GuHCl], ) occur at GuHCl concentrations -1.5 M lower for y-cyt c than for h-cyt c (Table I). The observed [GuHCl]^ and m values for h-cyt à and y-cyt at 22.5 °C are consistent with values reported in prior determinations at similar conditions using identical (or nearly identical) proteins (35-37). The same is true for the values found for y-cyt cP at 22.5 °C (37). D

ired

ox

fred

ox

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f

f

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201

AGf, ox P

U,ox

•

PF.C

-nFE

-nFE


u

F

AAG

f > r e d

.

=-nF(AE .u)

o x

F

Figure 1. Thermodynamic cycle illustrating the relationship between folding free energies (AG) and reduction potentials (E) for a redox protein (Ρ), η is the number of electrons transferred and F is the Faraday constant. Subscripts represent the states of the protein: U = unfolded, F = folded; ox = oxidized, red = reduced.

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3

4

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5

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Figure 2. Equilibrium unfolding data for h-cyt c and y-cyt c at 22.5 °C, pH 7. (A) Unfolding curves determinedfromcorrected fluorescence intensity data at 350 nm (y-cyt c, inverted triangles; h-cyt c, diamonds), y-cyt (long dashes); y-cyt (dashes); h-cyt (dotted); h-cyt c° (solid). (B) Plots of calculated AG vs. [GuHCl] curves based on the data set out in Table I. f


202 Table L Parameters describing the thermodynamics and kinetics of h-cyt c and y-cyt c folding and unfolding.


Protein

Τ (°Q

-AG ° [GuHCl]* (M) (kJmol ) (kJ mol M" ) (kJ mol" M" ) f

1

1

h-cyt J* 22.5(5)

2.8(1)

40(1)

14.3(4)

h-cyt c" 22.5(5)

5.3(1)

74(3)

13.8(4)

h-cyt (F 40.0(5)

2.4(1)

30(1)

12.2(4)

h-cyt c" 40.0(5)

4.7(1)

61(10)

13.1(20)

1.3(1)

24(1)

18.9(5)

22.5(5)

3.8(1)

59(4)

15.7(10)

y-cyt c" 40.0(5)

0.8(1)

15(1)

18.1(8)

y-cyt c° 40.0(5)

3.3(1)

45(3)

13.7(8)

1

y-cyt c" 22.5(5) 11

y-cyt c

1

1

1

1

5.2(5)

0.38

5.5(5)

0.42

5.4(5)

0.34

5.8(5)

0.42

Error estimates are given in parentheses.

The stability differences between y-cyt c and h-cyt c must arisefromside chain variations. Despite the nearly identical backbone folds of h-cyt c and y-cyt c (about 80% of the main chain to main chain hydrogen bonds are conserved between h-cyt c and y-cyt c, and the average deviation between main chain atoms is just 0.45 Â) (38), the two sequences are only 60% identical (59). Examination of the X-ray crystal structures of the two proteins reveals that the majority of the changes occur at surface residues, whereas the most highly conserved side chains are those that form the heme environment or are near the exposed heme edge (38,40,4Ί). This is likely due to the fact that the latter residues play a key role in determining the redox potential of the heme, which allows the protein to carry out its physiological ET function. Six distinct domains can be identified in cytochrome c: three α-helical regions (amino terminus, residues 1-13; carboxy terminus, 86-104; 60's helix, 61-69), and three omega loops (residues 20-35,36-60, and 70-85) (42). Only two of these regions, the 20-35 and 70-85 loops, display a high degree of sequence conservation (>80% identical) between y-cyt c and h-cyt c (39). The amino- and carboxy-terminal helices exhibit the lowest degree of similarity (58% and 37%, respectively). We also have measured the absorption spectral changes that accompany unfolding of cyt c by GuHCl. The primary change observed for oxidized h-cyt c at 22.5 °C and pH 7 is a slight shift of the Soret band to higher energy with an accompanying increase in extinction coefficient (e). Changes for the reduced protein (22.5 °C, pH 7) include a small shift to lower energy with an increase in e for the Soret band, and a very slight shift to lower energy with a decrease in e for the peaks at 520 and 550 nm. The electronic spectrum of cyt c is dominated by the heme group and the observed absorption changes reflect either differences in axial ligation or heme



203

environment that occur upon unfolding (43). The axial histidine remains bound to the heme upon unfolding of the oxidized protein at pH values above -2.5 (44); the axial methionine, however, is thought to be displaced by non-native histidine residues upon denaturation by GuHCl at pH 7 (8,45). Reduced unfolded cyt c is less well characterized but, presumably, the axial histidine remains bound and the methionine is replaced by another ligand at pH 7. Difference absorption spectra corresponding to formation of unfolded reduced (cyt eg - cyt c^) and folded reduced (cyt c$ - cyt c%) proteins exhibit similar overall shapes but differing extinction coefficients at the wavelengths used for most kinetics measurements (400, 420, and 550 nm) (20). Absorption spectral profiles of oxidized y-cyt c (in 0.18 M GuHCl) obtained upon thermal unfolding closely resemble those determined by denaturation with GuHCl. The transition is sufficiently steep and occurs at a high enough temperature (T - 51 °C) that the extinction coefficients at 22.5 and 40.0 °C are nearly identical (l-ms timescale. For these experiments, a reduction scheme based on the photochemistry of Co (ox) ~ is used (ox = ΟχΟ?~ ) (20). UV-laser excitation of Co (ox) " solutions rapidly generates Co° and a strong reductant, probably C0 " (E°(C(yC

Spectroscopic Studies of Ferrocytochrome c Folding - ACS Publications

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