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Secondary structure in ribonuclease
J. Mol. Biol.
(1982)
157. 357-371
Secondary II.
Structure
in Ribonuclease
Relations between Folding Kinetics and Secondary Structure Elements ALEXANDER M. LABHARDT Chemie A bteilung Biophysikalische der Universitd, Klingelbergstrasw CH 4056 Basel. Switzerland
Biozentrum
(Received
10 August
1981, and in, revised form
70
4 January
1982)
The kinetics of regain of 2’.CMP binding are monitored during renaturation of RNAase S. Experiments were performed by mixing equimolar amounts of Speptide with S-protein. The S-protein fragment was incubated initially (i.e. before mixing with S-peptide) at pH 6.2 or 1.7 and various guanidine hydrochloride (GuHCl) concentrations. Three well-resolved phases are observed. The fastest phase is second-order. The reciprocal half-time increases linearly with fragment concentration and is independent of initial conditions for the S-protein fragment. An apparent on rate of k,,, = 2 x lo5 M- ’ s- ’ is measured in 0.5 M-GuHCl (pH 6.2) and 20°C. Identical association kinetics are observed by changes in tyrosine absorbance. The fraction of native RNAase S formed in this second-order reaction strictly equals the fraction of S-protein molecules with intact b-sheet in initial conditions. The relation holds for different pH values, GuHCl concentrations and temperatures. The fraction of apparent helical content of S-protein in initial conditions may also vary but this is not reflected by the association reaction. We interpret this to mean that the B-sheet but not the a-helices must be preformed in initial conditions in order to generate the high-affinity peptide binding site of Sprotein. Furthermore, it is concluded that the S-protein moiety p-sheet forms or unfolds in a single one-step reaction. 2’.CMP binding reports, additionally, two slower phases of renaturation. These are produced by S-protein molecules that have their p-sheet unfolded in initial conditions. It is observed that a unique dependence of these two folding rates exists for RNAase A, RNAase S and S-protein as function of t,, the temperature of half-completion of thermal denaturation as measured by unfolding of the B-sheet in the respective compound in jinal conditions. The t, value varies with changing pH, with GuHCl concentration and (for R?iAase 6) with changing fragment concentration. The findings are interpreted to argue in favor of a sequential mechanism of folding, where the stability of a structural precursor determines the rate of folding.
1. Introduction Three independent spectroscopic elements have been detected in unfolding/refolding experiments with ribonuclease (RNAase) by amide circular dichroism (Labhardt. 1982). An approach was used that involves the decomposition of the c.d.t diff ewncc spectrum of unfolding or folding into loss or used : cd.. circular dichroism : GuHCl, guanidine hydrochloride. Xi 2836/82/l 40357-16 $03.00/O 10 1982 Academic Press Inc. (London)
t Abbreviations
Ltd.
35A
A. M. LABHARUT
gain of fractions of helical and pleated-sheet type conformations. When using this method it turns out that the cd. spectrum of native RNAase S differs from that of thermally denatured RNAase S or S-protein by a fraction of some 36O/,, of fl-pleat,ed sheet and some 9% of a-helix. From the crystal structure it is known that some 40% of the residues of the S-protein fragment of RNAase S part’icipatr in t)htl antiparallel p-pleated sheet (Wyckoff et al., 1970), which agrees favorably with the c.d. estimate for unfolding. The change of 9% n-helical content during thermal denaturation has been attributed to the unfolding of the S-peptide (residues 3 to 12 are helical in the folded state; Levitt & Greer, 1977). The assignment rests on thr fact that no change in r-helical content can be detected during thermal denaturation when the S-peptide is omitted from the solution. It had been noted that thermal denaturation of S-protein in the absence of S-peptide produces a c.d. difference spectrum with an isodichroic point at 225 nm (Labhardt. 1981/r). This indicated that the apparent helical c.d. (which is characterized by two strong bands near 225 nm and 109 nm; Brahms 8: Brahms, 1980) of S-protein itself does not change significantly during thermal denaturation, although this fragment accountjs for more than 60% of the helix content of folded RNAase S (Wyckoff et al., 1970). A c.d. difference spectrum consistent with the loss of about this amount of apparent helical type struct,ure is observed for t)rypticb digestion of thermall!, denatured S-protein or by exposing it to 5 M-GuHCl (Labhardt, 1982). Abbreviating the S-peptide helix by I,, the S-protein helices by x2 and the Sprotein F-sheet by j3 allows one to summarize these results by the following multistate scheme : K12 110128
’
“1 P
z
KS
1 1219
’
’
(1)
‘B
KS
1 G
L
,I I
If any of the three elements unfolds, its symbol is omitted in the above scheme. Below pH 2 S-peptide dissociates and unfolds (Richards & Logue, 1962). This destroys xl but leaves ‘x2 unaffected. Thermal denaturation destroys a1 and /3 but does not change the c.d. intensity of a2. It is not’ known whether the same structure generates the helical cd. of 3~~after thermal denaturation of the p-sheet as does in the native state. Therefore a2 is characterized by an asterisk in these conditions. 1; converts to the all random state (denoted by T) in 5 M-CuHCl. In the accompanying paper (Labhardt, 1982) it has been shown that all states except n1/3 in equation (1) can be present at equilibrium using appropriate conditions of temperature and GuHCl concentration. In order to assess the function of the three elements in relation to folding we have investigated the renaturation kinetics and their variation when starting from the different states shown in scheme (1). We report here the kinetics observed when mixing S-peptide with S-protein. Folding is monitored by the restoration of binding ability for the specific inhibitor 2’.CMP.
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The individual refolding kinetics of the p-sheet and of the S-peptide n-helix measured by fast c.d. stopped-flow will be published elsewhere. The different states in equation (1) display different folding kinetics. Three wellseparated phases are observed in the final conditions used here (0.5 M-GuHC’I. pH 62). The fastest phase is second-order and corresponds to the association between S-peptide and a moderately folded form of S-protein. The main observation is that the fraction of active RNAase S produced in this second-order reactlion exactly equals the fraction of S-protein molecules in the states /3 or azp of equation (1) in initial conditions. i.e. the fraction of S-protein with intact p-sheet. It, is shown that this finding implies that the p-sheet unfolds or refolds as a single cooperative unit. The two slower phases correspond to the folding reactions of unfolded S-peptidrS-prot)ein complexes. These t,wo phases are observed with a constant amplitude ratio. The log of both time constants are linear functions of the melting temperat,ure (t,) in widely varying final conditions of pH. GuHCl concentration and protein concentration. A unique dependence is observed for RNAase A. RSAase S and S-protein. Prom this observation we draw the conclusion that folding is a sequential process controlled by a, structural “on the pathway” intermediate. Circumstantial evidence suggests that the /l-sheet or a p-sheet precursor is (or is part of) the structure of this intermediate. The interpretation is consistent, with the findings of a recent c.d. stopped-flow investigation where the folding kinetics of the p-sheet and the ix-helix were monitored independently: t,he folding of the p-sheet is observed to precede the folding of the rest of the molecule (unpublished results).
2. Materials and Methods (a) Matmizls RNAase A. RNAase S, S-protein, S-peptide, pepsin, trypsin and .Z’-CMY were purchased from the Sigma Biochemical Co. I’ltra-pure guanidinr hydrochloride and urea were from SchaarziMann.
(I,) Methods Routine enzymatic activity assays of RNAase S were done according to Crook et al. (1960). Kinetic measurements were carried out with a modified Gibbs Durrum stopped-flow instrument with split beam optics. In GuHCl dilution experiments variable dilution ratios were achieved using various Hamilton syringes. In order to prevent Schlieren artifacts or uncontrolled premixing the densities of both solutions used in such dilution experiments were matched by sucrose. It is known, that sucrose (up to SOsl/o,w/w) does not affect the folding kinetics of RNAase A (Tsong & Baldwin, 1978). The data presented in Figs 2 and 4 show that the same holds true for RNAase S. The kinetic progress curves were stored on a Datalab transient recorder DLSOB interfaced to a PDF’ 11/40. Multiphasic progress curves were analyzed using an interactive graphic program (DIALOG).
3. Results (a) Second-order fmgwtmt
nssocintim
kin&s
It has been shown that S-peptide dissociates completely
from S-protein below
:Ml
pH 2 (Richards denoted by 1,:
A. M. LABHARDT
& Logue,
1962). A fraction,
,fi. of acidified
f,=ITji.
S-protein
molecules
is
(2)
CO
where co is the total concentration of S-protein. because it has the abilit!. to associate with S-peptide in a strictly second-order reaction with an on-rate k,,,, =7xlOS M-l s-l (at 20°C’. 0 X-GuHCl, pH 64) when the pH is rapidly raised in a stopped-flow spectrophotometer (Labhardt, & Baldwin, 1979a,(‘,). The kinetics have been interpreted by the scheme: 1,sp
“: PI, 682= ps,
GO
where I, symbolizes the fraction of S-protein capable of binding H-peptide (the index 3 is used here in order to be consistent with Labhardt, & Baldwin. 1979n). p stands for the S-peptide in any conformation and ~1, is the initial association complex at neutral pH. Its formation is detected by changes in tyrosine absorbance at 289 nm. pN is native RNAase S capable of binding the competitive inhibitor 2’. +pN is silent when measured by tyrosine absorbarrctl. CMP. The t’ransition p13 but at 265 nm and pH 6.2 the binding of 2’.C’MP to pN (which is a rapid reaction) can be monitored. The high association rate (Ic,,) of p and I, and the short conversion time of p1, to pN (68 ms at 2O”C’, Labhardt & Baldwin, 1979~~) suggest, that I3 may be partly structured in initial conditions. Various partly folded equilibrium intermediates have been detected and characterized (Labhardt. 1982). These states are summarized in equation (1). Here we measure t’he association reaction (3) as a function of temperat,ure. pH and GuHC’l concentration in order to establish whether or not the fraction fi of I, molecules correlat’es with the frac+on of any of the partly folded equilibrium intermediates (eqn (1)) as function of the same three variables. This will allow us to determine the S-protein structure that is necessary to mediate the rapid fragment association. Figure 1 shows three representative examples for kinetic progress curves. l’h(x fraction native. defined as:
where c,, is again the total concentration of RNAase S’. is measured by the extent of 2’.CMP binding. f”(t) is plotted against the logarithm of time after initiating refolding. Association and folding was started by mixing S-peptide with S-protein in a 1 : 1 molar ratio and adjusting the pH to neutral. if necessary. In tra,ce I Sprotein was equilibrated before mixing with S-peptide at pH 6.2. in trace 2 at pH 1.7 and in trace 3 again at neutral pH. however, in 1 M-GuHCl. Final conditions (i.e. after mixing S-peptide wit,h S-protein) are identical (see the legend to Fig. 1 for details). Except for trace 1 the kinetics are triphasic. The slower phase parameters were determined independently and are discussed in section (1~). below. The extrapolation of these slow changes into t,he “fast” time range (from 5 ms to ;i s: see Fig. 1) serves as a “baseline” to determinef,, (‘) . the fraction of native RNAase S
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50 60 70 80 .
90 100
I
0.005
I
0.5
0.050
5
I
50
t(s) PI<:. 1. The native fraction!,(t) plotted versus the logarithm of t.!.(t) is measured by the extent of 2’CMP binding as monitored at 265 nm. t is the time in seconds after mixing S-peptide with S-protein in a 1 : 1 molar ratio in a stopped-flow spectrophotometer. Refolding conditions are in all cases (traces 1. 2 and 3) 50 PM-RNAase 8, 130 ~~-2’-cbfP, 05 M-GuHCl. VC. pH 6.2. Before mixing, S-protein was equilibrated in X M-GuHCl at pH = Y. The buffers used and the values of X and Y corresponding to the labels 1. 2 and 3 are listed below. S-peptide before mixing was always at neutral pH, @I x-sodium cacodylate buffer containing 260 p~-2’-cMP and (in order to produce identical final conditions) 1 - X MGuHCI and varying amounts of sucrose as described in Materials and Methods. Isolated S-peptide is not folded and the Initial GuHCl concentration in the S-peptide solution has no effect. The progress curves were fitted by the sum of 2 slower exponential terms (f~” x ni exp (-l/7*): i = 1.2) and one faster second-order hyperbolic term (f$/[l +t/~~!~]). The native fraction produced in a fast second-order reaction fi” and the hyperbolic half-times. ~li2, are plotted for various temperatures in Fig. 2. The parameters for the slow exponentials were determined independently (see Figs 3 and 4). These slowphase parameters were used as a baseline for the above traces (- - - ). The labels in Figs 1. 2 and 3 indicate that S-protein was equilibrated in X k~-GuHCl at pH = Y: Curve
X (M-GuHCI)
Y (pH)
1 2
0 0 1 05 1
6.2 1.7 62 1.7 1.7
3
4 5
Huffer 01 0.1 0.1 0.1 0.1
-
M-sodium cacodylate M-NaCIO,
x-sodium
cacodylate
M-SaClO,
M-NaClO,
produced in the fast rea&ion. As can be seen. this fraction varies strongly with changing initial conditions for the S-protein fragment. Figure 2 summarizes the results for the different starting conditions : (a) refers t’o 2’7”MP binding assays and (b) shows that identical behavior is seen when monitoring the changes in tyrosine absorbance. The upper panels in Figure 2 give a semilogarithmic plot of the association half-times 7 1,Z against temperat’ure. They are independent of both initial conditions and method of detection (2’.CMP binding or changes in tyrosine absorbance). From the slope found with temperature one calculates an activation energy AH* = 26 kJ/mol. The inset gives the concentration dependence at 20°C. If in final conditions and on the time-scale of association the S-protein subclass I, is in either f&t or A~OIPequilibrium with thr
A. M. LARHARDT
-.
*-.... so .+A.., l *‘. t I 80
\ 2’CMP \
-
I
5
IO
15
20
25
30
35
40
’
Temperature (a)
5
IO
15
20
25
30
35
40
(“C) (b)
FIG. 2. (a) Lower panel: The native fractionf,!‘) produced in a second-order reaction. Experimental procedure and conditions are as described in the legend to Fig. 1 except for the temperature, which is variable. Filled symbols refer to neutral initial pH of the sulution containing the S-protein fragment. open symbols to acidic pH. Circles (0) refer to 0 M, triangles (A) to 0.5 M and squares (0) to 1 M-initial GuHCl concentration in the syringe containing the S-protein fragment. (. .) Drawn through the fj2) data points, represent the normalized fraction of B-sheet fa/0.36 of S-protein at neutral pH taken from )fs/0.36 withfB taken Fig. 5 of Labhardt (1982). Native RNAaae S contains around 36% p-sheet. (--from the left panel of Fig. 6 of Labhardt (1982). llpper panel: Semilogarithmic plot of the half-times. T,,~. in ms ZIWSUS temperature (scale below) and versus l/T (T = absolute temperature, scale above). The fact that all initial conditions produce the same half-time with the same activation energy (26 kJ/mol) shows that the same reaction has been monitored in each case. Varying the amounts of sucrose present during refolding (see Materials and Methods) had no effect on the kinetics. (b) an identical pattern is observed when measuring the changes in tyrosine absorbance at 289 nm instead of 2’.CMP binding at 265 nm. Experimental procedure and conditions are identical to those for (a). except for the absence of 2’-CMP. Lower panel : Plot of 2.2 ‘a@’ versus temperature. n (‘) is the fractional amplitude of the fast association reaction measured at 289 nm. In tyrosine absorbance 3 slower reactions are observed and the relative amplitude a”) is less than the native fraction@*’ as measured by 2’-CMP binding. The factor 2.2 was chosen to match the f,!“’ values for corresponding conditions. Labels and symbols have the same meaning as in (a). Upper panel: association half-times measured at 289 nm. The inset gives 7,,* W-W~Y final RNAase S concentration (c) in a double logarithmic plot. The slope of - 1 establishes that the reaction is second-order. ( - - - ) The native fraction of RNAase S in final conditions as measured b> equilibrium melting experiments in the presence (a) and in the absence (b) of 130 PM-2’-CMP.
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other S-protein states the association half-time 7112
=
c,2
x
STRUCTlTRE.
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363
in equation (3) is:
In (2 -fi”) 1 -fj”
(5)
with
fP +2 = fi x Lkonx co]- 1, The second t,erm in equation (5) describes the transition from a true second-order reaction (fn(‘) = 1) to pseudo first-order (f,‘) e 1) and varies from 1 to 0.7. Note that’ S-peptide and S-protein are in a 1 : 1 molar ratio. In the inset In ‘T~,~is plotted against In co. The solid line through the data points has a slope of - 1. which demonstrates that the fast phase is, within experimental error, a second-order reaction of the type of equation (3) also in the mildly denaturing final conditions used here (pH 6.2,0.5 M-GuHCl). The apparent on-rate k,, calculated from the data according to equation (6) (ji and fj2) were set equal) is 2 x 10’ M-I se1 in 05 MGuHCl at 20°C. The slight variation in 71,2 as predicted by equation (5) could not be detected. In general, the fast-phase component of the progress curves was fitted by a hyperbolic term as described in the legend to Figure 1. For j’:” < @l an exponential decay was assumed. The observation that the half-times are the same, independent of the degree of initial unfolding, meets theoretical expectations : kinetic time constants depend only on final conditions. unless the change in initial conditions alters the pathway (Eigen & De Maeyer, 1963). Hence the amplitudesfj2’ in Figure 2 refer to the same kinetic pathway, and their variation with GuHCl concentration, pH and temperature reflects the variation of the fraction of S-protein participating in equation (3). It should be emphasized that the kinetic amplitudes in Figure 2 are not affected by incomplete folding of RNAase S in final conditions with increasing temperature. The broken lines in the lower panels give the native fractionf,( 00) in 0.5 M-GuHCl (pH 6.2) in the presence of 130 p~-2’-cMP (Fig. 2(a)) and its absence (Fig. 2(b)): the stability of RNAase is larger in the presence of 2’-CMY (Barnard. 1964). which increases t, by some 5 deg.C; as can be seen all kinetic data have been collected in a temperature range with fn (co) > 0.9. The most important observation is that j’,j” as a function of initial GuHCI concentration. pH rind temperature strictly correlates with the normalized fraction of p-sheet of S-protein for those conditions. This is demonstrated by the solid and dotted lines representing the fractions fD/0.36 taken from Labhardt (1982). The fraction of apparent a-helical content fa of S-protein also varies in the conditions summarized in Figure 2; however, in a way uncorrelated to the f,j2) fraction (compare Figs 5 and 6 of Labhardt, 1982). (b) “First-order”?
folding
reactions
The remaining fraction : p
= 1 -fA”
(7)
t The 2 reactions termed “first-order” folding phases display a concentration dependence in the case of RSAase S, but not for S-protein or RNAase A. A unifying ikerpretation of the two phases is given in the text for all three molecular species. Hence we refer to them as first-order throughout.
A. M. LABHARI)T
a2
60
t
._
oo-
c
Temperature
PC)
FIG. 3. Renaturation experiments in the presence of 2’.CMP and 05 M-GuH(‘1 were carried out and analyzed as described in the legend to Fig. 1. Progress curves were monitored in the temperature range from 5 to WC” for 023 h after mixing of the 2 fragments, Between 5 and 900 s the progress curves arc strictly biphasic. The Figure shows that the relative amplitude of the slower phase asloWvaries only slightly with initial conditions and temperature. For the definition of labels see the legend to Fig. 1. The time constants are plotted in Fig. 4 for comparison with the folding kinetics of S-protein and RNAase A.
of S-protein molecules refolds with first-order kinetics. Progress curves (f”(t) ZWSU~St) were collected during 900 seconds in the temperature range from 5’Y and to 35°C for the same final conditions (05 M-GuHCl. pH 6.2, 130 FM-~‘-CMI-‘) various initial conditions. The experiments were again carried out as described in the legend to Figure 1. Two well-separated phases were detected in this time-range (a slow phase. labeled 1 and a fast phase, labeled 2) and they were analyzed as a biexponential change according to :
(8) The pre-exponential factors a, = qlOW and a1 = 1 -aSlOW are the relative slow and for various fastphase amplitudes. (lSIOWis plotted in Figure 3 ~c1rsus temperature initial conditions. As seen, the relative amplitude aslow remains constant for different temperatures and for initial conditions involving low pH. and almost constant for initial condition 3 ((a,,,,) = 73+5”i,). I n order to appreciate this one should note t’hat the kinetic amplitudes themselves (j:” x ai) change dramatically as a function of initial conditions and temperature (by more than a factor of 8: see Fig. 2 and eqn (7)). This indicates that in the present final conditions refolding in the two firstorder react’ions ((a i. 7i) and (az. TV)) proceeds largely independently and unaffected by the presence of partly folded material (I3). The folding kinetics starting from the present. only moderately denaturing, conditions are consistent with earlier investigations (Labhardt & Haldwin. 19796). In summary, the known facts are : (1) both unfolded RNAase 8 and unfolded S-
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protein exist in a way analogous to RNAase A as a mixture of two forms. a moderately fast (IT,) and a slow-folding form (U,). The two classes are found in a 25 : 75 ratio. independent of temperature higher than 20°C (Labhard& 1980). (2) In refolding the two forms T-1,and U, give rise to two well-separated kinetic phases. The slow phase is- complex when monitored by absorbance of tyrosine OI fluorescence. but not in 2’-CMP binding assays. As a molecular basis for the existence of the slow-folding form I’, of RNAase A prolyl peptide bond isomerization has been proposed (Brandts et nl.. 1975). (1) In unfolding (RNAase -+ I’,) the species CT, is formed from I:, in a SIOU equilibrium. The kinetic characteristics of this process are typical for free X-Pro peptide bond isomerization in model compounds. One observes (i) the high activation energy of 85 kJ/ mol and (ii) acid catalysis by strong acids (Schmid & Baldwin, 1978). (2) In refolding the involvement of proline isomerization is less easily assessed (Stellwagen. 1979: Kato et ~1.. 1981; Ridge et al.. 1981 : Julien 8r Baldwin, 1981). Proteins containing no prolines may lack the slow phase (Brandts et rrl., 1977) but may also have slightly modified slow-phase characteristics (Lin &’ Brandts. 1978). In the refolding of RNAase A the kinetic parameters of free prolyl peptide bond isomerization are no more reflected by the slow phase : solution conditions strongly favoring the native state considerably speed up the slow phase over free proline isomerization. The activation energy drops from 85 to 10 kJ/mol (Nail et ul., 1978). The slow phase in RNAase S at pH 6.8 has at 13 pM an activation energy of 75 k,J/mol. By increasing the concentration of both fragments tenfold this phase is speeded up almost threefold (Labhardt & Baldwin. 1979h). measured in the present final The t’ime constants 71 and 72 for refolding conditions (0.5 M-GuHCl. pH 6.2, [RNAase S] = 50 PM. t, = 37°C) are plotted in Figure 4 against t, together with data for RNAase A and S-protein. t,, t’he temperature of half-completion of thermal denaturation. measures the thermal stabilit)y of the folding product in final conditions. The Figure shows that the rates (TV. (a): TV (b)) are correlated with the thermal stability of the respective species in final conditions. This relation holds for a wide range of pH values and GuHCl concentrations, The t, value of RNAase A varies when changing t,he concentration of GuHCl (von Hippel & Wong. 1965) or pH (Tsong et al.. 1970: I’rivalov et nl.. 1973), and that of RNBase S. in addition, when changing the concentration of the fragments (Labhardt. 1981). It is known, both from kinetic (Labhardt B Baldwin, 19793) and equilibrium studies (Labhardt, 1981) t’hat (“low” -affinity) fragment association does not’ require complete folding. The existence of a unifying parameter (t,), which allows us to describe the folding kinetics of the three molecular species, is surprising. One must keep in mind that the folding rates, e.g. of RNAase A and S-protein. differ six-fold (TV) and over 500fold (TV) if they are compared in identical solution conditions (e.g. at pH 6.5. 3OY’. 0 31. GuHC’l) instead of at identical values oft,. \Z’e note that some ambiguity exists as to the assignment of one out of the three faster relaxation times of S-protein to the sinyIP fast-folding reaction of RNAase S or A. The reason for this is that no inhibitor binding assays are possible with S-protein. As observed by tyrosine in low to neut’ral pH jumps. changes (at pH 6%. 0 M-(:uHCI. 25°C) S-protein,
A. M. LABHARDT
366
0 35
\ I
I
I
I
I
40
45
50
55
60
FIN:. 4. The time constants measured at neutral pH for the slow ((b), 30 (‘) and fast ((a), 25’(‘) refolding reactions of RNAase A (0, 0). RNAase S (0, @ ) and S-protein (0) are correlated with the thermal stability of the folding product in final conditions (as reflected by the temperature t, of halfcompletion of thermal denaturation). The rates determined here for initial conditions 2.3 and 5 (see the legend to Fig. 1) at pH 6.2 and 0.5 M final GuHCl (@) are plotted together with relaxation time data taken from Labhardt & Baldwin (1979a,b) for RNAase Y and H-protein (pH 6%. 30°C. 0 M final [GuHCl]). and from Nail et nl. (1978) for RNAase .4. Their slow-phase time constants 71 were extrapolated from 25°C to 30°C using the activation enthalpies given in Fig. 6 of Xall et nl. (1978). 72 for KNAase S does not vary significantly between 20 and 31 ‘C (see Fig. 2 of Labhardt,. 1980) and 111, temperature correction was applied to the +z data of Nail et (I/. (1978). The folding time constants were varied in the case of RNAase S by changing the fragment ooncentration from 12 pM to 100 P’M. in the case of RNAase A by changing the final GuHCl concentration from 002 M to 2 M (0) or by lowering the final pH from 7 to 3 (a). The variation in t, with changing fragment concentration was taken from Labhardt (1981). with QuHCl concentration from van Hippel & \liong (1965) and with pH from Tsony rf nl. (1970).
displays a spectrum of four well-separated kinetic phases: (1) 100 seconds, (2) 25 seconds. (3) 2.5 seconds (Labhardt & Baldwin. 19796) and (4) 25 milliseconds (Labhardt & Baldwin, 1979a). The 100 second phase undoubtedly corresponds to the slow-folding phase of RNAase A or S as measured by the regain of 2’.OW’ binding (see Fig. 4(b)). The three faster phases observed for S-protein are plotted in Figure 4(a).
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4. Discussion (a) Folding
of ribotzuclease is rapid once the A-protein @-sheet has formed in a co-operative transition
moiety
ITsing c.d. it has been possible to show that partly folded states, summarized in equation (1). can be populated at equilibrium (Labhardt, 1982). Comparison of these results with the kinetics of 2’-CMP binding allows one to draw several detailed conclusions. The fragment association half-time in Figure 2 (upper panels) is t)he same, independent of the presence or absence of 2’-CMP. This is expected from t,he observation of Barnard (1964) that substrate analogues stabilize ribonuclease by decreasing the rate of unfolding in concentrated urea but leave the rate of refolding unaffected. It is consistent with the molecular picture emerging from nuclear magnetic resonance studies that the active-site region (as monitored by the residues His12 and 119) unfolds in an early stage both in urea-induced and thermal-unfolded conformations at low pH (Benz & Roberts, 1975a,b). Hence we identify the 2’.CMP binding state pN of equation (3) with the all-native conformation a1a2/? of scheme (1). (i) The folded p-sheet is necessary to mediate rapid fragment
association
The association reaction of scheme (3) has been measured for various initial conditions as function of temperature. These different conditions cause a large variation of fn(2). the fraction of native RNAase S formed in a second-order reaction (Fig. 2). For the same conditions the fractions of a-helix vaa)and p-pleated sheet( fP) have been determined by c.d. (Labhardt, 1982). The normalized fractions of b-sheet in initial conditions, fa/O’36, are also plotted in Figure 2. The two quantities correlate well in all cases. No correlation is observed betweenf, and fj”. This can be explained if the p-sheet of S-protein must be preformed in initial conditions to allow native RNAase S to be produced by peptide association. From a mechanistic point of view there are two alternatives. (1) Rapid association occurs between S-peptide and trll S-protein molecules with intact p structure. With reference to equations (1) to (3), the fraction offi of I, molecules is fi = fs/0.36. In the presence of o1r,fragment complexes with incomplete a2 content are unstable (Labhardt, 1982), and folding therefore proceeds rapidly towards the all native state a1 a2/?. The latter is detected by 2’.CMP binding such that fA2) =fi =f@36. (2) The alternative is that both element’ x2 and the p-sheet are necessary for rapid fragment association. It must then be assumed that a-helix formation on S-protein (element a2) can equilibrate rapidly in final conditions also in the absence of bound S-peptide, such that again ft2’ = &JO.36. IM ech anistically just S-proteins in conformation a2fl of equation (1) ;articipate in rapid association, which reduces the fraction fi of I, molecules to J 5 fs/0.36/(1 + K34) (the equality holds if transition ~~/I--fi in scheme (1) is 2state). This causes the association half-time to increase (see eqn (6)). The observed change of the apparent association rate from 7 x lo5 Me1 s-l to 2 x 10’ M-l s-l, when going from 0 M to 0.5 M final GuHCl concentration. is consistent with such a rapidly equilibrating destabilization of the high-affinity S-peptide binding site. In an earlier investigation the second-order association amplitude fi2) had been
X8
A. M. LABHARDT
measured in 0 M-GuHCl, pH 68 (Labhardt & Baldwin, 1979,). In these final conditions and between 5°C and 25°C the amplitude fi2’ is slightly larger than the normalized fraction of preformed p-sheets and shows a different t’emperature dependence (cf. Fig. 7 of Labhardt, 1980).
(ii) The entire /l-sheet
rcnfoldslrefolds
as a single co-operntioe
mit
S-protein displays an intermediate degree of ,&sheet at, low pH and in the presence of denaturants (fs/0.36, labeled 2, 3.4,5 in Fig. 2). This means one of two things. (1) Part of the @structure is formed on all (or most) S-protein molecules. This part could be. e.g. the central piece of the sheet with His48 presumed t)o be more stable (Labhardt, 1982), whereas the C-terminal, more floppy part remains unstructured. (2) Two classes of molecules exist. one without jl structure, the other with all (or most) of the p structure formed. The established quantitative coincidence of the normalized fp fractions with fj’) (Fig. 2) as a function of all three variables (pH, temperature and GuHCl concentration) shows that the second alternative prevails. Let us assume the contrary for the moment. i.e. t,hat stepwise unfolding of the p-sheet is possible: (a) if the more stable part of the p-sheet is sufficient to make S-protein an I, species of equat,ion (3). the native fraction fA2’ would be larger than f,JO.36: and (b) if the entire sheet (or the less stable part) is necessary for an 1, species, f;” would be smaller than fP/036. Hence, we conclude that the entire p-sheet forms and dissolves as a single COoperative unit. It should be emphasized that the c.d. results show an apparent “premelting” of the /3 structure of S-protein at pH 6.8 (see Fig. 2. label 1). The same premelting is detected by the kinetic quantityf, (2): t,his demonstrates that the observed decrease in F-sheet content that is well below the co-operative transition is not an artifact of the c.d. decomposition procedure. At 20°C (pH 6.8) a fraction of about 15% of S-protein appears to be unfolded (Fig. 2). This finding is consist,ent with the observation of Shindo & Cohen (1976), who detected unfolded material by nuclear magnetic resonance studies in solutions of S-protein at all pH values from 2 to 4.5 and 20°C. Folded and unfolded states are in slow exchange on the nuclear magnetic resonance time scale. (b) The thermal stability of the folding product as reflected by thr unfolding of th,e /3-sheet in final conditions determines the rate of refolding of both. the fa,st and the slow-folding
The thermal stability of S-protein. Rh’Aase S and RKAase A in various final conditions is reflected by the t, value, the temperature of half-completion of thermal denaturation. Structurally thermal denaturation of all three species involves unfolding of the S-protein moiety, which may (in RNAase A or RNAase S at high concentration) or may not (in S-protein or RNAase S at low concentration) be paralleled by a thermal transition of the helical S-peptide moiety. It is known that at low concentrations RNAase S shows different t, values for the two moieties (unpublished results). We correlate the data with the t, value of the S-protein moiety. Thermal unfolding of the latter has been shown to be incomplete
RIBONI~CLEASE
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(Labhardt, 1982; see the transition X$+X; in eqn (I)). Spectroscopically the t,ransition is dominated by a p-sheet to random coil conversion. In Figure 4 fast and slow-folding times are plotted against t,. They refer to the folding of material with denatured p-sheet in initial conditions (see Fig. 2 and eqns (7) and (8)). The relationship between folding rates and final stability as established in Figure 4 has several implications that bear on the mechanism of folding. In order to rationalize the deviation of the slow refolding phase from the characteristics of free proline isomerization the reaction scheme :
k 12
I,--
’ 12----N
was proposed (Nall et al., 1978). U, and U, are the slow and fast-folding species as characterized earlier (Hagerman & Baldwin? 1976), and I1 and I, are partly folded states with the same proline isomers as U, and U,, respectively; k,, and k,, are t,he apparent, average rates of isomerization of incorrect X-Pro peptide bonds to t’he correct configuration in the U and I states respectively. @ is the degree of conversion of U, to I, in final conditions. A large number of other schemes can explain biphasic kinetics. The rationale for introducing equation (9) is that the existence of two unfolded forms of ribonuclease is well-documented (Brandts et 01.. 1975; Hagerman & Baldwin, 1976; Schmid & Baldwin, 1978). If folding proceeds in solution conditions strongly favoring the native state, a direct conversion from U, to a quasi-N state (denoted by IN) has been observed (Cook et al., 1979: Schmid. 1986,1981; Schmid & Blaschek, 1981) and scheme (9) is no longer an adequat,e description. This is not the case for the data presented in Figure 4. If all folding steps in equation (9) are rapid compared to the rates k,, and k, 2 and if folding goes to completion, the expression for the rate of the slow-refolding reaction reads : h]-’
= k,,(t).[l
-@(t,
t,)]+k12(t).@(t.
t,)
(10)
(cf. Nall et al.. 1978; Tsong & Baldwin, 1978). The relaxation data 7i collected in Figure 4 have been measured at various final pH values (xi), final GuHCl concentrations (x2) and (in the case of RNAase S) at various fragment concentrations (2s). The t, values have been measured as a function of the same three variables. Plotting the two quantities In 7i and t, against each other produces. within experimental error. a unique curve with a common slope for all three variables xj. This implies:
(11) with one and the same proportionality constant, a(&,), for any of the three variables xj. Hence In 7i should be considered a direct function oft, and not of the individual xj values, as is seen when inserting equation (11) into the expression for the total differential of In ri :
370
A. M. LABHARLIT
x dxj = I
x dt,.
(12)
With reference to equation (10) this suggests, that the rates k,, and k,* cannot be strong functions of GuHCl concentration and pH. In model compounds this has been shown independently: the rate of proline isomerization is (except for dipeptides) independent of pH (Brandts et al.. 1975) and GuHCl concentration @all et al.. 1978). The same appears to hold true for the degree of conversion @(t, t,) : the data can be explained if CD is not a strong direct function: (1) of pH : (2) of GuH(‘I concentration: (3). in the case of RXAase 8. of fragment concentration co. (1) Our observat,ion has an analogue in equilibrium folding: Privalov CQ Khechinashvili (1974) showed that the heats of denaturation. AHd,-of RNAase A. lgsozyme and three other globular proteins are direct functions of t,(pH). but not of the pH itself. (2) Similarly the independence of the heat of denaturation from the concentration of GuHCl has been shown for lysozyme by Pfeil & Privalov (1976). (3) The variation of the fraction of native RNAase S with fragment) concentrat,ion c0 and the relation between t, and In c0 has been given recently (Labhardt. 1981). The surprising finding that In T, and the degree of conversion (@) of IT, t’o the intermediate I, (see eqn (9)) depend only on the t, value of RNAase S in ,@n/ conditions and not explicitly on the fragment concentration has a considerable conceptual implication. It indicates that the structure of I,. to which @ refers and which causes the rate enhancement from k,, to klz. has a S-peptide-modulat& stability. The structure of I 1 appears, however. not to involve the S-peptide itself as evidenced both by the absence of a direct dependence of In TV on fragment concentration and by the fact that the slow refolding time of S-protein in t)he absence of S-peptide falls on the line with RNAase A and S at the appropriate 1,. This suggests that I, is a precursor of the thermolabile structure of the S-probin moiety. which is (as mentioned) spectroscopically dominated by the /3-sheet. This conclusion is consistent with the findings that in equilibrium experiments binding of the S-peptide shields portions of the p-sheet from rapid amide proton exchange at places distant from the direct peptide-protein contact site (Rosa NE Richards, 1981). Kinetically early hydrogen-bonding of the backbone of RNAase A during refolding in “strongly native” solution conditions has been observed (Schmid & Baldwin, 1979). The S-peptide moiety is necessar? in order to stabilize this intermediate to the extent of being detected by hydrogen exchange experiments. I II a recent kinetic c.d. stopped-flow investigation the folding of t,he p-sheet has been observed directly : it precedes complete folding (unpublished results). The expert technical assistance by I. Bartoldus and E. l’schopp-#effen Financial support, by Dr ,J. Engel is gratefully acknowledged.
REFERENCES Barnard, Benz, F. Benz, F. Brahms,
E. A. (1964). J. Mol. Rio!. 10, 263-281. W. & Roberts. G. C. K. (1975a). J. &‘ol. Rio/. 91. 34-365. W. & Roberts. G. C. K. (1975h). .I. Mol. Rio/. 91. 367-387. S. & Brahms, J. (1980). J. ,Vol. Rio/. 138. 149-178.
was very helpful.
RIBONlTCLEASE
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37 I
Brmdts. J. F.. Halvorson, H. R. & Brennan, M. (1975). Biochemistry, 14, 4953-4963. Brandts, J. F., Brennan, M. & Lin, L.-N. (1977). Proc. *Vat. had. Ski., LT.S.A 74, 417% 4181. Cook, K. H.. Schmid, F. X. & Baldwin, R. L. (1979). Proc. Sat. zlcud. Sci., U.S.A. 76,6157~6161. (‘rook, E. M., Mathias, A. P. & Rabin, B. R. (1960). Biochem. J. 74, 234-238. Eigen, M. & De Maeyer, L. (1963). In Techniques of Organic Chemistry, (Friess. S. I,.. Lewis. E. S. & Weissberger, A., eds), vol. 8, part 2, p. 895, Wiley Interscience, Sew York. Hagerman, P. J. & Baldwin. R. L. (1976). Biochemistry, 15, 1462-1473. Julien, M. Br Baldwin, R. L. (1981). J. Mol. Biol. 145, 265-280. Kato, S.. Okamura, M., Shimamoto, N;. & Utiyama. H. (1981). Biochemistry, 20, 108C-1085. Labhardt, A. M. (1980). In Protein Folding (Jaenicke, R., ed.), pp. 401-425, Elsevier/Sort,hHolland. Amsterdam. Labhardt. A. ikl. (1981). Biopolymers. 20. 1359-1480. Labhardt, A. RI. (1982). J. Mol. Biol. 157. 331-35.5. Labhardt,, A. M. & Baldwin, R. L. (1979a). J. Mol. Biol. 135, 245-254. Labhardt, A. M. & Baldwin, R. L. (19796). J. Mol. Biol. 135, 231-244. Levitt. M. 8: Greer, .J. (1977). J. Mol. Rio/. 114, 181-239. Lin. L.-N. & Brandts, J. F. (1978). Biochemistry, 17, 4102-4110. Nail. B. T., Garel, J.-R. 8r Baldwin, R. I,. (1978). J. Mol. Biol. 118, 317-330. Pfeil. W. & Privalov, P. L. (1976). Biophys. Chem. 4, 23-50. Privalov. P. I,. & Khechinashvili, N. S. (1974). J. Mol. Biol. 86, 665-684. Privalov. P. L.. Tiktopulo, E. I. & Khechinashvili, S. N. (1973). Int. J. Protein Res. 5, 229237. Richards. F. M. & Logue, A. D. (1962). J. Biol. Chem. 237, 3693-3697. Ridge, *J. A.. Baldwin, R, L. & Labhardt, A. M. (1981). Biochemistry, 20, 1622-1630. Rosa, ,J. ,J. & Richards, F. M. (1981). J. Mol. Biol. 145, 835-851. Srhmid, F. X. (1980). In Protein Folding (Jaenicke. R., ed.), pp. 387-400, Elsevier/NorthHolland, Amsterdam. Schmid. F. X. (1981). Eur. J. Biochem. 114, 105-109. Schmid, F. X. & Baldwin, R. L. (1978). Proc. Sat. Acad. Sci., CTS.rl. 75. 4764-4768. Schmid, F. X. & Baldwin, R. L. (1979). J. Mol. Biol. 135, 199-215. Schmid. F. X. & Blaschek, H. (1981). Eur. J. Biochem. 114, 111-117. Shindo. H. & (‘ohen. J. S. (1976). J. Biol. Chrm. 251. 2648-2652. Stellwagen, E. (1979). J. Mol. Biol. 135, 217-229. Temussi, P. A., Tancredi, T. & Quadrifoglio, F. (1969). J. Phys. Chcm. 73, 4227-4232. Tsong. T. Y. & Baldwin, R. L. (1978). Biopolymers, 17, 1669-1678. Tsong. T. Y., Hearn, R. P.. Wrathall. D. I’. & Sturtevant, J. M. (1970). Biochemistry, 9, 2666-2677.
van Hippel, 1’. H. 8r Wong, K.-Y’. (1965). J. Biol. Chem. 240, 3909-3928. Wyckoff, H. W.. Tsernoglou, D.. Hanson. A. W.. Knox. J. R., Lee, B. & Richards, F. M. (1970). J. Biol.
(‘hem. 245, 305-328.
(1982)
157. 357-371
Secondary II.
Structure
in Ribonuclease
Relations between Folding Kinetics and Secondary Structure Elements ALEXANDER M. LABHARDT Chemie A bteilung Biophysikalische der Universitd, Klingelbergstrasw CH 4056 Basel. Switzerland
Biozentrum
(Received
10 August
1981, and in, revised form
70
4 January
1982)
The kinetics of regain of 2’.CMP binding are monitored during renaturation of RNAase S. Experiments were performed by mixing equimolar amounts of Speptide with S-protein. The S-protein fragment was incubated initially (i.e. before mixing with S-peptide) at pH 6.2 or 1.7 and various guanidine hydrochloride (GuHCl) concentrations. Three well-resolved phases are observed. The fastest phase is second-order. The reciprocal half-time increases linearly with fragment concentration and is independent of initial conditions for the S-protein fragment. An apparent on rate of k,,, = 2 x lo5 M- ’ s- ’ is measured in 0.5 M-GuHCl (pH 6.2) and 20°C. Identical association kinetics are observed by changes in tyrosine absorbance. The fraction of native RNAase S formed in this second-order reaction strictly equals the fraction of S-protein molecules with intact b-sheet in initial conditions. The relation holds for different pH values, GuHCl concentrations and temperatures. The fraction of apparent helical content of S-protein in initial conditions may also vary but this is not reflected by the association reaction. We interpret this to mean that the B-sheet but not the a-helices must be preformed in initial conditions in order to generate the high-affinity peptide binding site of Sprotein. Furthermore, it is concluded that the S-protein moiety p-sheet forms or unfolds in a single one-step reaction. 2’.CMP binding reports, additionally, two slower phases of renaturation. These are produced by S-protein molecules that have their p-sheet unfolded in initial conditions. It is observed that a unique dependence of these two folding rates exists for RNAase A, RNAase S and S-protein as function of t,, the temperature of half-completion of thermal denaturation as measured by unfolding of the B-sheet in the respective compound in jinal conditions. The t, value varies with changing pH, with GuHCl concentration and (for R?iAase 6) with changing fragment concentration. The findings are interpreted to argue in favor of a sequential mechanism of folding, where the stability of a structural precursor determines the rate of folding.
1. Introduction Three independent spectroscopic elements have been detected in unfolding/refolding experiments with ribonuclease (RNAase) by amide circular dichroism (Labhardt. 1982). An approach was used that involves the decomposition of the c.d.t diff ewncc spectrum of unfolding or folding into loss or used : cd.. circular dichroism : GuHCl, guanidine hydrochloride. Xi 2836/82/l 40357-16 $03.00/O 10 1982 Academic Press Inc. (London)
t Abbreviations
Ltd.
35A
A. M. LABHARUT
gain of fractions of helical and pleated-sheet type conformations. When using this method it turns out that the cd. spectrum of native RNAase S differs from that of thermally denatured RNAase S or S-protein by a fraction of some 36O/,, of fl-pleat,ed sheet and some 9% of a-helix. From the crystal structure it is known that some 40% of the residues of the S-protein fragment of RNAase S part’icipatr in t)htl antiparallel p-pleated sheet (Wyckoff et al., 1970), which agrees favorably with the c.d. estimate for unfolding. The change of 9% n-helical content during thermal denaturation has been attributed to the unfolding of the S-peptide (residues 3 to 12 are helical in the folded state; Levitt & Greer, 1977). The assignment rests on thr fact that no change in r-helical content can be detected during thermal denaturation when the S-peptide is omitted from the solution. It had been noted that thermal denaturation of S-protein in the absence of S-peptide produces a c.d. difference spectrum with an isodichroic point at 225 nm (Labhardt. 1981/r). This indicated that the apparent helical c.d. (which is characterized by two strong bands near 225 nm and 109 nm; Brahms 8: Brahms, 1980) of S-protein itself does not change significantly during thermal denaturation, although this fragment accountjs for more than 60% of the helix content of folded RNAase S (Wyckoff et al., 1970). A c.d. difference spectrum consistent with the loss of about this amount of apparent helical type struct,ure is observed for t)rypticb digestion of thermall!, denatured S-protein or by exposing it to 5 M-GuHCl (Labhardt, 1982). Abbreviating the S-peptide helix by I,, the S-protein helices by x2 and the Sprotein F-sheet by j3 allows one to summarize these results by the following multistate scheme : K12 110128
’
“1 P
z
KS
1 1219
’
’
(1)
‘B
KS
1 G
L
,I I
If any of the three elements unfolds, its symbol is omitted in the above scheme. Below pH 2 S-peptide dissociates and unfolds (Richards & Logue, 1962). This destroys xl but leaves ‘x2 unaffected. Thermal denaturation destroys a1 and /3 but does not change the c.d. intensity of a2. It is not’ known whether the same structure generates the helical cd. of 3~~after thermal denaturation of the p-sheet as does in the native state. Therefore a2 is characterized by an asterisk in these conditions. 1; converts to the all random state (denoted by T) in 5 M-CuHCl. In the accompanying paper (Labhardt, 1982) it has been shown that all states except n1/3 in equation (1) can be present at equilibrium using appropriate conditions of temperature and GuHCl concentration. In order to assess the function of the three elements in relation to folding we have investigated the renaturation kinetics and their variation when starting from the different states shown in scheme (1). We report here the kinetics observed when mixing S-peptide with S-protein. Folding is monitored by the restoration of binding ability for the specific inhibitor 2’.CMP.
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The individual refolding kinetics of the p-sheet and of the S-peptide n-helix measured by fast c.d. stopped-flow will be published elsewhere. The different states in equation (1) display different folding kinetics. Three wellseparated phases are observed in the final conditions used here (0.5 M-GuHC’I. pH 62). The fastest phase is second-order and corresponds to the association between S-peptide and a moderately folded form of S-protein. The main observation is that the fraction of active RNAase S produced in this second-order reactlion exactly equals the fraction of S-protein molecules in the states /3 or azp of equation (1) in initial conditions. i.e. the fraction of S-protein with intact p-sheet. It, is shown that this finding implies that the p-sheet unfolds or refolds as a single cooperative unit. The two slower phases correspond to the folding reactions of unfolded S-peptidrS-prot)ein complexes. These t,wo phases are observed with a constant amplitude ratio. The log of both time constants are linear functions of the melting temperat,ure (t,) in widely varying final conditions of pH. GuHCl concentration and protein concentration. A unique dependence is observed for RNAase A. RSAase S and S-protein. Prom this observation we draw the conclusion that folding is a sequential process controlled by a, structural “on the pathway” intermediate. Circumstantial evidence suggests that the /l-sheet or a p-sheet precursor is (or is part of) the structure of this intermediate. The interpretation is consistent, with the findings of a recent c.d. stopped-flow investigation where the folding kinetics of the p-sheet and the ix-helix were monitored independently: t,he folding of the p-sheet is observed to precede the folding of the rest of the molecule (unpublished results).
2. Materials and Methods (a) Matmizls RNAase A. RNAase S, S-protein, S-peptide, pepsin, trypsin and .Z’-CMY were purchased from the Sigma Biochemical Co. I’ltra-pure guanidinr hydrochloride and urea were from SchaarziMann.
(I,) Methods Routine enzymatic activity assays of RNAase S were done according to Crook et al. (1960). Kinetic measurements were carried out with a modified Gibbs Durrum stopped-flow instrument with split beam optics. In GuHCl dilution experiments variable dilution ratios were achieved using various Hamilton syringes. In order to prevent Schlieren artifacts or uncontrolled premixing the densities of both solutions used in such dilution experiments were matched by sucrose. It is known, that sucrose (up to SOsl/o,w/w) does not affect the folding kinetics of RNAase A (Tsong & Baldwin, 1978). The data presented in Figs 2 and 4 show that the same holds true for RNAase S. The kinetic progress curves were stored on a Datalab transient recorder DLSOB interfaced to a PDF’ 11/40. Multiphasic progress curves were analyzed using an interactive graphic program (DIALOG).
3. Results (a) Second-order fmgwtmt
nssocintim
kin&s
It has been shown that S-peptide dissociates completely
from S-protein below
:Ml
pH 2 (Richards denoted by 1,:
A. M. LABHARDT
& Logue,
1962). A fraction,
,fi. of acidified
f,=ITji.
S-protein
molecules
is
(2)
CO
where co is the total concentration of S-protein. because it has the abilit!. to associate with S-peptide in a strictly second-order reaction with an on-rate k,,,, =7xlOS M-l s-l (at 20°C’. 0 X-GuHCl, pH 64) when the pH is rapidly raised in a stopped-flow spectrophotometer (Labhardt, & Baldwin, 1979a,(‘,). The kinetics have been interpreted by the scheme: 1,sp
“: PI, 682= ps,
GO
where I, symbolizes the fraction of S-protein capable of binding H-peptide (the index 3 is used here in order to be consistent with Labhardt, & Baldwin. 1979n). p stands for the S-peptide in any conformation and ~1, is the initial association complex at neutral pH. Its formation is detected by changes in tyrosine absorbance at 289 nm. pN is native RNAase S capable of binding the competitive inhibitor 2’. +pN is silent when measured by tyrosine absorbarrctl. CMP. The t’ransition p13 but at 265 nm and pH 6.2 the binding of 2’.C’MP to pN (which is a rapid reaction) can be monitored. The high association rate (Ic,,) of p and I, and the short conversion time of p1, to pN (68 ms at 2O”C’, Labhardt & Baldwin, 1979~~) suggest, that I3 may be partly structured in initial conditions. Various partly folded equilibrium intermediates have been detected and characterized (Labhardt. 1982). These states are summarized in equation (1). Here we measure t’he association reaction (3) as a function of temperat,ure. pH and GuHC’l concentration in order to establish whether or not the fraction fi of I, molecules correlat’es with the frac+on of any of the partly folded equilibrium intermediates (eqn (1)) as function of the same three variables. This will allow us to determine the S-protein structure that is necessary to mediate the rapid fragment association. Figure 1 shows three representative examples for kinetic progress curves. l’h(x fraction native. defined as:
where c,, is again the total concentration of RNAase S’. is measured by the extent of 2’.CMP binding. f”(t) is plotted against the logarithm of time after initiating refolding. Association and folding was started by mixing S-peptide with S-protein in a 1 : 1 molar ratio and adjusting the pH to neutral. if necessary. In tra,ce I Sprotein was equilibrated before mixing with S-peptide at pH 6.2. in trace 2 at pH 1.7 and in trace 3 again at neutral pH. however, in 1 M-GuHCl. Final conditions (i.e. after mixing S-peptide wit,h S-protein) are identical (see the legend to Fig. 1 for details). Except for trace 1 the kinetics are triphasic. The slower phase parameters were determined independently and are discussed in section (1~). below. The extrapolation of these slow changes into t,he “fast” time range (from 5 ms to ;i s: see Fig. 1) serves as a “baseline” to determinef,, (‘) . the fraction of native RNAase S
RIBONI’CLEASE
i
SECONDARY
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50 60 70 80 .
90 100
I
0.005
I
0.5
0.050
5
I
50
t(s) PI<:. 1. The native fraction!,(t) plotted versus the logarithm of t.!.(t) is measured by the extent of 2’CMP binding as monitored at 265 nm. t is the time in seconds after mixing S-peptide with S-protein in a 1 : 1 molar ratio in a stopped-flow spectrophotometer. Refolding conditions are in all cases (traces 1. 2 and 3) 50 PM-RNAase 8, 130 ~~-2’-cbfP, 05 M-GuHCl. VC. pH 6.2. Before mixing, S-protein was equilibrated in X M-GuHCl at pH = Y. The buffers used and the values of X and Y corresponding to the labels 1. 2 and 3 are listed below. S-peptide before mixing was always at neutral pH, @I x-sodium cacodylate buffer containing 260 p~-2’-cMP and (in order to produce identical final conditions) 1 - X MGuHCI and varying amounts of sucrose as described in Materials and Methods. Isolated S-peptide is not folded and the Initial GuHCl concentration in the S-peptide solution has no effect. The progress curves were fitted by the sum of 2 slower exponential terms (f~” x ni exp (-l/7*): i = 1.2) and one faster second-order hyperbolic term (f$/[l +t/~~!~]). The native fraction produced in a fast second-order reaction fi” and the hyperbolic half-times. ~li2, are plotted for various temperatures in Fig. 2. The parameters for the slow exponentials were determined independently (see Figs 3 and 4). These slowphase parameters were used as a baseline for the above traces (- - - ). The labels in Figs 1. 2 and 3 indicate that S-protein was equilibrated in X k~-GuHCl at pH = Y: Curve
X (M-GuHCI)
Y (pH)
1 2
0 0 1 05 1
6.2 1.7 62 1.7 1.7
3
4 5
Huffer 01 0.1 0.1 0.1 0.1
-
M-sodium cacodylate M-NaCIO,
x-sodium
cacodylate
M-SaClO,
M-NaClO,
produced in the fast rea&ion. As can be seen. this fraction varies strongly with changing initial conditions for the S-protein fragment. Figure 2 summarizes the results for the different starting conditions : (a) refers t’o 2’7”MP binding assays and (b) shows that identical behavior is seen when monitoring the changes in tyrosine absorbance. The upper panels in Figure 2 give a semilogarithmic plot of the association half-times 7 1,Z against temperat’ure. They are independent of both initial conditions and method of detection (2’.CMP binding or changes in tyrosine absorbance). From the slope found with temperature one calculates an activation energy AH* = 26 kJ/mol. The inset gives the concentration dependence at 20°C. If in final conditions and on the time-scale of association the S-protein subclass I, is in either f&t or A~OIPequilibrium with thr
A. M. LARHARDT
-.
*-.... so .+A.., l *‘. t I 80
\ 2’CMP \
-
I
5
IO
15
20
25
30
35
40
’
Temperature (a)
5
IO
15
20
25
30
35
40
(“C) (b)
FIG. 2. (a) Lower panel: The native fractionf,!‘) produced in a second-order reaction. Experimental procedure and conditions are as described in the legend to Fig. 1 except for the temperature, which is variable. Filled symbols refer to neutral initial pH of the sulution containing the S-protein fragment. open symbols to acidic pH. Circles (0) refer to 0 M, triangles (A) to 0.5 M and squares (0) to 1 M-initial GuHCl concentration in the syringe containing the S-protein fragment. (. .) Drawn through the fj2) data points, represent the normalized fraction of B-sheet fa/0.36 of S-protein at neutral pH taken from )fs/0.36 withfB taken Fig. 5 of Labhardt (1982). Native RNAaae S contains around 36% p-sheet. (--from the left panel of Fig. 6 of Labhardt (1982). llpper panel: Semilogarithmic plot of the half-times. T,,~. in ms ZIWSUS temperature (scale below) and versus l/T (T = absolute temperature, scale above). The fact that all initial conditions produce the same half-time with the same activation energy (26 kJ/mol) shows that the same reaction has been monitored in each case. Varying the amounts of sucrose present during refolding (see Materials and Methods) had no effect on the kinetics. (b) an identical pattern is observed when measuring the changes in tyrosine absorbance at 289 nm instead of 2’.CMP binding at 265 nm. Experimental procedure and conditions are identical to those for (a). except for the absence of 2’-CMP. Lower panel : Plot of 2.2 ‘a@’ versus temperature. n (‘) is the fractional amplitude of the fast association reaction measured at 289 nm. In tyrosine absorbance 3 slower reactions are observed and the relative amplitude a”) is less than the native fraction@*’ as measured by 2’-CMP binding. The factor 2.2 was chosen to match the f,!“’ values for corresponding conditions. Labels and symbols have the same meaning as in (a). Upper panel: association half-times measured at 289 nm. The inset gives 7,,* W-W~Y final RNAase S concentration (c) in a double logarithmic plot. The slope of - 1 establishes that the reaction is second-order. ( - - - ) The native fraction of RNAase S in final conditions as measured b> equilibrium melting experiments in the presence (a) and in the absence (b) of 130 PM-2’-CMP.
RIRONCCLEASE
SECONDARY
other S-protein states the association half-time 7112
=
c,2
x
STRUCTlTRE.
II
363
in equation (3) is:
In (2 -fi”) 1 -fj”
(5)
with
fP +2 = fi x Lkonx co]- 1, The second t,erm in equation (5) describes the transition from a true second-order reaction (fn(‘) = 1) to pseudo first-order (f,‘) e 1) and varies from 1 to 0.7. Note that’ S-peptide and S-protein are in a 1 : 1 molar ratio. In the inset In ‘T~,~is plotted against In co. The solid line through the data points has a slope of - 1. which demonstrates that the fast phase is, within experimental error, a second-order reaction of the type of equation (3) also in the mildly denaturing final conditions used here (pH 6.2,0.5 M-GuHCl). The apparent on-rate k,, calculated from the data according to equation (6) (ji and fj2) were set equal) is 2 x 10’ M-I se1 in 05 MGuHCl at 20°C. The slight variation in 71,2 as predicted by equation (5) could not be detected. In general, the fast-phase component of the progress curves was fitted by a hyperbolic term as described in the legend to Figure 1. For j’:” < @l an exponential decay was assumed. The observation that the half-times are the same, independent of the degree of initial unfolding, meets theoretical expectations : kinetic time constants depend only on final conditions. unless the change in initial conditions alters the pathway (Eigen & De Maeyer, 1963). Hence the amplitudesfj2’ in Figure 2 refer to the same kinetic pathway, and their variation with GuHCl concentration, pH and temperature reflects the variation of the fraction of S-protein participating in equation (3). It should be emphasized that the kinetic amplitudes in Figure 2 are not affected by incomplete folding of RNAase S in final conditions with increasing temperature. The broken lines in the lower panels give the native fractionf,( 00) in 0.5 M-GuHCl (pH 6.2) in the presence of 130 p~-2’-cMP (Fig. 2(a)) and its absence (Fig. 2(b)): the stability of RNAase is larger in the presence of 2’-CMY (Barnard. 1964). which increases t, by some 5 deg.C; as can be seen all kinetic data have been collected in a temperature range with fn (co) > 0.9. The most important observation is that j’,j” as a function of initial GuHCI concentration. pH rind temperature strictly correlates with the normalized fraction of p-sheet of S-protein for those conditions. This is demonstrated by the solid and dotted lines representing the fractions fD/0.36 taken from Labhardt (1982). The fraction of apparent a-helical content fa of S-protein also varies in the conditions summarized in Figure 2; however, in a way uncorrelated to the f,j2) fraction (compare Figs 5 and 6 of Labhardt, 1982). (b) “First-order”?
folding
reactions
The remaining fraction : p
= 1 -fA”
(7)
t The 2 reactions termed “first-order” folding phases display a concentration dependence in the case of RSAase S, but not for S-protein or RNAase A. A unifying ikerpretation of the two phases is given in the text for all three molecular species. Hence we refer to them as first-order throughout.
A. M. LABHARI)T
a2
60
t
._
oo-
c
Temperature
PC)
FIG. 3. Renaturation experiments in the presence of 2’.CMP and 05 M-GuH(‘1 were carried out and analyzed as described in the legend to Fig. 1. Progress curves were monitored in the temperature range from 5 to WC” for 023 h after mixing of the 2 fragments, Between 5 and 900 s the progress curves arc strictly biphasic. The Figure shows that the relative amplitude of the slower phase asloWvaries only slightly with initial conditions and temperature. For the definition of labels see the legend to Fig. 1. The time constants are plotted in Fig. 4 for comparison with the folding kinetics of S-protein and RNAase A.
of S-protein molecules refolds with first-order kinetics. Progress curves (f”(t) ZWSU~St) were collected during 900 seconds in the temperature range from 5’Y and to 35°C for the same final conditions (05 M-GuHCl. pH 6.2, 130 FM-~‘-CMI-‘) various initial conditions. The experiments were again carried out as described in the legend to Figure 1. Two well-separated phases were detected in this time-range (a slow phase. labeled 1 and a fast phase, labeled 2) and they were analyzed as a biexponential change according to :
(8) The pre-exponential factors a, = qlOW and a1 = 1 -aSlOW are the relative slow and for various fastphase amplitudes. (lSIOWis plotted in Figure 3 ~c1rsus temperature initial conditions. As seen, the relative amplitude aslow remains constant for different temperatures and for initial conditions involving low pH. and almost constant for initial condition 3 ((a,,,,) = 73+5”i,). I n order to appreciate this one should note t’hat the kinetic amplitudes themselves (j:” x ai) change dramatically as a function of initial conditions and temperature (by more than a factor of 8: see Fig. 2 and eqn (7)). This indicates that in the present final conditions refolding in the two firstorder react’ions ((a i. 7i) and (az. TV)) proceeds largely independently and unaffected by the presence of partly folded material (I3). The folding kinetics starting from the present. only moderately denaturing, conditions are consistent with earlier investigations (Labhardt & Haldwin. 19796). In summary, the known facts are : (1) both unfolded RNAase 8 and unfolded S-
RIRONCCLEASE
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protein exist in a way analogous to RNAase A as a mixture of two forms. a moderately fast (IT,) and a slow-folding form (U,). The two classes are found in a 25 : 75 ratio. independent of temperature higher than 20°C (Labhard& 1980). (2) In refolding the two forms T-1,and U, give rise to two well-separated kinetic phases. The slow phase is- complex when monitored by absorbance of tyrosine OI fluorescence. but not in 2’-CMP binding assays. As a molecular basis for the existence of the slow-folding form I’, of RNAase A prolyl peptide bond isomerization has been proposed (Brandts et nl.. 1975). (1) In unfolding (RNAase -+ I’,) the species CT, is formed from I:, in a SIOU equilibrium. The kinetic characteristics of this process are typical for free X-Pro peptide bond isomerization in model compounds. One observes (i) the high activation energy of 85 kJ/ mol and (ii) acid catalysis by strong acids (Schmid & Baldwin, 1978). (2) In refolding the involvement of proline isomerization is less easily assessed (Stellwagen. 1979: Kato et ~1.. 1981; Ridge et al.. 1981 : Julien 8r Baldwin, 1981). Proteins containing no prolines may lack the slow phase (Brandts et rrl., 1977) but may also have slightly modified slow-phase characteristics (Lin &’ Brandts. 1978). In the refolding of RNAase A the kinetic parameters of free prolyl peptide bond isomerization are no more reflected by the slow phase : solution conditions strongly favoring the native state considerably speed up the slow phase over free proline isomerization. The activation energy drops from 85 to 10 kJ/mol (Nail et ul., 1978). The slow phase in RNAase S at pH 6.8 has at 13 pM an activation energy of 75 k,J/mol. By increasing the concentration of both fragments tenfold this phase is speeded up almost threefold (Labhardt & Baldwin. 1979h). measured in the present final The t’ime constants 71 and 72 for refolding conditions (0.5 M-GuHCl. pH 6.2, [RNAase S] = 50 PM. t, = 37°C) are plotted in Figure 4 against t, together with data for RNAase A and S-protein. t,, t’he temperature of half-completion of thermal denaturation. measures the thermal stabilit)y of the folding product in final conditions. The Figure shows that the rates (TV. (a): TV (b)) are correlated with the thermal stability of the respective species in final conditions. This relation holds for a wide range of pH values and GuHCl concentrations, The t, value of RNAase A varies when changing t,he concentration of GuHCl (von Hippel & Wong. 1965) or pH (Tsong et al.. 1970: I’rivalov et nl.. 1973), and that of RNBase S. in addition, when changing the concentration of the fragments (Labhardt. 1981). It is known, both from kinetic (Labhardt B Baldwin, 19793) and equilibrium studies (Labhardt, 1981) t’hat (“low” -affinity) fragment association does not’ require complete folding. The existence of a unifying parameter (t,), which allows us to describe the folding kinetics of the three molecular species, is surprising. One must keep in mind that the folding rates, e.g. of RNAase A and S-protein. differ six-fold (TV) and over 500fold (TV) if they are compared in identical solution conditions (e.g. at pH 6.5. 3OY’. 0 31. GuHC’l) instead of at identical values oft,. \Z’e note that some ambiguity exists as to the assignment of one out of the three faster relaxation times of S-protein to the sinyIP fast-folding reaction of RNAase S or A. The reason for this is that no inhibitor binding assays are possible with S-protein. As observed by tyrosine in low to neut’ral pH jumps. changes (at pH 6%. 0 M-(:uHCI. 25°C) S-protein,
A. M. LABHARDT
366
0 35
\ I
I
I
I
I
40
45
50
55
60
FIN:. 4. The time constants measured at neutral pH for the slow ((b), 30 (‘) and fast ((a), 25’(‘) refolding reactions of RNAase A (0, 0). RNAase S (0, @ ) and S-protein (0) are correlated with the thermal stability of the folding product in final conditions (as reflected by the temperature t, of halfcompletion of thermal denaturation). The rates determined here for initial conditions 2.3 and 5 (see the legend to Fig. 1) at pH 6.2 and 0.5 M final GuHCl (@) are plotted together with relaxation time data taken from Labhardt & Baldwin (1979a,b) for RNAase Y and H-protein (pH 6%. 30°C. 0 M final [GuHCl]). and from Nail et nl. (1978) for RNAase .4. Their slow-phase time constants 71 were extrapolated from 25°C to 30°C using the activation enthalpies given in Fig. 6 of Xall et nl. (1978). 72 for KNAase S does not vary significantly between 20 and 31 ‘C (see Fig. 2 of Labhardt,. 1980) and 111, temperature correction was applied to the +z data of Nail et (I/. (1978). The folding time constants were varied in the case of RNAase S by changing the fragment ooncentration from 12 pM to 100 P’M. in the case of RNAase A by changing the final GuHCl concentration from 002 M to 2 M (0) or by lowering the final pH from 7 to 3 (a). The variation in t, with changing fragment concentration was taken from Labhardt (1981). with QuHCl concentration from van Hippel & \liong (1965) and with pH from Tsony rf nl. (1970).
displays a spectrum of four well-separated kinetic phases: (1) 100 seconds, (2) 25 seconds. (3) 2.5 seconds (Labhardt & Baldwin. 19796) and (4) 25 milliseconds (Labhardt & Baldwin, 1979a). The 100 second phase undoubtedly corresponds to the slow-folding phase of RNAase A or S as measured by the regain of 2’.OW’ binding (see Fig. 4(b)). The three faster phases observed for S-protein are plotted in Figure 4(a).
RIBONVCLEASE
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4. Discussion (a) Folding
of ribotzuclease is rapid once the A-protein @-sheet has formed in a co-operative transition
moiety
ITsing c.d. it has been possible to show that partly folded states, summarized in equation (1). can be populated at equilibrium (Labhardt, 1982). Comparison of these results with the kinetics of 2’-CMP binding allows one to draw several detailed conclusions. The fragment association half-time in Figure 2 (upper panels) is t)he same, independent of the presence or absence of 2’-CMP. This is expected from t,he observation of Barnard (1964) that substrate analogues stabilize ribonuclease by decreasing the rate of unfolding in concentrated urea but leave the rate of refolding unaffected. It is consistent with the molecular picture emerging from nuclear magnetic resonance studies that the active-site region (as monitored by the residues His12 and 119) unfolds in an early stage both in urea-induced and thermal-unfolded conformations at low pH (Benz & Roberts, 1975a,b). Hence we identify the 2’.CMP binding state pN of equation (3) with the all-native conformation a1a2/? of scheme (1). (i) The folded p-sheet is necessary to mediate rapid fragment
association
The association reaction of scheme (3) has been measured for various initial conditions as function of temperature. These different conditions cause a large variation of fn(2). the fraction of native RNAase S formed in a second-order reaction (Fig. 2). For the same conditions the fractions of a-helix vaa)and p-pleated sheet( fP) have been determined by c.d. (Labhardt, 1982). The normalized fractions of b-sheet in initial conditions, fa/O’36, are also plotted in Figure 2. The two quantities correlate well in all cases. No correlation is observed betweenf, and fj”. This can be explained if the p-sheet of S-protein must be preformed in initial conditions to allow native RNAase S to be produced by peptide association. From a mechanistic point of view there are two alternatives. (1) Rapid association occurs between S-peptide and trll S-protein molecules with intact p structure. With reference to equations (1) to (3), the fraction offi of I, molecules is fi = fs/0.36. In the presence of o1r,fragment complexes with incomplete a2 content are unstable (Labhardt, 1982), and folding therefore proceeds rapidly towards the all native state a1 a2/?. The latter is detected by 2’.CMP binding such that fA2) =fi =f@36. (2) The alternative is that both element’ x2 and the p-sheet are necessary for rapid fragment association. It must then be assumed that a-helix formation on S-protein (element a2) can equilibrate rapidly in final conditions also in the absence of bound S-peptide, such that again ft2’ = &JO.36. IM ech anistically just S-proteins in conformation a2fl of equation (1) ;articipate in rapid association, which reduces the fraction fi of I, molecules to J 5 fs/0.36/(1 + K34) (the equality holds if transition ~~/I--fi in scheme (1) is 2state). This causes the association half-time to increase (see eqn (6)). The observed change of the apparent association rate from 7 x lo5 Me1 s-l to 2 x 10’ M-l s-l, when going from 0 M to 0.5 M final GuHCl concentration. is consistent with such a rapidly equilibrating destabilization of the high-affinity S-peptide binding site. In an earlier investigation the second-order association amplitude fi2) had been
X8
A. M. LABHARDT
measured in 0 M-GuHCl, pH 68 (Labhardt & Baldwin, 1979,). In these final conditions and between 5°C and 25°C the amplitude fi2’ is slightly larger than the normalized fraction of preformed p-sheets and shows a different t’emperature dependence (cf. Fig. 7 of Labhardt, 1980).
(ii) The entire /l-sheet
rcnfoldslrefolds
as a single co-operntioe
mit
S-protein displays an intermediate degree of ,&sheet at, low pH and in the presence of denaturants (fs/0.36, labeled 2, 3.4,5 in Fig. 2). This means one of two things. (1) Part of the @structure is formed on all (or most) S-protein molecules. This part could be. e.g. the central piece of the sheet with His48 presumed t)o be more stable (Labhardt, 1982), whereas the C-terminal, more floppy part remains unstructured. (2) Two classes of molecules exist. one without jl structure, the other with all (or most) of the p structure formed. The established quantitative coincidence of the normalized fp fractions with fj’) (Fig. 2) as a function of all three variables (pH, temperature and GuHCl concentration) shows that the second alternative prevails. Let us assume the contrary for the moment. i.e. t,hat stepwise unfolding of the p-sheet is possible: (a) if the more stable part of the p-sheet is sufficient to make S-protein an I, species of equat,ion (3). the native fraction fA2’ would be larger than f,JO.36: and (b) if the entire sheet (or the less stable part) is necessary for an 1, species, f;” would be smaller than fP/036. Hence, we conclude that the entire p-sheet forms and dissolves as a single COoperative unit. It should be emphasized that the c.d. results show an apparent “premelting” of the /3 structure of S-protein at pH 6.8 (see Fig. 2. label 1). The same premelting is detected by the kinetic quantityf, (2): t,his demonstrates that the observed decrease in F-sheet content that is well below the co-operative transition is not an artifact of the c.d. decomposition procedure. At 20°C (pH 6.8) a fraction of about 15% of S-protein appears to be unfolded (Fig. 2). This finding is consist,ent with the observation of Shindo & Cohen (1976), who detected unfolded material by nuclear magnetic resonance studies in solutions of S-protein at all pH values from 2 to 4.5 and 20°C. Folded and unfolded states are in slow exchange on the nuclear magnetic resonance time scale. (b) The thermal stability of the folding product as reflected by thr unfolding of th,e /3-sheet in final conditions determines the rate of refolding of both. the fa,st and the slow-folding
The thermal stability of S-protein. Rh’Aase S and RKAase A in various final conditions is reflected by the t, value, the temperature of half-completion of thermal denaturation. Structurally thermal denaturation of all three species involves unfolding of the S-protein moiety, which may (in RNAase A or RNAase S at high concentration) or may not (in S-protein or RNAase S at low concentration) be paralleled by a thermal transition of the helical S-peptide moiety. It is known that at low concentrations RNAase S shows different t, values for the two moieties (unpublished results). We correlate the data with the t, value of the S-protein moiety. Thermal unfolding of the latter has been shown to be incomplete
RIBONI~CLEASE
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(Labhardt, 1982; see the transition X$+X; in eqn (I)). Spectroscopically the t,ransition is dominated by a p-sheet to random coil conversion. In Figure 4 fast and slow-folding times are plotted against t,. They refer to the folding of material with denatured p-sheet in initial conditions (see Fig. 2 and eqns (7) and (8)). The relationship between folding rates and final stability as established in Figure 4 has several implications that bear on the mechanism of folding. In order to rationalize the deviation of the slow refolding phase from the characteristics of free proline isomerization the reaction scheme :
k 12
I,--
’ 12----N
was proposed (Nall et al., 1978). U, and U, are the slow and fast-folding species as characterized earlier (Hagerman & Baldwin? 1976), and I1 and I, are partly folded states with the same proline isomers as U, and U,, respectively; k,, and k,, are t,he apparent, average rates of isomerization of incorrect X-Pro peptide bonds to t’he correct configuration in the U and I states respectively. @ is the degree of conversion of U, to I, in final conditions. A large number of other schemes can explain biphasic kinetics. The rationale for introducing equation (9) is that the existence of two unfolded forms of ribonuclease is well-documented (Brandts et 01.. 1975; Hagerman & Baldwin, 1976; Schmid & Baldwin, 1978). If folding proceeds in solution conditions strongly favoring the native state, a direct conversion from U, to a quasi-N state (denoted by IN) has been observed (Cook et al., 1979: Schmid. 1986,1981; Schmid & Blaschek, 1981) and scheme (9) is no longer an adequat,e description. This is not the case for the data presented in Figure 4. If all folding steps in equation (9) are rapid compared to the rates k,, and k, 2 and if folding goes to completion, the expression for the rate of the slow-refolding reaction reads : h]-’
= k,,(t).[l
-@(t,
t,)]+k12(t).@(t.
t,)
(10)
(cf. Nall et al.. 1978; Tsong & Baldwin, 1978). The relaxation data 7i collected in Figure 4 have been measured at various final pH values (xi), final GuHCl concentrations (x2) and (in the case of RNAase S) at various fragment concentrations (2s). The t, values have been measured as a function of the same three variables. Plotting the two quantities In 7i and t, against each other produces. within experimental error. a unique curve with a common slope for all three variables xj. This implies:
(11) with one and the same proportionality constant, a(&,), for any of the three variables xj. Hence In 7i should be considered a direct function oft, and not of the individual xj values, as is seen when inserting equation (11) into the expression for the total differential of In ri :
370
A. M. LABHARLIT
x dxj = I
x dt,.
(12)
With reference to equation (10) this suggests, that the rates k,, and k,* cannot be strong functions of GuHCl concentration and pH. In model compounds this has been shown independently: the rate of proline isomerization is (except for dipeptides) independent of pH (Brandts et al.. 1975) and GuHCl concentration @all et al.. 1978). The same appears to hold true for the degree of conversion @(t, t,) : the data can be explained if CD is not a strong direct function: (1) of pH : (2) of GuH(‘I concentration: (3). in the case of RXAase 8. of fragment concentration co. (1) Our observat,ion has an analogue in equilibrium folding: Privalov CQ Khechinashvili (1974) showed that the heats of denaturation. AHd,-of RNAase A. lgsozyme and three other globular proteins are direct functions of t,(pH). but not of the pH itself. (2) Similarly the independence of the heat of denaturation from the concentration of GuHCl has been shown for lysozyme by Pfeil & Privalov (1976). (3) The variation of the fraction of native RNAase S with fragment) concentrat,ion c0 and the relation between t, and In c0 has been given recently (Labhardt. 1981). The surprising finding that In T, and the degree of conversion (@) of IT, t’o the intermediate I, (see eqn (9)) depend only on the t, value of RNAase S in ,@n/ conditions and not explicitly on the fragment concentration has a considerable conceptual implication. It indicates that the structure of I,. to which @ refers and which causes the rate enhancement from k,, to klz. has a S-peptide-modulat& stability. The structure of I 1 appears, however. not to involve the S-peptide itself as evidenced both by the absence of a direct dependence of In TV on fragment concentration and by the fact that the slow refolding time of S-protein in t)he absence of S-peptide falls on the line with RNAase A and S at the appropriate 1,. This suggests that I, is a precursor of the thermolabile structure of the S-probin moiety. which is (as mentioned) spectroscopically dominated by the /3-sheet. This conclusion is consistent with the findings that in equilibrium experiments binding of the S-peptide shields portions of the p-sheet from rapid amide proton exchange at places distant from the direct peptide-protein contact site (Rosa NE Richards, 1981). Kinetically early hydrogen-bonding of the backbone of RNAase A during refolding in “strongly native” solution conditions has been observed (Schmid & Baldwin, 1979). The S-peptide moiety is necessar? in order to stabilize this intermediate to the extent of being detected by hydrogen exchange experiments. I II a recent kinetic c.d. stopped-flow investigation the folding of t,he p-sheet has been observed directly : it precedes complete folding (unpublished results). The expert technical assistance by I. Bartoldus and E. l’schopp-#effen Financial support, by Dr ,J. Engel is gratefully acknowledged.
REFERENCES Barnard, Benz, F. Benz, F. Brahms,
E. A. (1964). J. Mol. Rio!. 10, 263-281. W. & Roberts. G. C. K. (1975a). J. &‘ol. Rio/. 91. 34-365. W. & Roberts. G. C. K. (1975h). .I. Mol. Rio/. 91. 367-387. S. & Brahms, J. (1980). J. ,Vol. Rio/. 138. 149-178.
was very helpful.
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37 I
Brmdts. J. F.. Halvorson, H. R. & Brennan, M. (1975). Biochemistry, 14, 4953-4963. Brandts, J. F., Brennan, M. & Lin, L.-N. (1977). Proc. *Vat. had. Ski., LT.S.A 74, 417% 4181. Cook, K. H.. Schmid, F. X. & Baldwin, R. L. (1979). Proc. Sat. zlcud. Sci., U.S.A. 76,6157~6161. (‘rook, E. M., Mathias, A. P. & Rabin, B. R. (1960). Biochem. J. 74, 234-238. Eigen, M. & De Maeyer, L. (1963). In Techniques of Organic Chemistry, (Friess. S. I,.. Lewis. E. S. & Weissberger, A., eds), vol. 8, part 2, p. 895, Wiley Interscience, Sew York. Hagerman, P. J. & Baldwin. R. L. (1976). Biochemistry, 15, 1462-1473. Julien, M. Br Baldwin, R. L. (1981). J. Mol. Biol. 145, 265-280. Kato, S.. Okamura, M., Shimamoto, N;. & Utiyama. H. (1981). Biochemistry, 20, 108C-1085. Labhardt, A. M. (1980). In Protein Folding (Jaenicke, R., ed.), pp. 401-425, Elsevier/Sort,hHolland. Amsterdam. Labhardt. A. ikl. (1981). Biopolymers. 20. 1359-1480. Labhardt, A. RI. (1982). J. Mol. Biol. 157. 331-35.5. Labhardt,, A. M. & Baldwin, R. L. (1979a). J. Mol. Biol. 135, 245-254. Labhardt, A. M. & Baldwin, R. L. (19796). J. Mol. Biol. 135, 231-244. Levitt. M. 8: Greer, .J. (1977). J. Mol. Rio/. 114, 181-239. Lin. L.-N. & Brandts, J. F. (1978). Biochemistry, 17, 4102-4110. Nail. B. T., Garel, J.-R. 8r Baldwin, R. I,. (1978). J. Mol. Biol. 118, 317-330. Pfeil. W. & Privalov, P. L. (1976). Biophys. Chem. 4, 23-50. Privalov. P. I,. & Khechinashvili, N. S. (1974). J. Mol. Biol. 86, 665-684. Privalov. P. L.. Tiktopulo, E. I. & Khechinashvili, S. N. (1973). Int. J. Protein Res. 5, 229237. Richards. F. M. & Logue, A. D. (1962). J. Biol. Chem. 237, 3693-3697. Ridge, *J. A.. Baldwin, R, L. & Labhardt, A. M. (1981). Biochemistry, 20, 1622-1630. Rosa, ,J. ,J. & Richards, F. M. (1981). J. Mol. Biol. 145, 835-851. Srhmid, F. X. (1980). In Protein Folding (Jaenicke. R., ed.), pp. 387-400, Elsevier/NorthHolland, Amsterdam. Schmid. F. X. (1981). Eur. J. Biochem. 114, 105-109. Schmid, F. X. & Baldwin, R. L. (1978). Proc. Sat. Acad. Sci., CTS.rl. 75. 4764-4768. Schmid, F. X. & Baldwin, R. L. (1979). J. Mol. Biol. 135, 199-215. Schmid. F. X. & Blaschek, H. (1981). Eur. J. Biochem. 114, 111-117. Shindo. H. & (‘ohen. J. S. (1976). J. Biol. Chrm. 251. 2648-2652. Stellwagen, E. (1979). J. Mol. Biol. 135, 217-229. Temussi, P. A., Tancredi, T. & Quadrifoglio, F. (1969). J. Phys. Chcm. 73, 4227-4232. Tsong. T. Y. & Baldwin, R. L. (1978). Biopolymers, 17, 1669-1678. Tsong. T. Y., Hearn, R. P.. Wrathall. D. I’. & Sturtevant, J. M. (1970). Biochemistry, 9, 2666-2677.
van Hippel, 1’. H. 8r Wong, K.-Y’. (1965). J. Biol. Chem. 240, 3909-3928. Wyckoff, H. W.. Tsernoglou, D.. Hanson. A. W.. Knox. J. R., Lee, B. & Richards, F. M. (1970). J. Biol.
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