Comparative thermodynamic study of pepsinogen and pepsin structure

.J. Mol.

Hid

(19X1 ) 152, 445-464

Comparative

Thermodynamic Study of Pepsinogen and Pepsin Structure

PETFCKI,. PRIVALOV, PEDRO L. MATEO~, NIKOLAI N. KHECHINASHVIIJ Institute =Icadumy

c#’ Rriences

of Protein Research P oustch,ino, Moscou~

of the l’.S.S.R..

\T~~~~~~~~ M. STEPAN~V Departmrnt

(Receiwd

AND

LYCDMILA P.

Region,

U.S.S.Iz.

RWINA

of Chemistry

I7 April

1981)

Pepinogen. pepsin and its (:-terminal fragment have been studied thermodynamically in solution by a sranning microcalorimetric method at various pH and salt content values. It has been shown that: (I ) thermal denaturation of pepsinogen is a highly reversible process that takes place within a narrow temperature range depending upon the prevalent conditions, but under no condition does it correspond to a two-state transition. This process can be approximated by two quasi-independent transitions. indicating that the pepsinogrn molecule consists of t,wo slightly interacting, co-operative structural blocks of different size. (2) Thermal denaturation of pepsin is a complex process that proceeds in two distinct stages occurring at different temperatures, only the second being completely reversible. These stages correspond to separat,e meltings of two independent parts of the molecule, these being the N-terminal lobe and t,he more stable Ct’erminal lobe. Neither of these stages represents a two-state transit,ion. Analysis of t’hese transitions shows that both parts of t,he pepsin molecule consist of two quasi-independent, co-operative units. (3) All four co-operative units of pepsin have a rompart, structure with a welldeveloped hydrophobic Core, and therefore these units should be regarded as stjructural domains of the molerule. (‘onsequent,ly, each lobe of the pepsin molecule represents a struct)ural block ronsisting of two domains. In the presence of pepstatin, the two domains in the K-terminal lobe co-0perat.e to form a single q&em. (1) In pcpsinogen, the above-mentioned blocks are murh less independent than the co-operative units in pepsin, and it, is likely that each of them consists of two mr,rgrd domains. Therefore, release of the 44.residue N-terminal polypeptide upon activation of pepsinogen leads to a loosening of the domain struct,ure and to an inc,rrase of interdomain motilit,y of the molerulc.

t Perrnanmt address: (~wnatla, Spain.

Ihqxartamento

de Quimira

F&a.

Facultad

de (‘iencias.

,$? 1981 Academic

Press Inc.

[Tniversidad

de

(London)

I,td.

446

P.

I,.

PRI\‘;\I,O\-

ET

AL

1. Introduction Although crysballography is indisputably the main SOUI‘W ofstruct~ural informat~ion in studying proteins, some conclusions c*onc*erning t,heir submolecular organizat,ion can be obtained from t,he thermodynamic* analysis of the process of unfolding of their compact’ native structure: i.e. of denaturation. This becomes rspe~~ially important when the molecule is very large and its submolecular parts arr not ver> distinct, or when crystallographic information on it)s struc%ure does not exist at all. as is the case with most of the prot~eins. As has been shown recently. the most efficient experimental method for t)hr quant,itative thermodynamic study of macromolecular unfolding is scanning microcalorimetry (Privalov K; Nlimonov. 1978), since a knowledge of t,he t)emperature drpendencsr of the enthalpy function temperature-induced process without any (I permit)s a strict analysis of the complex priori assumptions (Freire & IWonen, 197X). In this paper we demonstrate the possibility of this approach for studying t)hr submolrc~ular organization of proteins. using as an example two relat,ed proteins, pepsinogen and pepsin. At present, t’hc rt crystallographic: struct’ure is known only for pepsin at 3.0 A4 resolution (;\ndrerra ~1.. 1978). It’ is expected that the structure of pepsinogrn will bc revraled soon (Kao of nl.. 1977). Therefore, t,he correctness of the structural conclusions drawn from thtx tjhermodyna,mic study of these systems might be (*hecked within a short period of’ t’ime. I)mat,uration of these t,wo proteins has been extjrnsively shudird by various researchers (I’erlmann, 1963; Frattali et al., 1965: Ahmad 62 McPhie. 1978a,6,1979). Unfortunately. no quantitative thermodynamics information can be drawn from thr published results on equilibrium without. an n priori assumption concaerniny thtb two-state character of the studied procbess, but t,he crux of the problem lies preciseI> here, and that is what, we wish to consider in this paper (see also Mat,eo & Privalov. 1981).

2. Materials (a)

and Methods Matrriats

(irade 1 porcine pepsinogen, chromatographically free of pepsin activity, was purchased from Sigma as a lyophilized powder and used without further purification. The porcine pepsin used throughout these studies was purified from the commercial preparation by ion-exchange chromatography on aminosilochrom (Matyash et al.. 1975). The homogeneity of the enzyme was checked by activity measurements against haemoglobin. by affinity chromatography on bacitracin-Sepharose 4B and by disc electrophoresis in 103;, (w/v) polyacrylamide gel at pH 5.5 and 9.4 in the presence of 6 M-urea. The C-terminal fragment of the diazoacetyl-inhibited pepsin (DDE-Ft) was prepared according to the procedure described in detail by Revina et al. (1975). Pure porcine pepsin was inactivated by ~~-diazoacetyl-,~‘-tL,4-dinitrophenylethylenediaminr in the presence of Cu2+ (Stepanov ct al., 1968). Each molecule of t,he pepsin (DDE-P), completely inactivated by this treatment, contained the residue of one inhibitor attached to aspartic acid residue 21s: (‘H,COOCH,CONHCH2(‘H2NH~(:,H,(N0,), I -NH-CH-(‘0 t Abbreviations used: DDE-F and diszoacetyl-N’-2,4-dinitrophenylethylenediamine.

DDE-P.

fragment and pepsin,

respectively,

treated

with

LV

PEPSINOGEN

ANI)

PEPSIN

STRI‘(‘TI’RE

117

was incubated at pH 645 and 33°C for 15 min with active Inhibited pepsin (DDE-P) liepsin (weight ratio 30: 1). The mixture was then applied t.o a DEAE-cellulose column etluilibrated wit)h acetate buffer (pH 5.1). Elution of the column with a concentration of the roloured peak III, which gradient of Na(‘l (0 t,o 1 M: pH 5.1) allowed the separation was further purified by passage through Sephadex G-75 to yield peak 111-l. This material ret,ained the oovalently bound inhibitor, judging by its behaviour during Sephadex G-75 gel tiltration, and corresponded to globules with a molecular weight of about 20,000. It gave one bantl when subjected to disc electrophoresis at pH 9.4 in the presence of 6 M-urea. Tt,s (‘terminal amino acid residue, alanine, roincided with that of porcine pepsin. The presence in this pepsin fragment of both the arginine residues and a unique lysine residue known t,o be situated at the (‘-terminal part of the sequence indicated that t)his peptide corresponds to the (‘-terminal moiety of t,he enzyme. The terminal amino acid sequence dekrmined by an automated Edman procedure was found to coincide with the sequenre 180 t,o 204 of porcine pepsin. Therefore. it was evident that fragment III-1 corresponds to the C-terminal moiety 01’ pepsin covering the sequence 180 to 327 of the enzyme. Two other relatively stable fragments. III-2 and TTI-3, found among the products of inhibited pepsin-limited proteolysis c,orrrspond to sequenc*es 180 to 314 and 188 to 314. The S-terminal moiety of porcine pepsin, tieing obviously less stable, gives a mixture of relatively short peptides when submit,ted to (~xteritled proteolysis. I’epst,atin used in this study was purchased from the Protein Research Foundation. Protein solutions were prepared by dissolving dry samples in buffer solutions. Before (.alorimrtric, experiments, sample solutions were dialyzed for 24 h at 4°C against a large volume of buffer solution with 3 changes of dialysis fluid. The buffers used were 5 mM-sodium phosphate (monobasic) for pH values 65, 7.1, 7.5 and 8.0, and 5 m,n-sodium cacodylate for pH 6G. The pH values were determined at 20°C’ with a glass electrode. The ionic strength ot the solutions was varied by the addition of XaCI up to 100 rnbr. In experiments with pepst,atin. the protein solutions were mixed with an appropriate amount of saturat.ed pepstatin solution. The choncentrations of pepsin and pepsinogen solutions were determined spectrophotometrically at 278 nm using the respective molar absorption coefficients of 5.10~ 104 (&Ahmad & McPhie. 197%) and 5.17 x lo4 1 cm-’ mall’ (A-non 8: Perlmann. 1963). Difference spectra between active pepsin and DDE-P showed that there is practically no difference between their respective absorption values at 278 nm. The value for the absorption coefficient of DDE-F at, 278 nm was found to be 2% x lo4 1 cm-’ mol- ‘, according t’o the nitrogen content in the sample determined by combustion on a Hitachi C’HN-analyzer. For all measurements. the prot)ein concentrations in solut,ions were in the range 0.50 to 2.1Omgml-‘. The possible self-proteolysis of active pepsin samples was checked by gel electrophoresis in the presence of sodium dodecyl sulphate before and after the catorimet,ric experiments. ,4t pH values of 65 and above. there was no evidence of accumulation of pepsin self-digestion product,s. either in initial or in heated solutions, while at pH values below 6.5. more than one zone in the gels was found. The evidence of self-digestion precluded a quantitative thermodynamic study of pepsin at pH values below 6.5, where it is in the active stat.e. .1ggregation of protems on denaturation was another serious obstacle that precluded the study. especially of inhibited pepsin, at pH values below fi+.

(b) ,Wrthods (‘alorimetric measurements were carried out’ in a differential adiabatic microcalorimeter DASM-l?UI (Priralov, 1980) at a scan rate of 2 K min-’ with 1 ml cells. Degassing during heating was prevented by using an extra constant pressure of 15 atm. over the liquids in the <*ells. The reversibility of the thermally induced transition was checked by reheating the protein solution in the calorimeter cell immediately after t,he cooling from the first run. Fig. 1 shows

148

I’. 1,. PKI\‘.4I,OV

ET AL

1

Ij

80 FIG. 1. Original calorimetric recordings of difference heat capacity function of pepsinogen at :! consecutive beatings of the same sample. Curve 1. first heating: curve 2. second heating. Concentration of protein in solution. OS! mg/ml. pH 60. 5 mw-cawdylk buffer. d consrwtive runs for a pepsinogen solution as an example of the reversibility of the t,ransition, as well as the reproducihilit,y of the experimental measurements. as a function of The partial specific heat capacity of proteins. Cp,,“, was calculated t,emperature by the deviation of the calorimetric. recording for solution from the base-line dC,(t)“PP (see Fig. 1) using the equation :

(1) where mpr is the amount of prot,ein in t,he calorimet,ric cell. and lir and I;,, are the partial specific volumes of thr protein and solvent,, respe&ively (Privalov & Khechinashvili, 1974). for pepsin and pepsinogen. The values for Vpr used here were 0.726 and (b730 ml g-l respect,ively (Williams & Rajagopalan, 1966). The increase of the partial heat caparit? at melting of the active structure, A,,$‘, (Fig. I ) was estimated by linear extrapolation of imtial and final heat capacity values to the middle of t,he process. as described elsewhere (Privalov, 1979). The calorimetric. or real enthalpies of melting, A,Hca’. were c-alculated from the area under the heat absorption peak using an adequat,e calibration mark. Values for the effect’irc, or ran’t Hoff, enthalpies were obtained by the equation :

P-” ---!. A mH’h = 4K7’= A m

Q, ’

(2)

where ACF’ is the height of the heat capacity peak at the melting temperature T,,,. and 0, is the area of the peak in energy units (Privalor & Khechinashrili, 1974). In the calculations of molar thermodynamic quant,ities, the molecular weight’s used were 39,687, 34,644 and 14,644 for pepsinogen, pepsin and DDE-F, respectively, estimated from the known sequence of these proteins (Moravek 8r Kostka, 1974: Tang et al., 1973: Pedersen 8 Foltman. 1975).

PEPSINOGES

ASI)

PEPSIN

RTRVCTURE

119

3. Results (a) Pepsinogen As shown in Figure 1, pepsinogen thermal denaturation takes place in a narrow t)emperature range wit’h an intensive heat absorption. and results in an essential heat capacity increase. This process is highly reversible under all coriditions st’udied. judging by the reproducibility of the peak on reheating the sample. Figure 2 shows the t,emperature dependence of the specific partial heat capacity of pepsinogen under different experimental conditions. At. 20°C it is and with the (0.39 +OW) oal K- ’ 8-l for native protein under any condition, increase of temperat,ure the specific partial heat capacity increases linearly with a slope of 22 x 10P3 cal K-* g-l. On denaturation. the specific heat capacity function shifts by a A$,, value of (0~145$_MlO) cal K-’ g-l. The calorimetric, A,Hca’ and ran’t Hoff enthalpy values d,Hvh for pepsinogen drnaturation at various conditions are given in Table I. The last column of the ‘l’ablt~ givcv t’he extent of reproducibility (Repr.) of the heat effect on reheating t)hr samples. As seen. the reproducibility. i.e. reversibility of the process, somewhat) drcarcxases with increase of pH and salt content.

Figures 3 and 1 show the temperature dependence of the specific partial heat c.apacity for active pepsin at different pH and ionic strengt,h values. Thermal denaturation of the protein manifests itself in int)ensivr heat absorption, which appears as one rather broad peak in salt-free solution at pH 6.5 and at lower pH values. \vhivh are not considered here because of self-proteolysis at these values. IVith increase of pH and ionic strength, this heat absorption peak splits into two Mcll-defined peaks (-4 and R in Fig. 4). The observed splitting of the denaturational heat absorption is thr result of t)he different effects of pH and ionic st’rength on the temperature of appearance of its two constituent peaks. The increase of pH leads to in this temperature for bot,h peaks, especially for t,he first one, resulting a tlewrase

FIG:. 1. Sptvitic partial heat capacity functions of 1qGmgrn

at various pH values of solution

(‘ondition

pH pH pH pH pH pH pH pH pH

6-O 65, 7.1 X4 7.1. 65, 7.1. 7.1. 7.1.

1,( ‘(‘l

A,H’“’ (kwl mol - ‘)

A,P (kval mol - ‘)

Kt

h?pr.:

I .fi

100 mwNa(‘l

20 1.i

I ,.i I .1 I ,!4 1.7 1+i 1.x

100 ITIM-Nd’l I.0 JI-urea 14 JI-urea 1.0 M-IITCB 34) wuwa

J Repr.. ‘?,, of reproducibility

090 I

of enthalpy

value at reheating of solution.

,# la/ n

I

ib)

n

FIN:. 3. Specific partial heat capacity functions of pepsin at various conditions. Solid line. tirst heating: broken line. second heating of the same solution. (a) pH 6.5: (b) pH 7.1 : (c) pH 7.1. 10 mw SaCI : (d) pH 7.1. 100 mu-N&l.

in an increase of the distance between the peaks on the temperature scale. The ionic strength also affects this temperature for both peaks in an opposite manner. decreasing the temperature in the case of t,he first peak and increasing it for the second one (Fig. 5). The broken lines in Figures 3 and 4 show the results of reheating ofthe previously heated samples. As can be seen. peak A does not appear at all on reheating, while peak B is perfectly reproducible (not less than 8&,), indicat,ing that the process corresponding to this peak is reversible.

PEPSISOGEN

20

AND

30

40

PEPSIS

STKl’CTl~RE

50

60

151

70

80

t PC)

l+1(:. 1. Specitic partial heat capacity function of pepsin in solution. pH 6.5. 100 mwNaCI. Solid line iirst heat,ing: dotted line, second heating of the same sample. Broken line shows extrapolation of heat rapacity of native. half-denatured and denatured states. Hatched area rorresrwnds to the heat effect of the second st,agr at, denaturation.

70

60

50 2 z

40

30

20

I1 5.5

6

/ 6.5

I 7

I 7.5

PH

FTC;. 5. Dependence of temperature of maximum of peaks A and H of pepsin denaturation various values of salt content. (0) Peak A in the absent of NaCl : (0) peak A in the presence Sa(‘l: (A) peak B in the absence of NaCl: (A) peak R in the presence of I00 mM-NaCl.

on pH at of 100 rnM

The slopes of the partial heat capacity function before and after denaturational heat absorption are very similar in all cases (including reheating experiments), and very close to the value of 22 x 10-j cal Km2 g-’ found for other compact globular prot,eins (Privalov & Khechinashvili, 1974). The value of the specific partial heat capacit,y at 20°C’. c,,,,(ZO”C), seems t,o be the same for the native protein in all

452

P.

L.

P$IVALOV

ET

AL

experimental conditions and is equal to (0.34 kO.02) cal K- ’ gP ‘, while after the first heating of the protein solution it becomes (0.42 + @02) cal K- ’ g5- ‘. Complete denaturation leads to an increase of the protein partial heat capacity by (0~13OfO~010) cal K-’ g-l. This d,C’!,(A, B) value seems to be independent of pH and ionic strength and therefore independent of tjransition temperature. Of this total increase of heat capacity, about’ @OS0 pal K- ’ g- ’ takes placsr at stage A, and 0.050 cal K - ’ g - * at stage B (see Fig. 4). From the area of the heat absorption peaks, it is possible to estimate the real or calorimetric> enthalpy for overall melting of t)he prot,ein structure, d,H”“‘(A, B). as well as the enthalpy for each stage, dJP’(A) and d,,$P’(B). The total area of the peak is limited from above by the heat absorption curve and from below by the lines that, are obtained by linear ext,rapolation of the specific heat capacity of native, reheated and denatured prot,ein to the middle of stages A and B. The area of the second peak B is obtained in a similar manner (Fig. 4, hatched part), and that of the first peak A by subtraction of the second peak area from t,he total. van’t Hoff enthalpies for both peaks, D,Hvh(A) and d,Hvh(B), were estimated from the peak heights oCrX(A) and oC”r(B). Calorimet,ric and ran’t Hoff enthalpies for bot,h stages of pepsin denaturation under different experimental conditions are given in Table 2. Here we do not discuss the results obtained at pH values below 6.0 because of a marked self-proteolysis of the sample. A slight effect of self-proteolysis is observed also at pH 60. and manifests itself in a lower reversibility of t,he system on cooling.

Peak

Peak A

/,( Y’)

(‘ondition

A,H’“’ (kral nd-

pH pH pH pH pH pH pH pH pH pH

5.9 5.9. 6.5 65, 7.1 7.1. 7.1, 7.5 7.5, 6.5,

I)

Kt

t,(’ (‘)

A,H’“’

A,H”h

(kcal mol-‘)

(kc-al mol-‘)

Kt

105

I ,A

844)

10 mix-N&l 100 miwNaC1

x0 80 71 70 65 1

I G-3 1.6 1.7 1.5 1.5 -

100 miwN&l 100 mwXaC1 and pepstatin

644 554 62.5 52.0 55.0 60.5 51.0 600

108 100 x3 104 74 87 97 75 92

55 5.5 45 55 46 50 47 40 46

I ,9 I9 19 20 1.6 1.7 2.0 1.9 PO

140

0.x5

62.5

109

56

2.0

100 mM-Xd‘l 100 mwSaC1

t R = AJPIJA,HVh i Values

A,.JYh (kcal IMK’)

13

uncertain.

PEPSINOGEN

AND

PEPSIN

ATRlrCTI’RE

(c) Pepsin fragment In order to assign the thermal transitions found in pepsin to certain regions of the molecule, calorimetric experiments were carried out on the C-terminal fragment of inhibited pepsin (DDE-F) and on t)he inhibited pepsin itself (DDE-P) as a direct, reference. (lalorimet,ric records of DDE-P were very similar in all cases to those of act,ivr pepsin for a given set of conditions, showing the split of the heat absorption curve at increasing pH and ionic strength into two well-defined peaks, the first of which does not appear on reheating (Fig. 6(a)). The difference in the calorimetric behaviour of these two proteins is in the higher transition temperature of the second peak in DDE-P by about, six degrees. The situation is the same under the conditions where only t)he second peak is observed from the very beginning (Fig. 6(b)). On reheating the DDE-P. however, the temperat,ure of the remaining peak becomes very close to t.hat of pepsin under t,he corresponding conditions. (‘alorimetric experiments on DDE-F under all studied conditions show only one peak, with the melting temperature region very much resembling the second DDEI’ transition (Fig. 6(a) and (lo), u pI )er curves). This peak repeats itself also on reheating but’ the temperature is less. as in t,he case of DDE-P, and becomes veq &)SP to the temperature of the second transition in pepsin. The ohscrved decrease of t.emperat.ure for t,he DDE-F peak and for t.he second

11 IO

I

I

I

I

I

I

I

I

20

30

40

50

60

70

80

90

I CT‘)

FIN:. 6. CMorimetric recordings of heat absorption on heating inhibited pepsin (DDE-P) and inhibited pepsin fragment (DDE-F) in solutions at pH 7.1 and (a) 10 mwNaCI and (b) 100 mwSaC1. Solid line. first heating: broken line. second heating.

454

P.

I,.

PRIVALOV

ET

AI,

peak of DDE-I’ on reheating is evidently connected to the release of the label at the elevat’ed temperatures during the first run of the sample. To prove this. the heated fragment solut,ion was dialyzed TLWSUXbuffer and its light. absorption was measured spectrophot,ometrically. After dialysis. the absorption at 360 nm caused by the charaoterist,ic yellow colour of the label has disappeared completely. In calorimet,ric experiments of this dialyzed fragment solution, a transit,ion is observed with thermodynamic characteristics similar to that of non-dialyzed DDE-F solution on reheating. It, follows from a comparison of the calorimetric recordings on heating of pepsin and its fragment (Figs 2 and 6) that, melting of the (J-terminal fragment corresponds t,o the second stage (B) in denaturation of the intact pepsin molecule. The calorimetric and van’t Hoff enthalpies of melting of DDE-F at the first, and second heatings under different solvent condit’ions are given in Table 3.

Pepstatin has a striking effect on the melting profile of the pepsin molecule. In its presence (see Fig. 7). peak A markedly shifts to a higher t,emperature. but peak B does not move at all. This is especially clear from the reheating experiment (Fig. 7, broken line). It does not. affect the thermal stability of the C-t.erminal pepsin fragment either (Fig. 7. broken and dotted line). It follows then that Ijepstatin interacts mainly with the N-terminal half of t,he pepsin molecule. It, is worth noting also that peak A in the presence of pepst,atin becomes much sharper than in its absence but its area, i.e. melting ent,halpy. does not, increase, notwithstanding an process on temperature scale (see essential shift of this temperat,ure-dependent Table 1). The other notable fact is that, the denatlurational heat, capac:it,y change, A,,,(‘,. of pepsin with pepstatin is about 0.3 kcal K-l mol-’ great,er t,han that of pepsin alone, being 1% instead of 45 kcal K - ’ mol -I.

First

1,(T)

(‘ondition

A,Hcal (kcal

pH pH pH pH pH

6.5. 100 mwNaCl 85 7.1 7.1, 10 mwXaC1 7.1. 100 mwNaC1

pH 65.

100 mwN&Cl and pepstatin

t R 3 A,Hca’JA,H”h

heating

A,H’”

Second

R-t

cd ‘(‘1

A,H’“’ (kcal

mol-‘)(kcal

heating

A,H’”

Rt

molt’)

mol-l)(kcal

mot-‘)

67.0 62.5 60.0 61.0 645

90 81 77 68 90

45 44 40 38 45

2.0 1.8 1.9 1.8 2.0

67-O 57.0 53-O 54-O 59.5

80 69 64 58 52

42 39 36 35 37

2.0 14 1% 1.7 I .7

67.0

95

46

2.1

64.0

85

42

2.0

PEPSINOGEN

ASI)

PEPSIN

STRlT(‘T17KE

FII:. 7. Heat capacity function of pepsin with pepstatin in solution at pH 6.5. 100 mu-Na(‘1. Solid line. first heating: broken line. second heating. Dotted line represents heat capacity of pepsin without iwpstatin: dashed-and-dotted line represents the heat capacity of the C-terminal fragment of pepsin without the label in the presence of pepstatin at the same solvent conditions.

4. Discussion (a) Thrrmodynamic

characteristi’cs

From the results presented above. it is evident that thermal denaturation, i.e. melting of t)he native struct,ure. of pepsinogen and pepsin proceeds in a veq different manner. While pepsinogen denaturation is a highly reversible sharp pro(Aess that, takes place in a narrow temperature range. pepsin denatures in two distinct stages. each occurring at two distinct’ temperatures. That’ these two stages are independent is clear from the results obtained under different pH and salt (sontent conditions (Fig. 5). Therefore. they cannot be two consecutive steps in the trmprrat,ure-induced transformation of a single system, but proceed in more or less independent subsystems, which differ in the quantity of ionizable groups, in stability and in the ability to fold back into the initial conformation. This is in agreement with crystallographic information, according to which, pepsin has a deep cleft dividing the molecule into two distinct lobes (.4ndreeva rt al., 1978). Therefore. the complex character of the observed melting curves might be a consequence of a different stability of the two lobes of pepsin. The correctness of t)his assumption is confirmed by the results obtained on melting the (‘-terminal fragment of pepsin. DDE-F, which includes residues 180 to 327. and which c*orresponds to the C-terminal lobe of pepsin. As has been shown, t’he melt,ing of this fragment proceeds in one stage, which corresponds to the second stage of pepsin denaturation. Thus, the second stage of pepsin denaturation is nothing more than the melting of the C-terminal lobe of the molecule. This is confirmed also by the results of limited proteolysis of the pepsin molecule, at which the molecule is cleaved at, the stretch connecting the two lobes. But the N-terminal lobe, being less stable than the C-terminal lobe, is rapidly digested into small fragments (see .Mat,rrials and Methods).

45ti

P.

I,.

PRIVALOV

E7’

AL

It is logical that the C-terminal part of the molecule is more stable than the i% terminal part. since it remains intact upon activation of the zymogen. when 44 residues are removed from the N-terminal end of the polypeptide chain. Neither is it surprising that after the loss of 44 residues the N-terminal part of t,he polypeptide which it has inherit,ed from (*annot’ fold back int,o its initial conformat,ion, pepsinogen. To explain this, it has to be assumed only that t’hc removed fragment plays the role of a nucleus for the folding of the polypeptide chain int,o a compact structure. According to the crystallographic model of pepsin (see Fig. 8), the X-terminal part of the molecule consists of residues 1 to 180. However, it seems that the C’terminal hairpin, which includes the last 20 residues, is also attached t,o the IC terminal part, forming with it one compact structure (X-. S. Andreeva. personal communication). This crystallographic information is confirmed by the fact that the loss of the last 13 (‘-terminal residues does not essentially influence the existence of the compact conformation of the (‘-t,erminal fragment (fragments III-2 and 111-3: see Materials and Methods). Therefore, the N-terminal lobe of the molecule includes 199 residues (1 to 179 and 307 to 327) and the (‘-terminal lobe includes 126 residues (180 to 396). Correspondingly, the actual molecular weight of these lobes is 21,450 and 13,200. Thus one (an calculate from the obtained calorimetric: data t,he specific thermodynamics characteristics of melting of these the value of’ lobes. For the specifk heat capacity change A,Pp. we obtain 0.130 Cal K-l 8-l for bot,h lobes. The specific melting enthalpies are presented in Figure 9 as functions of corresponding transition temperatures. As seen, these functions are very similar for both lobes. and the slope dA,h/dt, is close to 0.130 cal K-’ g -’ in both cases. It follows from the correspondence of this value

@‘I(:. 8. Scheme of pepsin structure according to Andreeva 8z Gustchina (1979). Reproduced with t,he author’s permission. Broken lines encircle the compact regions with hydrophobic cores. The arrow indicates the place of cleavage of the polypeptide chain at fragmentation (residues 179 t,o 180).

PEPSINOGEN

I 40

AND

PEPSIS

I 50

STRI’C’TITRE

157

I --1 60 t, PC)

PIG:. 9. Temperature dependences of specitic~ heat of melting (A) lobs of pepsin and that of the whole pepsinogen molecule to spwitic heat of melting of N-terminal lohe in the presence

ofthe S-terminal (0) and the (‘-terminal (0) without urea. Point (@) cwrrrsponds of pepstatin.

with t)hat found by direct calorimetric measurements of heat’ capacity change that the influenc~e of pH and ionic strength on the stability of both lobes of the pepsin molecule is of an entropic and not of an enthalpic nature. while the temperaturt> dependence of t)he melting enthalpy is complet’ely determined by the heat’ capacit’! change at the unfolding of the protein compact structure, as is the case with the 1979). The agreement bet,ween the estimated other globular proteins (Privalov, values caan be regarded as an addit,ional confirmation of the correctness of thr assignment of the (‘-terminal hairpin to the N-terminal lobe of the pepsin molecule. For prpsinogen, the specific melting enthalpy is very close to that found for bot,h being only a somewhat steeper function of temperature lobes of pepsin, (dd,h/dt, = 0.115 cal K-’ g-l) with lesser values at 50 to 60°C (see Fig. 9). What is remarkable. however, is that all these three functions are extrapolat’ed to the same point (13+ 1) ~a1 g-l at llO”(‘. As has been found in denaturation studies of globular proteins, t,his is a charact,eristic value for t,he melt,ing of compact globular wit,h structures maximally saturated internal hydrogen bonds (about 0.7 bond/residue). while the slope of t,he specific enthalpg function is determined by the c.onc~erit,ratioli of non-polar caont,acts : i.e. pairs of non-polar groups located at a distance of 4 A (Privalov. 1979). A comparison with thfx result’s obtained for other globular proteins shows that there are about 120 non-polar contacts per residue in each of the pepsin molecule lobes. while in pepsinogen the number of contactas is somewhat greater: 1.30 per residue. Recalculating it for the whole structure. wt’ observe that’ there are 140, 140 and 450 non-polar conta&s in t,he (‘-terminal and Sterminal lobes of pepsin, and in pepsinogen, respectively. Therefore. pepsinogen has aboutj 70 more non-polar contacts than pepsin. Assuming that these extra contacts are created by the N-terminal fragment, which is removed a,t activation. one can c*oncalude that this fragment) is att)acahrd to the main body of the molecule mostly 1)) hydrophobic bonds. As has t)cscn shown (see Results). pepstatin, a specific substrate-like inhibit,or of pepsin, interacts mainly with the S-terminal lobe of the molecule. great]? stabilizing it. Hut it is remarkable t)hat the enthalpy of melt,ing of t,his N-terminal

&ix

P.

L.

PRIVALOV

ET

AL.

lobe with the at,tached pepstatin is much lower than the value t)hat it should have at the corresponding temperature without pepst,atin (see Fig. 9). The observed enthalpy deficit. which amounts t,o 60 kcal molt ’ _ can be explained only by the negative enthalpy of the specific interaction of pepstatin with the N-terminal lobe of pepsin. It follows that pepstatin is also attached to pepsin mainly 1~) hydrophobic bonds, as is the removable S-terminal fragment in pepsinogen. This is confirmed also by the observed increase of the A,(“, value in t,hr presence of pepstatin. &Assuming that the observed change of A,~‘,. 0.30 kcal K-’ mol--‘, is cbonnected with the N-terminal lobe. we will find that A,(‘, for this lobe is 3.10 kcal K - 1 mol - ’ inst,ead of 2+X) kcal K - ’ mol - ’ m the absence of pepstatin: i.e. the specific partial heat capacity change for this lobe in the presence of pepstatin is 0.143 cal K ’ g- ’ t:sing this value for rxt,rapolatjion of the specificmelting enthalpy of the Y-terminal lobe in the presence of pepstatin to the characteristic: point 1 lO”(‘, we will find the same value of 13 cal p-l that has been obtained wit’hout prpstatin. Therefore, the total number of’ hydrogen bonds seems t’o be conserved in this s@em.

(b) Huhmolrcrslur

.vtrtrctrrrex

The remarkable fact) established calorimetrically is that the calorimetric enthalpy for all transitions observed in pepsinogen, pepsin and its fragment without pepstatin essentially deviates from the van’t Hoff ent,halpy (see Tables 1. 2 and 3). Under some conditions. the ratio AHca’/AHvh reaches the value of 24). From this experimental fact it, immediately follows that, neither qf the ohserced melting processrs Indeed, t’he van-t Hoff enthalpy, which is rq7rexrnt.s n two-et& trnnsitiorr calculated on the assumpt,ion that the considered process is a reversible t,wo-st,ate transition. should be equal to the real enthalpy measured calorimetrically if t’he assumption made is correct. The great deviation of AHvh form AHC”’ usually means that the system under st’udy consists of several subsystems. For a system consisting of two identical and independent subunits, the ratio AHc”‘/AHvh should be equal t’o 20. but it will decrease with increase of interaction between the units. Therefore. t,hr result’s obtained on the pepsin fragment (DDE-F) and each lobe of the pepsin molecule can be int,erpreted as an indication that, each of the lobes consists of two. more or less independent. subst’ructures that behave as individual co-operative units in a melting process. This conclusion seems t,o be valid also for the irreversible melting of the N-terminal lobe of pepsin. not only for t’he (‘-terminal one, since irreversibility itself could only decrease the apparent ratio AH’*‘/AHvh. sharpening t,he curve of the temperature-induced process. Assuming t’hat) the pepsinogen structure cannot be qualitatively different from that of pepsin, one can expect that’ it also should consist of at least four structural subunits, as pepsin does. But the problem is that pepsinogen exhibits only one sharp peak at denaturation, for which t,he ratio AHca’/AHVh does not exceed 20. Therefore. if there are four structural subunits in the pepsinogm molecule, they should interact strongly with each other. To solve this problem of co-operative regions in pepsinogen and pepsin we would need a much more detailed analysis of the calorimet’rically obtained melting curves.

PEPSIXOGEN

ANI)

PEPSIS

STRY(‘TITKE

As has Iwen shown by Freire & Biltonen (1978). the excess heat capacity function irwlu~lrs all the information required to deconvolute the complex temperat’urcsinducwi prowss into constituent’ two-st’ate t,ransitions. and this can be done wit holIt any /I priori assumption even if t,hese transitions are non-independent. I‘sing for cwml)uter analysis of the excess heat capacity function the sequential prowdl~re suggested by these aut’hors. with an optimizajtion at each step t)o impro\.r t I)(%awuracy (Matveev rt ~1.. lSSO), we decwnroluted the t’emperature-indwed 1)rowss in prpsinogen and pepsin into a set of two-state t,ransitions (see Fig. 10, and ‘I’altks 1 and 5). It has been found that : (1 ) in all studied cases excess heat capacit? function can kw approximat~ed with high accuracy (deviation less than lC?O) by the similar stat of two-stat’e t)ransition functions, even when the melting curve is not

I,-

(a)

III-

Fit:. 10. (‘omputrr drconvolution of the excess heat, capacity function of pepsinogen (CL)and pepsin (1,) at the same solvent condition: pH M, 100 mwXaC’1. Crosses indicate calculated excess heat capwit> function. which ahnost completely coincides with t,he experimental one.

Xl !)i 4!bO 52.0

71 93

PEPSINOGEX

AND TABLE

PEPSIN

llil

STRUC’TL’RE

5 (continued)

Lequence c;

4(“(‘)

A,H (kral mol-‘)

1

pH 7.1. 100 mwNd’1

2

pH 7.3

pH 7.5. 100 mwNa(“l

reproduced csompletely on reheating : therefore. the observed irreproducibility of peak X in pepsin might be the result of the slow kinetics of the first stage on folding which is the last stage of its unfolding. (2) of t*he corresponding structure, Pepsinogen denaturation under any condition studied (even in the presence of 3 Murea) is represented by two quasi-independent transitions with different enthalpies. (3) Pepsin denaturation is represented by four two-state transitions grouped into t,wo independent, pairs of quasi-independent’ transit,ions : therefore, it is likely that two pairs of substructures, which are more or less independent’ in pepsin. are merged into two larger co-operative blocks in pepsinopen. Bearing in mind that the t’otal enthalpies of the first pair of transitions in pepsin relating to the N-t,erminal lobe are somewhat larger than t’hose of the second pair, it is tempting to assume that in pepsinogen the structure that, melts with larger enthalpy is formed by t’he S-terminal part of the polypeptide chain. As seen from Figure 11, enthalpy of all the four transitions in pepsin is a function increasing with temperature. Since each of the t)ransitions corresponds t,o the melting of some of the submolecular structure, one can assume that this process leads to an essential increase of the partial heat capacity of t’his structure. and therefore t’o the exposure of internal non-polar groups to water. It follows then that ench of these four suhmolrc&w strtrctwrs itl pepsir~ must harv a wll-dwe2oprd h;ydrophobic cow and thus it is n strrrcture of a globular type, which folds independrntly of’ thr

rvst

of thr

molecule:

i.c.

it is nothing

~1s~ than

a structural

domain.

This

I

1

I

,

J

I

‘W 1. (“C I l+k. 11. The plot of the tmnsit.ion enthalpy against wxwsponding transition temprrature for pepsin (a) and pspsinogan (b). (0) and (0) Transitions in t,hr S-terminal lobe of pepsin: (A) and (A) transitions in the (‘-terminal lobe of pepsin: (m) and (0) transitions in pepsinogen. 30

50

70

conclusion is valid not only for neutral pH values. where the exist,ence of four cooperat,ive units in pepsin has been shown exlterimrnt~ally, hut for acidic pH regions as well. where this enzyme is in an active st,ate and its X-ray structure has been studied. Indeed. on decrease of pH. the st,ability of the thermodynamically revealed co-operative units increases (see Fig. 5). Therefore, these structural units will always appear in a denaturation experiment as more stable elements of protein structure, even if a decrease of pH leads also to an increase of interaction between t,hem. Knowing that pepsin has four domains grouped in pairs in two blocks corresponding to the two lobes of this molecule, it is tempting to identify them on the model. This is not easy to do. since they are not as distinct as the two lobes that were usually considered as structural domains of acid proteases. But a structural domain, by definition, cannot consist of several co-operative units, as it represents the region of protein structure where t.he polypeptide chain is folded compactly and more or less independently of t,he rest of the molecule. A recent analysis of the configuration of the polypeptide chain in acid proteases revealed that, the first’ and second half of the chain have a similar pattern in each al., 1979). Therefore, each lobe of lobe (Andreeva & Gustchina, 1979: Blundell this molecule appears as a paired structure, an “intramolecular dimer”, built from t,wo structurally equivalent parts. This finding is very intriguing, since it evokes an idea ofgene duplication as a possible mechanism for the evolution of acid proteases (Blundell al., 1979; Tang, 1979). Nevertheless, when considering models of acid proteases, it. is rather unclear whether these topologically equivalent parts of the lobes can fold and unfold more or less independently, as we have observed experimentally. At, the same time, the polypeptide chain in these molecules is far from being uniformly packed throughout the interior of the lobes. After a careful examination of the pepsin model in collaboration with Dr K. S. Andreeva, we defined four rather separate hydrophobic regions in the molecule, which seem to

et

et

have more internal than external contacts. These regions are indicated by broken of the problem of localization ot lines in Figure 8. But the final solution tjhermodynamic*ally detected domains in pepsin will be possible only when these domains are isolated and tested individu&lly. According to the results presented in this paper. this is quit,e a realistic experimental possibilit,y. It is evident that pepsinogen should have the same domains as pepsin, but here the pairs of domains in the lobes are much more tightly connected with each ot)her. forming one cao-operative block. This might be the result of the removable Sterminal fragment filling the gap between the pairs of domains. In this context. it is very int’erest’ing to note t)hat the presencae of pepstatin also leads to the co-operation of t,wo domains in the N-terminal lobe of pepsin (see Table 5). Therefore, with the stoichiometrically attached pepstatin molecsule. this lobe is much more stable and presents a single co-operat,ive block. as is found in pepsinogen. But in contrast) t,o the removable N-terminal fragment in pepsinogen. pepstatin. which is a mucah shorter peptide. does not affect the domains in the (‘-terminal lobe of pepsin.

(c) Stability of structure ard its motility The stability of a protein is usually defined by the Gibbs energy required for the disruption of its co-operat,ive st,ructure (Privalov, 1979). If a macromolec~ule c*onsists of several co-operative subunits, t)hen in order to define its stability we obviously have to evaluate the energy required to disrupt each unit. Assuming t,hat int,eraction between the units is negligible, one can est,imate the Gibbs energy for a unit from c*alorimetric data using t)he equat,ion (Privalov K: Khechinashvili, 1974):

AC(T)

q-7’ = A,H 7’ t

-A,P,(T,-T)+3,P,Tlng.

For t,he proteins consider t, and A,H are given in Tables 4 and 5. and A,(‘, can he evaluated from the slopes of the corresponding enthalpy functions in Figure 1 I. Xt a st,andard t,emperature of 25°C’ in solution with ~JH 6.5 and 100 mnr-?l;a(‘l, we obt,ain the values 6.2 and 9.5 kcal mol-’ for G’(25Y’) for the two co-operat’ive blocks in pepsinogen. For pepsin we get 19 and 3.0 kcal mol-’ for the two co-operative units in t’he N-terminal lobe, and 3.9 and 2-O kcal mol- ’ for the two units in the (‘terminal lobe. Thus the total Gibbs energy amounts to 15.7 kcal mol-’ for pepsinogen and 10% kcal mol- I for pepsin. But clearly the stability of the native state of a macromolecule consist,ing of several independent subunits is not defined k)y t,he tot,al Gibbs energy value needed for complete disruption of its structure, but by t,he stability of the least stable subunit of this system. Therefore. t,he stability of pepsin is determined by the least st’able domain. with dU(25”V) = 19 kcal mol- ‘. which is in its N-terminal lobe. while t’hat, of pepsinogen by the less stable block u&h A1;(25”(‘) = 6.2 kcal mol- ’ : i.e. the stability of t,hese two molecules differs b> more t,han threefold. Another aspect of this problem is the following: pepsin is a less stable protein than pepsinogen, and it has a much looser domain struclture and is therefore a more motile molecule. This fact naturally gives rise t,o the quest)ion of whether the

lti4

P. I,. PRIPALOV

interdomain

motility

of pepsin

is important)

regard to this, the results obtained presence of pepstatin, the stability 6.4 kcal mol - ‘. approaching integrat’ion of two domains substrate-like its t,urn.

inhibitor

it is subject’ed

ET

AL.

to its function

on pepstatin action of the S-terminal

as an enzyme.

that, of pepsinogen. This stabilization of this lobe into a single co-operative

decreases

interdomain

motility

With

are very intriguing. In the lobe of pepsin increases to

in pepsin

is achieved by s.ystem: i.e. the and

therefore,

in

to stretching.

The authors express their deep gratitude to Dr N. S. Andreeva for consultation on the d&ails of pepsin structure and to S. V. Matverv and S. A. Potekhin for the computer program used in thermodynamic analyses.

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Konformatsionniye

Izmenenija

Biopolymerov v Rastvorakh. Proc. 5th Conf..

Telavi,

1980,

11. 74, Izdatel’stvo “Metsniereha”. Tbilisi. V. M. (197.5). Prikladruzya Matyash, I,. F., Vojushina, T. L.. Belyaev, S. V. & Stepanov. Biokhim. Mikrohiol. I’.S.S.K. 11, 604-607. Moravek, L. 8: Kostka, V. (1974). FEBS Lrttrrs, 43. 207~ 211. Pedersen, V. B. & Folt’man, B. (1975). Eur. J. Biochem. 55, 9.5103. Perlmann, G. E. (1963). J. Xol. Biol. 6, 452-464. Privalov, I’. L. (1979). Advan. Protsivc Chem. 33, 167 -241. Privalov. P. I,. (1980). J. PWP Appl. (‘hrm. 52, 479-497. Privalov. I’. I,. K: Filimonov, V. V. (1978). J. Mol. Biol. 122, 447-464. Privalov. P. L. dz Khechinashvili, N. S. (1974). J. ,Vo/. Riol. 86, 665-684. Rao, S. N., Koszelak, S. N. 8: Hartsuck. S. A. (1977), J. Riol. Chem. 252, 8728~8730. Kevina. L. P.. Vakhitova. E. A., Baratova, I,. A.. Belyanova, L. P. & Stepanov. \‘. M. (1975). Rioorgan. Khim., V.S.S.R. 1, 958-964. Stepanov, V. M., Lohareva, L. S. & Mal’tsev. N. I. (1968). Biochim. Biophys. Acta. 151, 719721. Tang, J. (1979). Mol. Cell. Biochem. 26, 93-I 10. Tang, ,J., Sepulveda, P., Marciniszyn, J., Chen, K. (‘. S.. Huang, W.-Y., Tao, N., Liu, I). h Lanier, .J. P. (1973). Proc. Nat. dcad. Sci.. r:.S’.rl_ 70, 3437-3439. Williams. R. C”. 8 Rajagopalan, T. (4. (1966). J. Biol. Chrm. 241, 4951-4954.