Thermodynamics of the Thermal Denaturation of Acid Molten Globule

Subscriber access provided by UNIV OF NEW ENGLAND

Article

Thermodynamics of the thermal denaturation of acid molten globule state of cytochrome c indicate a reversible high-temperature oligomerization process Shigeyoshi Nakamura, Tomonori Saotome, Akiko Nakazawa, Masao Fukuda, Yutaka Kuroda, and Shun-ichi Kidokoro Biochemistry, Just Accepted Manuscript • Publication Date (Web): 21 Apr 2017 Downloaded from http://pubs.acs.org on April 23, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Biochemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Thermodynamics of the thermal denaturation of acid molten globule state of cytochrome c indicate a reversible high-temperature oligomerization process Running Title: Reversible high-temperature oligomerization of cytochrome c

Shigeyoshi Nakamura1,2, Tomonori Saotome3, Akiko Nakazawa2, Masao Fukuda2, Yutaka Kuroda3, and Shun-ichi Kidokoro2* 1

Department of General Education, National Institute of Technology, Ube College,

2-14-1 Tokiwadai, Ube 755-8555, Japan 2

Department of Bioengineering, Nagaoka University of Technology, 1603-1 Kamitomioka,

Nagaoka 940-2188, Japan 3

Department of Biotechnology and Life Science, Tokyo University of Agriculture and

Technology, 2-24-15 Nakamachi, Koganei 184-888, Japan

*Corresponding author: Shun-ichi Kidokoro, Department of Bioengineering, Nagaoka University of Technology, 1603-1 Kamitomioka, Nagaoka 940-2188, Japan Phone/Fax: +81-258-47-9425; E-mail: [email protected]

1

ACS Paragon Plus Environment

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Funding Information This work was supported by a Grant-in-Aid for Scientific Research on Innovative Areas (KAKENHI: No. 23118707) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and by Grants-in Aid for Scientific Research (C) (Nos. 25400426 and 25460576) from the Japan Society for the Promotion of Science.

Abbreviations MG: molten globule N: native D: denatured I2: dimer I3: trimer I4: tetramer I5: pentamer DSC: differential scanning calorimetry PPC: pressure perturbation calorimetry CD: circular dichroism SXS: solution X-ray scattering DLS: dynamic light scattering

2


Page 2 of 31

Page 3 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Rh: hydrodynamic radius

3


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract In this study, we performed differential scanning calorimetry (DSC) and pressure perturbation calorimetry (PPC) analysis of the thermal transition of cytochrome c from an acidic molten globule (MG) state with the protein concentrations of 0.5-18.2 mg/mL. DSC profiles were highly reversible and showed clear protein-concentration dependence, indicating that reversible oligomerization occurred accompanying the thermal transition from the MG state. The DSC and PPC data required at

 → MG2 ←  → D ←  → 1 I 2 ←  → 1 I3 ←  → 1 I 4 ) including three new least a six-state model ( MG1 ←      2 3 4 oligomeric states: dimer (I2), trimer (I3), and tetramer (I4) in addition to the three monomeric states previously characterized. Dynamic light scattering confirmed the oligomerization during the thermal transition. The partial specific volumes of these oligomeric states were found to be smaller than those of the monomeric states, MG2 and D, indicating dehydration of hydrophobic surface and/or hydration of released anions may occur with the reversible oligomerization.

Keywords Cytochrome c, molten globule, reversible oligomerization

4


Page 4 of 31

Page 5 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Thermodynamic properties of proteins including their volumetric characteristics give us the important knowledge on the thermodynamic stability and kinetic folding of proteins [1-10]. Several proteins were reported to show an equilibrium molten globule (MG) state, a compact intermediate state with a native-like secondary structure but a disordered tertiary structure on protein folding [11-12]. The knowledges of the thermodynamics and structure of MG state are highly significant to comprehend the folding, stabilization, and functional mechanisms of globular proteins [12-18]. The volumetric character of the state, however, has not been reported in detail yet. The molten globule state of cytochrome c under the condition; low temperature, high salt concentration, and low pH, was the first MG state to be reported (here, we designate it as MG1 state) [11]. Several molten globule states of cytochrome c were identified traditionally under some special conditions, such as acidic pH and high salt condition [11, 15, 19-21]; alkaline pH and high salt condition [22-23]; moderate concentration of denaturant, surfactant, or alcohol [24-27]; or chemical modification of amino acid side chain [28]. The equilibrium molten globule state of horse cytochrome c under a native condition has been recently identified in the thermodynamic transition from N to D state by calorimetry, SXS, and CD spectroscopy [29-30]. The acid MG state at low temperature (we call this state MG1) of cytochrome c exhibits a cooperative transition [29-33]. Although initially the structural transition from MG1 to D was considered to be a two-state transition based on the results from differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC) analysis [31-32], in the case of low protein

5


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

concentration ( ≤ 1 mg/mL), however, the transition from MG1 to D state was found to be at least a three-state transition including an intermediate (I) state, which we designate the MG2 state, by CD and precise DSC [21, 30, 33-34]. As shown in the previous literatures, it is worthwhile that both the N-to-D and MG-to-D transition of cytochrome c were demonstrated to be highly reversible when the pH of the solution was less than 5, and the reversibility enabled us to determine the change of entropy and Gibbs energy directly by DSC [34]. Recently, it was reported that cytochrome c formed a domain-swapped oligomer under a high concentration of ethanol [35]. This domain-swapped oligomer was also detected in the refolding process from the acid MG state to the native state of cytochrome c [36]. A model was proposed in which the MG oligomer is formed at acidic pH and high chaotropic ion conditions. Cytochrome c also formed amyloid fibrils under special conditions, such as the presence of SDS [37] and ionic phospholipid [38]. The thermodynamic study of the oligomerization of cytochrome c is, therefore, essential to understand the stabilization mechanism of the oligomer state. While the concentration dependence for the reversible thermal transition of monomeric proteins is not checked in many cases, we have observed reversible oligomerization from monomeric protein, Dengue envelope protein domain 3, in high temperature [39] recently. In this study, we reported that the thermal transition of the acidic MG state of cytochrome c also show reversible oligomerization in high temperature. This is the first report for the volumetric characterization of the reversible oligomerization in high temperature.

6


Page 6 of 31

Page 7 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Experimental Section Preparation of sample solution Lyophilized powder of horse cytochrome c (c-2506; Sigma, St. Louis, MO) was dissolved in 50 mM glycine (171-09; Nacalai Tesque, Kyoto, Japan) buffer including 500 mM KCl at pH 2.5. The sample solution was dialyzed with the dialysis membrane described previously [21]. In all experiments, pH was adjusted by using the electrode made of glass and pH device (F23; Horiba, Kyoto, Japan). The calibration of pH meter was performed as previously reported [21]. The protein solution was filtrated by using a membrane filter (Minisart; Sartorius Stedim Japan, Tokyo, Japan), with a 0.2 µ m pore size and ultrafilter-unit (USY-20; Advantec, Tokyo, Japan) described previously [21]. The protein concentration was monitored spectrophotometrically by using a UB-35 spectrophotometer (Jasco, Tokyo, Japan) and with an extinction coefficient of ε

409

= 10.6 × 104 M-1cm-1. Before the

measurements, the sample solution was thoroughly degassed as previously reported [21].

Differential scanning calorimetry (DSC) All the DSC measurements were done by using a high precision calorimetric equipment, VP-DSC (Microcal, Northampton, MA). DSC experiments were conducted as described previously [21, 34]. DSC samples with cytochrome c at 0.5–18 mg/mL concentrations in 50 mM glycine including 500 mM KCl at pH 2.5 were used to evaluate the concentration dependence of the thermal transitions. The scanning-rate in this study was used for 1 K/min unless otherwise noted.

7


Biochemistry

In all measurements, the reversibility of the thermal transition was confirmed by repeated scans of the sample solution (see Fig.1B). It was also checked that the scanning rate dependence of DSC profile of the thermal transition with the protein concentration of 18.0 mg/mL. The down-scan was started from 353.15 K to 283.15 K with the scanning rate of -0.5 K/min, and the up-scan was done just after the down-scan from 283.15 K to 353.15 K with the scanning rate of 1 K/min (see Fig.1C).

A

10 kJ K-1mol-1

18 mg/ml 10 mg/ml ∆Cpapp

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 31

4 mg/ml 1 mg/ml 0.5 mg/ml

280

300

320

340

T/K

8


360

380

400

Page 9 of 31

B -1

-1

∆Cpapp

10 kJ K mol

280

290

300

310

320

330

340

350

360

370

C ∆Cpapp

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

4 mJ K-1

280

290

300

310

320

330 T/K

340

350

360

370

Fig. 1A Heat capacity profiles of the transition of cytochrome c. Open squares, circles, triangles, diamonds, and crosses show the heat capacity profiles at 18, 10, 4, 1, and 0.5 mg/mL cytochrome c, respectively.

Solid

lines

show

the

fitting

data

calculated

with

the

six-state

model

 → MG2 ←  → D ←  → 1 I 2 ←  → 1 I3 ←  → 1 I 4 ). ( MG1 ←      2 3 4 1B Reversibility of the thermal transition of the molten globule state of cytochrome c with the protein concentration of 18.0 mg/mL. Open circles show the first heating of DSC (scanning up to 353.15 K). Solid line shows the second heating of DSC. 1C Scanning rate dependence of DSC profile of the thermal transition of the molten globule state of cytochrome c with the protein concentration of 18.0 mg/mL. Both data of the down scan (broken lines) with the scanning rate of -0.5 K min-1and the up scan (Thick solid line) with 1 K min-1 were 9


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

indicated in the figure. The down scan was started from 353.15 K to 283.15 K, and the up scan was done just after the down scan in the same temperature range.

The thermodynamic parameters were evaluated by the double deconvolution method and refined precisely by the nonlinear least-squares fitting analysis with the transition model including the self-dissociation/association process [40-41] using SALS libraries [42].

Pressure perturbation calorimetry (PPC) PPC experiments were done with the VP-DSC with a PPC accessory (Microcal, Northampton, MA) [4, 43]. In this study, the experimental condition and analytical method of PPC measurements were same as described previously [21]. For the PPC experiments, 5.6 mg/mL and 11.2 mg/mL cytochrome c at pH 2.5 in 50 mM glycine and 500 mM KCl were used to evaluate the protein concentration dependence of the PPC profiles. The monomeric N and MG states of cytochrome c in highly concentration (17 mg/mL) at low temperature (10-25oC) by solution X-ray scattering [29]. We checked the reversibility of the 11 mg/mL cytochrome c solution in this thermal transition. PPC analysis was performed using the six-state analysis including reversible oligomerization. In this study, the thermal expansion coefficient change in the transition from the MG1 to the oligomeric states, such as dimer, trimer, and tetramer, were fixed to zero.

10


Page 10 of 31

Page 11 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Dynamic light scattering (DLS) measurement DLS measurements were done at both 10oC and 90oC with 1~12.2 mg/mL protein solution (pH 2.5, 500 mM KCl) and 1~5.5 mg/mL protein solution (pH 4.0) with a Zetasizer NanoZS (Malvern Instruments, Malvern, UK) as previously reported [30]. Before the measurements, all the samples were filtered by using a 0.2 µ m pore size MolCut ultrafilter unit (USY-20; Advantec, Tokyo, Japan) with a cutoff molecular weight of 200 kDa. The hydrodynamic radius, Rh, of native (pH 4.0) and MG states (pH 2.5, 500 mM KCl) of cytochrome c were evaluated with the Einstein Stokes equation.

11


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Results The heat capacity profiles of the thermal transition of cytochrome c in the MG state clearly indicated a protein concentration dependence (Fig. 1A). The peak temperature decreased when the protein concentration was increased. The thermal transitions at all protein concentrations were fully reversible as Fig.1B even at the highest sample concentration of 18 mg/mL. If the protein solution in the DSC cell is kept more higher temperature or more longer time, the reversibility will become the lower due to the irreversible reaction as observed in the case of many other proteins. It is clear that our thermal transitions observed in this condition are fully reversible and do not include the irreveisible aggregation process. The scanning rate dependence of DSC profiles was also checked with a set of down- and up-scan with scanning rate of -0.5 K/min for the down-scan and 1 K/min for the up-scan, respectively. The thermal transition profiles of cytochrome c in the MG state with different scanning rate showed almost same, indicating the independency of this thermal transition discussed in this paper on scanning rate (Fig. 1C). Therefore, this transition of cytochrome c was confirmed that this transition could be treated as an equilibrium reaction. The protein concentration dependence of the thermal transition occurring under equilibrium condition indicated that the thermal transition included a self-association/disassociation process [34, 41, 45]. Especially, this protein concentration dependence suggested that the oligomerization occurred at high temperature, because the peak temperature shifted lower when protein concentration was higher.

This protein concentration dependence is in sharp contrast to the

12


Page 12 of 31

Page 13 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

commonly reported one, where the midpoint temperature usually increases with increasing protein concentration [44-45]. Thus, the present results indicated that the thermal transition of the MG state of cytochrome c undergoes a complicated transition that necessarily includes a reversible oligomerization process. We analyzed the DSC data by using a global fitting analysis with four-, five-, and six-state models including a reversible oligomerization process （Fig. 2A). A dimeric state, I2, was added to the

three

monomeric

states,

MG1,

MG2,

and

D,

in

the

four-state

model

 → MG2 ←  → D ←  → 1 I 2 ). Then we added a trimeric state, I3 to the four-state model, ( MG1 ←    2  → MG2 ←  → D ←  → 1 I2 ←  → 1 I3 ). Finally, a which became a five-state model ( MG1 ←     2 3 tetrameric state, I4, was added to the five-state model, which turned to a six-state model  → MG2 ←  → D ←  → 1 I 2 ←  → 1 I3 ←  → 1 I 4 ). The ∆ Cp in the MG1-to-MG2 transition ( MG1 ←      2 3 4 was fixed at 0. The ∆ Cp in the transition from MG1 to D state was fixed at 3.6 kJ K-1mol-1 as reported previously by DSC and ITC measurements [30].

13


Biochemistry

18 mg/ml

5 mJ K-1

A 290

∆Cpapp

280

300

310

320

330

340

350

360

1 mJ K-1

370

380

4 mg/ml

B 280

290

300

310

320

330

340

350

360

0.25 mJ K-1

370

380

0.5 mg/ml

C 280

290

300

310

320

330

340

350

360

370

T/K

500 450

D

400

Residual / µJK-1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 31

350 300 250 200 150 100 50 0 2

3

4

5

6

State Number

14


7

380

Page 15 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Fig. 2 DSC profiles without the normalization of protein concentration. 2A, 2B, 2C show the DSC profiles of 18 mg/mL, 4 mg/mL, and 0.5 mg/mL, of cytochrome c. Open squares show the DSC profiles. Red, Green, Blue lines show the fitting data calculated with six-states model ( (

 → MG2 ←  → D ←  → 1 I 2 ←  → 1 I3 ←  → 1 I4 MG1 ←      2 3 4  → MG2 ←  → D ←  → 1 I2 ←  → 1 I3 MG1 ←     2 3

),

five-state

),

four-state

model model

 → MG2 ←  → D ←  → 1 I 2 ) including an association/disassociation process. Magenta and ( MG1 ←    2 brown lines show the theoretical fitting curves with the three-state model (MG1-to-MG2-to-D) and the two-state model (MG1-to-MG2-to-D) without an oligomerization process, respectively. 2D show the residuals of global fitting analysis of DSC data for 2- to 7-state transition model.

The four-state and five-state models could not fully explain the experimental data, especially at low protein concentrations such as 1 mg/mL and 0.5 mg/mL. On the other hand, theoretical curve fitting fully explained the experimental data when the six-state model was used. Though it is common sense that increasing the number of parameters improves the fitting, the fitting residual significantly decreased when the six-state model (MG1-to-MG2-to-D-to-I2-to-I3-to-I4) was used (Fig, 2B). In addition, we assessed a seven-state transition model, where the pentameric state, I5, was added to the above six-state model, but the residual of the seven-state model was almost the same

as

that

of

the

six-state.

We

thus

concluded

that

the

six-state

model

 → MG2 ←  → D ←  → 1 I 2 ←  → 1 I3 ←  → 1 I 4 ) was the simplest model that explained the ( MG1 ←      2 3 4 15


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

protein concentration dependence of the DSC data of the MG state of cytochrome c. The mole fraction of each state, defined as “molar fraction” in the previous analysis [41], by the global fitting analysis with the six-state model is illustrated in Fig. 3. At 18 mg/mL and high temperature, cytochrome c was mainly in the I4 state. The oligomeric states, such as I2, I3, and I4, were mainly observed at temperatures over 320 K. On the other hand, the monomeric states, such as MG1, MG2, and D, were mainly observed under 350 K in the 0.5 mg/mL cytochrome c. The thermodynamic parameters evaluated by the global fitting analysis with the six-state model indicated that the enthalpy changes, ∆H , of the MG1 state to any of the oligomeric states were larger than the

∆H of the MG1-to-MG2 transition (Table 1). Similarly, the ∆H of the MG1 to any of the oligomeric states was smaller than the ∆H in the transition from MG1 to D.

 ∂V  The  p  during the thermal transition of the molten globule state (MG1) of  ∂T  p cytochrome c was measured by PPC (Fig. 4) [45]. The experimental data were fully rationalized by the six-state model very. Table 1 shows the ∆V evaluated by PPC analysis at 5.6 mg/mL and 11.2 mg/mL cytochrome c and pH 2.5 in the presence of 500 mM KCl with the six-state model. ∆V MG1-to-MG2 was larger than ∆V MG1-to-D, which was very small. The partial volume, V, of the dimer

(I2) and that of the trimer (I3) were larger than that of the MG1. On the other hand, V of the tetramer (I4) was smaller than that of the MG1 and V of the dimer (I2), trimer (I3), and tetramer (I4) were smaller than that of the MG2.

16


Page 16 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Population fraction

Page 17 of 31

1.0 0.8 MG1 0.6 MG2 0.4 18 mg/ml 0.2 0.0 280

290

300

1.0 0.8 0.6 0.4 4 mg/ml 0.2 0.0 280

1.0 0.8 0.6 0.4 0.2 0.0

I4

I2 I3 310

320

330

340

A

D 350

360

370

B

290

300

310

320

330

340

350

360

370

C 0.5 mg/ml

280

290

300

310

320

330

340

350

360

370

T/K

Fig. 3 Mole fractions in the thermal transition of the molten globule states of 18 mg/mL (A), 4 mg/mL (B), and 0.5 mg/mL cytochrome c (C). Black, green, red, blue, brown, and magenta lines show the mole fractions of MG1, MG2, D, I2, I3, and I4, respectively.

17


Biochemistry

0.00065

0.00060

/ cm g-1K-1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 31

0.00055

0.00050

0.00045

0.00040 280

290

300

310

320

330

340

350

360

370

T/K  ∂V  Fig. 4 The temperature dependence of  p  of the MG state of 11 mg/mL cytochrome c. Open  ∂T  p  ∂V  squares show the  p  . Solid line shows the fitting data calculated by six-states analysis. Broken  ∂T  p

 ∂V  lines show the baselines of  p  .  ∂T  p

In order to confirm the reversible formation of oligomers in the thermal transition of cytochrome c, we measured the hydrodynamic radius (Rh) by DLS as a direct measure of cytochrome

c molecular size under a specified condition. Figure 5A shows the size distribution of the thermal transition of the MG state of cytochrome c. The size at 90oC was larger than at 10oC both at 1.2 mg/mL and 13.2 mg/mL, but the size increase was much larger at 13.2 mg/mL than at 1.2 mg/mL (Table 2). The larger size of cytochrome c at high protein concentration suggests the presence of 18


Page 19 of 31

oligomers at high temperature in line with DSC analysis. On the other hand, DLS measurements at pH 4 performed at 1.3 mg/mL and 5.5 mg/mL cytochrome c indicted that Rh was only slightly larger at 90oC than at 10oC, indicating that the size of the denatured state (D) was larger than that of the N state of cytochrome c. However, the size of cytochrome c at 1.3 mg/mL and 5.5 mg/mL, pH 4, and 90oC were almost the same, and thus no protein concentration dependence was observed under this condition, which suggests that the D state of cytochrome c at pH 4 is in a monomeric state and that no oligomers are present in the denaturation.

Intensity / %

25

A

20 15 10 5 0 25 0.1

Intensity / %

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

1

10

B

20 15 10 5 0 0.1

1

Radius / nm

10

Fig. 5A Size distribution of molten globule state of cytochrome c by DLS measurements. Black and red squares show the intensity profiles of the molten globule states of 1.2 mg/mL cytochrome c at 10 o

C and 90 oC, respectively. Open triangles and blue squares show the intensity profiles of the molten

19


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

globule states of 13.2 mg/mL cytochrome c at 10oC and 90 oC, respectively. 5B Size distribution of cytochrome c at pH 4.0 by DLS measurements. Black and red squares show the intensity profiles of 1.3 mg/mL cytochrome c at 10 oC and 90oC, respectively. Open triangles and blue squares show the intensity profiles of 5.5 mg/mL cytochrome c at 10 oC and 90 oC, respectively.

20


Page 20 of 31

Page 21 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Table 1 Thermodynamic parameters calculated by the six-state fitting analysis of the DSC / ℃

∆Cp / kJ K-1mol-1

∆H (Tm) / kJ mol-1

∆H (50℃) / kJ mol-1

∆V (50℃) / 10-3 nm3

50.8 40.5 40.7

0(fixed) 3.6 (fixed) 3.1

90 260 155

91 253 185

25.5 2.5 21.5

MG1-to- 1 I3

42.8

3.8

210

237

21.3

MG1-to- 1 I4

42.5

7.5

196

252

-4.5

MG2-to- 1 I2

94

-4.0

MG2-to- 1 I3

146

-4.2

MG2-to- 1 I4

161

-30

Transition MG1-to-MG2 MG1-to-D MG1-to- 1 I2

Tm

2

3

4

2

3

4

Table 2 Hydrodynamic radius, Rh, of cytochrome c

Solvent

Concentration

T / oC

Rh* / nm

pH 2.5, 500 mM KCl H 2.5, 500 mM KCl H 2.5, 500 mM KCl

1.2 mg/mL 1.2 mg/mL 13.2 mg/mL

10 90 10

1.8 2.5 1.8

H 2.5, 500 mM KCl pH 4.0 pH 4.0 pH 4.0

13.2 mg/mL 1.3 mg/mL 1.3 mg/mL 5.5 mg/mL

90 10 90 10

3.9 1.7 2.3 1.8

pH4.0

5.5 mg/mL

90

2.1

21


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Discussion Although irreversible aggregation upon denaturation occurs very frequently, the observation of a reversible oligomerization state at high temperature is rare. In a previous study, we observed the reversible thermal transition of Dengue envelope protein domain 3 (ED3), where ED3 undergo a transition from monomeric native state to the tetrameric intermediate state, and monomeric denatured state (the N-to-I4-to-D transition) [38]. This study revealed that the MG state of cytochrome c undergo a similar thermal transition that includes reversible oligomerization states at high temperature. Data fitting using a transition model that includes oligomeric states indicated that the residuals decreased significantly when the number of states was increased from four to six (Fig. 2B). However, the residuals with the six-state and seven-state analyses were almost the same. Therefore,

 → MG2 ←  → D ←  → 1 I 2 ←  → 1 I3 ←  → 1 I4 ) as mentioned above, the six-state model ( MG1 ←      2 3 4 was the simplest one that explained the thermal transition of the acidic MG state of cytochrome c. We also evaluated the partial volume change, ∆V , at 50oC in the thermal transition including oligomerization by using PPC measurements (Table 1). The ∆V during the thermal transition are determined by the hydrophobic hydration effect and the hydration effect of anion binding to the cytochrome c molecule. The ∆ V at 50oC in the MG1-to-MG2 transition was larger than that in the MG1-to-D transition. This large positive volume change in the MG1-to-MG2 transition is presumably derived from the increased exposure of hydrophobic residues, the increased

22


Page 22 of 31

Page 23 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

size of the cavity in the MG2 state, and the anion binding to the MG2 state. The partial volumes of oligomeric states, such as dimer, trimer, and tetramer were smaller than that of the MG2 state. The negative volume change in the transition from the MG2 to the oligomeric state is thought to be derived from the decrease in the exposure of the hydrophobic region and/or the anion-release effect from the MG2 state. The C-terminal helix of cytochrome c is incorporated in the other molecule in the structure of the domain-swapped dimer and tetramer of cytochrome c [35]. The similar intermolecular interaction may be occurred in case of the reversible oligomerization process of cytochrome c in our study. Although farther study will be required to evaluate the structure of the oligomers reversibly formed in high temperature, it seems reasonable that the reversible oligomerization is driven by decreasing the hydrophobic surface and the number of the binding anions to the protein surface decreases by decreasing the exposed surface. The monomeric acidic MG state of cytochrome c in the equilibrium condition was considered to be the kinetic intermediate on the protein folding [11-13, 15, 29-30]. On the other hand, the monomeric alkali-MG state of cytochrome c was confirmed as the off-pathway species in the protein folding [23]. Although the reversible oligomeric state in our study is not considered the intermediate state in the monomeric protein folding, there is a possibility of the oligomeric intermediate state in the folding process from the monomeric acid-MG state to the domain-swapped oligomer of cytochrome c proposed previously [36].

23


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Acknowledgments The authors would like to dedicate this article to Dr. Akiyoshi Wada, Professor Emeritus, the University of Tokyo, on the occasion of his 88th birthday celebration, "Beiju" in Japanese. We thank Mr. Eiichi Tsuruta (Shoko Scientific Co., Ltd.) for his advice on DLS measurements and analyses.

24


Page 24 of 31

Page 25 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

References (1)

Privalov, P. L. (1979) Stability of proteins small globular proteins, Adv. Protein Chem. 33, 167-236.

(2)

Chalikian, T. V., and Breslauer, K. J. (1996) Volumetric properties of the molten globule state of cytochrome c in the thermal three-state transition evaluated by pressure perturbation calorimetry, Biopolymers 39, 619-629.

(3)

Chalikian, T. V. (2003) Volumetric properties of proteins, Annu. Rev. Biophys. Biomol. Struct. 32, 207-235.

(4)

Lin, L. N., Brandts, J. F., Brandts, J. M., and Plotnikov, V. (2002) Determination of the volumetric properties of proteins and other solutes using pressure perturbation calorimetry, Anal. Biochem. 302, 144-160.

(5)

Mitra, L., Smolin, N., Ravindra, R., Royer, C., and Winter, R. (2006) Pressure perturbation calorimetric studies of the solvation properties and the thermal unfolding of proteins in solution—experiments and theoretical interpretation, Phys. Chem. Chem. Phys. 8, 1249-1265.

(6)

Schweiker, K. L., Fitz, W., and Makhatadze, G. I. (2009) Universal convergence of the specific volume changes of globular proteins upon unfolding, Biochemistry 48, 10846-10851.

(7)

Fan, H. Y., Shek, Y. L., Amiri, A., Dubins, D. N., Heerklotz, H., Macgregor, Jr., R. B., and Chalikian, T. V. J. (2011) Volumetric characterization of sodium-induced G-quadruplex formation,

25


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Am. Chem. Soc. 133, 4518-4526. (8)

Roche, J., Caro J. A., Norverto, D. R., Barthe, P., Roumestand, C., Schlessman, J. L., Garcia, A. E., Garcia-Moreno, B. E., Royer, C. A. (2012)

Cavities determine the pressure unfolding of

proteins, Proc. Natl. Acad. Sci. U S A 109, 6945-6950. (9)

Tsamaloukas, A. D., Pyzocha, N. K., and Makhatadze, G. J. (2010) Pressure perturbation calorimetry of unfolded proteins, Phys. Chem. B. 114, 16166-16170.

(10)

Schweiker, K. L., and Makhatadze, G. I. (2009) Use of pressure perturbation calorimetry to characterize the volumetric properties of proteins, Methods Enzymol. 466, 527-547.

(11)

Ohgushi, M., Wada, A. (1983) 'Molten-globule state': a compact form of globular proteins with mobile side-chains, FEBS Lett. 164, 21-24.

(12)

Arai, M., Kuwajima, K. (2000) Role of the molten globule state in protein folding, Adv. Protein Chem. 53, 209-282

(13)

Ptitsyn, O. B. (1995) Molten globule and protein folding, Adv. Protein Chem. 47, 83-229.

(14)

Ikeguchi, M., Kuwajima, K., Mitani, M., Sugai, S. (1986) Evidence for identity between the equilibrium unfolding intermediate and a transient folding intermediate: a comparative study of the folding reactions of .alpha.-lactalbumin and lysozyme, Biochemistry 25, 6965-6972.

(15)

Colon, W. C., Roder, H. (1996) Kinetic intermediates in the formation of the cytochrome c molten globule, Nat. Struct. Biol. 3, 1019-1025.

(16)

Balbach, J., Forge, V., Lau, W. S., Van Nuland, N. A., Brew, K., Dobson, C. M. (1996) Protein

26


Page 26 of 31

Page 27 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

folding monitored at individual residues during a two-dimensional NMR experiment, Science

274, 1161-1163. (17)

Fink, A. L. (2005) Natively unfolded proteins, Curr. Opin. Struct. Biol. 15, 35-41.

(18)

Dunker, A. K., Cortese, M. S., Romero, P., Iakoucheva, L. M, Uversky, V. N., Flexible nets; The roles of intrinsic disorder in protein interaction networks, FEBS J. 272, 5129-5148.

(19)

Kuroda, Y.,

Endo, S., Nagayama, K., and Wada, A. (1995) Stability of α-helices in a molten

globule state of cytochrome c by hydrogen-deuterium exchange and two-dimensional NMR spectroscopy, J. Mol. Biol. 247, 682-688. (20)

Santucci, R., Bongiovanni, C., Mei, G., Ferri, T., Polizio, F., Desideri, A. (2000) Anion size modulates the structure of the A state of cytochrome c, Biochemistry 39, 2632-12638.

(21)

Nakamura, S., and Kidokoro, S. (2012) Volumetric properties of the molten globule state of cytochrome c in the thermal three-state transition evaluated by pressure perturbation calorimetry, J. Phys. Chem. B 116, 1927-1932.

(22)

Rao, D. K., Kumar, R., Yadaiah, M., Bhuyan, A. K. (2006) The alkali molten globule state of ferrocytochrome c: extraordinary stability, persistent structure, and constrained overall dynamics, Biochemistry 45, 3412-3420.

(23)

Bhuyan, A. K. (2010) Off-pathway status for the alkali molten globule of horse ferricytochrome c, Biochemistry 49, 7764-7773.

(24)

Bai, Y., Sosnick, T. R., Mayne, L., Englander, S. W. (1995) Protein folding intermediates:

27


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

native-state hydrogen exchange, Science 269, 192-197. (25)

Kamatari, Y. O., Konno, T., Kataoka, M., and Akasaka, K., (1996) Thermodynamic stability

of ribonuclease A in alkylurea solutions and preferential solvation changes accompanying its thermal denaturation: a calorimetric and spectroscopic study, J. Mol. Biol. 259, 512-523. (26)

Chattopadhyay, K., Mazumdar, S. (2003) Protein folding intermediates: native-state hydrogen exchange, Biochemistry 42, 14606-14613.

(27)

Qureshi, S. H., Moza, B., Yadav, S., Ahmad, F. (2003) Conformational and thermodynamic characterization of the molten globule state occurring during unfolding of cytochromes-c by weak salt denaturants, Biochemistry 42, 1684-1695.

(28)

Goto, Y., Nishikiori, S. (1991) Role of electrostatic repulsion in the acidic molten globule of cytochrome c, J. Mol. Biol. 222, 679-686.

(29)

Nakamura, S., Baba, T., and Kidokoro, S., (2007) A molten globule-like intermediate state detected in the thermal transition of cytochrome c under low salt concentration, Biophys. Chem.

127, 103-112. (30)

Nakamura, S., Seki, Y., Katoh, E., and Kidokoro, S. (2011) Thermodynamic and structural properties of the acid molten globule state of horse cytochrome c, Biochemistry 50, 3116-3126.

(31)

Potekhin, S., Pfeil, W. (1989) Microcalorimetric studies of conformational transitions of ferricytochrome c in acidic solution, Biophys. Chem. 34, 55-62.

28


Page 28 of 31

Page 29 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(32)

Biochemistry

Hamada, D., Kidokoro, S., Fukada, H., Takahashi, K., and Goto, Y. (1994) Salt-induced formation of the molten globule state of cytochrome c studied by isothermal titration calorimetry, Proc. Natl. Acad. Sci. U S A 91, 10325-10329.

(33)

Kuroda, Y., Kidokoro, S., Wada, A. (1992) Thermodynamic characterization of

cytochrome c at low pH. Observation of the molten globule state and of the cold denaturation process, J. Mol. Biol. 223, 1139-1153. (34)

Kidokoro, S., and Nakamura, S. (2016) IATC, DSC, and PPC analysis of reversible and multi-state structural transition of cytochrome c, Methods Enzymol. 567, 391-412.

(35)

Hirota, S., Hattori, Y., Nagao, S., Taketa, M., Komori, H., Kamikubo, H., Wang, Z., Takahashi, I., Megi, S., Sugiura, Y., Kataoka, M., and Higuchi, Y. (2010) Cytochrome c polymerization by successive domain swapping at the C-terminal helix, Proc. Natl. Acad. Sci. U S A 107, 12854-12859.

(36)

Deshpande, M. S., Parui, P. P., Kamikubo, H., Yamanaka, M., Nagao, S., Komori, H., Kataoka, M., Higuchi, Y., and Hirota, S. (2014) Formation of domain-swapped oligomer of cytochrome C from its molten globule state oligomer, Biochemistry 53, 4696-4703.

(37)

Haldar, S., Sil, P., Thangamuniyandi, M., and Chattopadhyay, K. (2015) Conversion of amyloid fibrils of cytochrome c to mature nanorods through a honeycomb morphology, Langmuir 31, 4213-4223.

(38)

Alakoskela, JM., Jutila, A., Simonsen, A. C., Pirneskoski, J., Pyhajoki, S., Turunen, R., Marttila,

29


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

S., Mouritsen, O. G., Goormaghtigh, E., and Kinnunen, KJ. P. (2006) Characteristics of fibers formed by cytochrome c and induced by anionic phospholipids, Biochemistry 45, 13447-13453. (39)

Saotome, T., Nakamura, S., Islam, M. M., Nakazawa, A., Dellarole, M., Arisaka, F., Kidokoro, S., Kuroda, Y., Unusual reversible oligomerization of unfolded Dengue envelope protein domain 3 at high temperature and its abolition by a point mutation, Biochemistry 55, 4469-4475.

(40)

Kidokoro, S., Wada, A. (1987) Determination of thermodynamic functions from scanning calorimetry data, Biopolymers 26, 213-229.

(41)

Kidokoro, S., Uedaira, H., Wada, A. (1988) Determination of thermodynamic functions from scanning calorimetry data. II. For the system that includes self‐dissociation/association process, Biopolymers 27, 221-225.

(42)

Nakagawa, T., and Oyanagi, T. Program system SALS for non-linear least-squares fitting in experimental sciences, Recent developments in statistical inference and data analysis, North Holland Publishing Company, Amsterdam, 1980, pp. 221-225.

(43)

Mitra, L., Rouget, J. B., Garcia-Moreno, B., Royer, C. A., Winter, R. (2008) Microcalorimetry of heat capacity and volumetric changes in biomolecular interactions—the link to solvation?, Chemphyschem. 9, 2715-21.

(44)

Kitakuni, E., Kuroda, Y., Oobatake, M., Tanaka, T., and Nakamura, H. (1994) Thermodynamic characterization of an artificially designed amphiphilic α-helical peptide containing periodic prolines: Observations of high thermal stability and cold denaturation, Protein Science 3,

30


Page 30 of 31

Page 31 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

831-837.

(45)

Ali, S. A., Iwabuchi, N., Matsui, T., Hirota, K., Kidokoro, S., Arai, M., Kuwajima, K., Schuck, P., Arisaka, F. (2003) Reversible and fast association equilibria of a molecular chaperone, gp57A, of bacteriophage T4, Biophys. J. 85, 2606-2618.

(46)

Dellarole, M., Kobayashi, K., Rouget, JB., Caro, J. A., Roche, J., Islam, M. M., Garcia-Moreno, B., Kuroda, Y., and Royer, C. A. J. (2013) Probing the physical determinants of thermal expansion of folded proteins, Phys. Chem. B 117, 12742-12749.

For Table of Contents Use Only

31


Thermodynamics of the Thermal Denaturation of Acid Molten Globule

Recommend Documents