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[7] Mutatational analysis of protein folding mechanisms
[7]
MUTATIONAL
ANALYSIS
OF PROTEIN
FOLDING
MECHANISMS
113
[7] M u t a t i o n a l A n a l y s i s o f P r o t e i n F o l d i n g M e c h a n i s m s By PATRICIA A. JENNINGS, SUSANNE M. SAALAU-BETHELL, BRYAN E . FINN, XIAOWU CHEN, a n d C. ROBERT MATTHEWS Introduction
Site-directed mutagenesis has proved to be a powerful technique for probing the relationship between the amino acid sequence of a protein and its folding, stability, structure, and function.l-4 In a previous contribution to this series, we discussed the application of mutagenesis to the investigation of the mechanism by which the sequence directs the rapid and efficient folding to the unique native conformation, that is, the mechanism of protein folding. 5 Although the data available at that time were certainly encouraging, it was left for future studies to determine the ultimate utility of the mutagenic approach to the solution of one of the most vexing problems in biochemistry. An increasing number of contributions to the literature has indicated that the initial promise was real. There are now a number of examples in which amino acid replacements in globular proteins have provided new insight into the molecular events involved in rate-limiting steps in folding reactions. 6-8 The purpose of this chapter is (1) to extend the approach to a three-state folding model involving a transient intermediate, (2) to demonstrate how the integration of kinetic and equilibrium folding data can provide a more comprehensive understanding of the effects of mutations on the folding mechanism, and (3) to summarize some of the recent findings from our laboratory on the folding of dihydrofolate reductase using this approach. 1 L. M. Gierash and J. King (eds.), "Protein Folding: Deciphering the Second Half of the Genetic Code." AAAS Press, Washington, D.C., 1990. 2 D. L. Oxender (ed.), "Protein Structure, Folding and Design." Alan R. Liss, New York, 1986. 3 C. S. Craik, R. Fletterick, C. R. Matthews, and J. Wells (eds.), "Protein and Pharmaceutical Engineering." Wiley-Liss, New York, 1990. 4 j. j. Villafranca (ed.), "Current Research in Protein Chemistry: Techniques, Structure, and Function." Academic Press, New York, 1990. 5 C. R. Matthews, this series, Vol. 154, p. 498. 6 E. P. Garvey and C. R. Matthews, Biochemistry 28, 2083 (1989). 7 A. Matouscheck, J. T. Kellis, L. Serrano, M. Bycroft, and A. R. Fersht, Nature (London) 346, 440 (1990). 8 N. B. Tweedy, M. R. Hurle, B. A. Chrunyk, and C. R. Matthews, Biochemistry 29, 1539 (1990).
METHODS IN ENZYMOLOGY, VOL. 202
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
114
PROTEINS AND PEPTIDES: PRINCIPLES AND METHODS
[7]
Methods A detailed description of the experimental techniques used to obtain both equilibrium and kinetic data on the reversible folding reaction can be found in our previous chapter. 5 To summarize briefly, one can determine the free energy difference, AGNu ,9a between the native and unfolded states in a two-state equilibrium reaction from chemical denaturation experiments. Formally, AGNu = - R T In KNU, where KNU = [U]/[N]. As the concentration of denaturant increases, the equilibrium shifts to favor the unfolded form. Consequently, the value of KNU increases while that for AGNu decreases. The value of AGNu at any denaturant concentration can be calculated from A ~H20
AGNu = ,-,t-/su + ANu[denaturant]
(1)
A ~H20
where ,~t/NU is the free energy difference in the absence of denaturant and ANU is a parameter which reflects the cooperativity of the transition (Asu < 0 in this formalism). From the kinetic data, one can calculate the activation free energy, AG t, which represents the difference in free energy between one of the species in the kinetic mechanism and the transition state separating it from other species in the reaction. The relevant equation is AG* = - R T ln[kh/kBT]
(2)
where k is the microscopic rate constant, h, kB, and R are the Planck, Boltzmann, and universal gas constants, respectively, and Tis the absolute temperature. The activation free energies for folding reactions have been observed to depend on the denaturant concentration according to Eq. (3)5,9 AG* = AG*H2° + A*[denaturant]
(3)
where AG*H2° and A* have interpretations similar to their equilibrium counterparts. For unfolding, A*u < 0 and for refolding A*R > 0. Analysis The free energy data from both equilibrium and kinetic studies of the folding reactions of mutant and wild-type proteins can be summarized in a reaction coordinate diagram. In this diagram, the free energies of the various species along the folding pathway are plotted as a function of the reaction coordinate. This coordinate does not correspond to any particular 9 B. Chen, W. A. Baase, and J. A. Schellman, Biochemistry 28, 691 (1989). 9~ The free energy difference is that between the native and unfolded forms of the protein at standard state conditions. This stipulation is typically denoted by a superscript zero which has been deleted in this and other equations to simplify the notation.
[7]
MUTATIONAL ANALYSIS OF PROTEIN FOLDING MECHANISMS
115
molecular dimension but rather reflects the progress of the folding reaction between the two end states, the native and the unfolded conformations. In our previous chapter, 5 we addressed the case of a two-state folding mechanism which involved a native, N, and an unfolded, U, conformation. We now extend the discussion to the case in which a transient intermediate, I, is also present: N.
kl " k_ I slow
I
k2 "U "k_2 fast
kl and k z represent microscopic rate constants for the unfolding reactions, and k_l and k_ z are rate constants for the refolding reactions. Let us further stipulate that the interconversion of the native and intermediate forms is rate limiting: k2, k_2 >>k~, k_ 1. This situation has been observed for a number of proteins and appears to be a common (if somewhat simplified) folding mechanism. Note that the identification of I as a transient intermediate means that it is not measurably populated at equilibrium; the equilibrium unfolding process would still follow the behavior expected for a simple two-state model. 5 Perturbations of this system from equilibrium result in two relaxation times.l° With the above restrictions on the rate constants, these relaxation times can be expressed in terms of the microscopic rate constants as follows: q'falt = k2 + k_2 (4) 'rsl-olw = kl + k_l[(kE/(k_2 + k2)] (5) alternatively, 7"sl-olw = kl +
k-l[(1/(1
+ KIU)]
(6)
where Kiu = k2/k-2 = [ U ] / [ I ] is the equilibrium constant for the fast reaction between I and U. If the folding intermediate, I, has spectroscopic properties which differ from N and U, the slow relaxation time will be observed in both unfolding and refolding experiments. It is coupled to the fast reaction by the 1/(1 + Kiu) term. Under strongly unfolding conditions where kl >> k_ 1 and Kiu >> 1, ~-~olw~ kl. A similar simplification for the refolding reaction under strongly folding conditions where k_ 1 >> kl and Kiu < 1 shows that Cslow1 k_ 1. These equations provide a means of determining the microscopic rate constants, kl and k_ l, in the native and unfolded baseline regions, respectively. These data can be converted to activation free energies using Eq. (2) and the activation energy at any denaturant concentration calculated with Eq. (3). l0 C. F. Bernasconi, "Relaxation Kinetics." Academic Press, New York, 1976.
116
PROTEINS AND PEPTIDES: PRINCIPLES AND METHODS
[7]
One can also fit the slow relaxation time data in the transition region to Eq. (6). In this case, the equilibrium constant Kio should also reflect the urea dependence of the I ,~ U reaction: . • ~H20
Kit; = exp[-[/xL/itj
+ Aio[denaturant])/RT]
(7)
For the two-state equilibrium model, Kio is greater than unity in the transition region, that is, [U] >> [I]. The contribution of this term to the observed relaxation time is relatively small, and one obtains rather poor estimates of its value from the nonlinear least-squares fits. This procedure does have the advantage of using a greater portion of the data set for fitting and of avoiding the possibility of a change in the nature of the rate-limiting step from folding to isomerization in the native baseline region• For a number of proteins, the dependence of the slow relaxation time on the denaturant concentration under strongly folding conditions changes markedly from its dependence in the transition zone. n-13 The denaturantindependent relaxation times observed for folding under these conditions have been suggested to reflect proline isomerization.14 With the rate constants for the interconversion of N and I in hand, the related equilibrium constant, KNI = [I]/[N], at any urea concentration can be calculated from KNI = kl/k_l. The free energy difference between the native and intermediate conformations is then determined from AGNI = - R T In KNI. Equivalently, A G N I = AGI - AG*_1. One can also position the transition state energy for the N ~ I reaction on the reaction coordinate diagram relative to the energy of the native conformation from the value of the activation free energy of the unfolding reaction, namely, AGI. Thus, kinetic studies on the rate-limiting step in this three-state folding reaction permit the measurement of the free energy of the transition state and intermediate relative to that of the native conformation. The fast relaxation time can only be detected in refolding experiments if the free energy of the intermediate is less than that of the unfolded form under these conditions. If the free energy of the intermediate was always higher than that of the unfolded form, the intermediate would never become sufficiently populated to contribute to the observed signal. For the case in which the intermediate is not significantly populated in the transition zone (two-state equilibrium model), one would expect to detect the fast phase only under strongly refolding conditions. In this instance, 'rf~slt = k _ 2 . The activation free energy between the unfolded form and the transition state leading to the intermediate can be obtained at any urea n C. R. M a t t h e w s a n d M. M. Crisanti, Biochemistry 20, 784 (1981). 12 M. R. Hurle, C. B. M a r k s , P. A. K o s e n , S. A n d e r s o n , and I. D. K u n t z , Biochemistry 29, 4410 (1990). 13 R. F. Kelley and E. Stellwagen, Biochemistry 23, 5095 (5095). 14 j. F. Brandts, H. R. H a l v o r s e n , and M. B r e n n a n , Biochemistry 14, 4953 (1975).
[7l
MUTATIONAL ANALYSIS OF PROTEIN FOLDING MECHANISMS
117
concentration as described above for the slow phase. In this case, the energy of the transition state is measured relative to that of the unfolded form. The combination of the equilibrium and kinetic data allows one to calculate the relative energies of all these species in the above threestate mechanism. Hypothetical reaction coordinate diagrams under three different conditions are shown in Fig. 1. Figure 1A reflects the relative energies of these species in the absence of the perturbation required to shift the equilibrium between these species, that is, chemical denaturant. The reaction coordinate diagram at the midpoint of the transition is shown in Fig. lB. The energies of the native and unfolded forms are equal whereas that of the intermediate must be higher to satisfy the requirements of the two-state equilibrium model. Figure 1C summarizes the conditions which favor the unfolded form. Applications
Reaction Coordinate Diagram for Wild-Type Dihydrofolate Reductase As an example, this analysis can be applied to the urea-induced unfolding of wild-type dihydrofolate reductase (DHFR) from Escherichia coli. The equilibrium unfolding reaction follows a two-state model in which the free energy of the native conformation is 5.9 kcal/mol lower than that of the unfolded form at pH 7.8, 15°) 5 Kinetic studies show that the reaction is actually more complex; multiple phases are observed in both unfolding and refolding. The two phases detected in unfolding have counterparts which are also seen in refolding studies. Additional faster phases are also detected in refolding. A semilog plot of the dependence of these relaxation times on the final urea concentration is shown in Fig. 2. The inverted V or chevron shape observed for the two slowest reactions, z i and T2, is characteristic of folding reactions and reflects the kinetic consequences of varying the denaturant concentration. 5The fact that there are two such reactions for D~£FR has been interpreted in terms of a folding model in which there are two native conformations in slow equilibrium that unfold and refold by parallel pathways. 15 The TI and z2 phases are presumed to reflect the interconversion of these native forms with a pair of unstable intermediates. These intermediates are then linked to a corresponding pair of unfolded forms by a pair of much faster reactions, which have similar relaxation times. These later reactions are designated by their relaxation times as the z 5 folding phases (Fig. 2). The unfolded forms equilibrate with two additional unfolded forms, resulting in a manifold 15 N. A. T o u c h e t t e , K. M. Perry, and C. R. M a t t h e w s ,
Biochemistry 25,
5445 (1986).
118
PROTEINS
AND
PEPTIDES:
PRINCIPLES
AND
TS 1 I-'N I t
TS 2 I I
I I I I f I I I I I I
t I I t I t I I I
m
Z m m m
I t t t I I t t
t m
U
!
m
N
TS 1 I
t
I
t
I # I
~e
B
I I
Z
T S2
t i t
r"N J t
I t I I
I f I f
I
..~
t
t J I J
I
t t
J
I I t
][
t
N
U
TS r...~ 1 r i
~e
|
i t
i i
i
t
I f
| t
I r I
Z I
m
N
TS 2 r-'t I t I I I
! I t t t
I I I I
I
1 t L./ !
I I I t I I I t 1
i ! !
!-_ u
REACTION
COORDINATE
METHODS
[7]
[7]
MUTATIONAL ANALYSIS OF PROTEIN FOLDING MECHANISMS I
I
I
I 2
I 3
119
I
I
I
I
I
I 4
I 5
I 6
I 7
I 8
1000
100
g
u~ v ¢J
10
n-
Y _.....I ~5
0.1 0
I 1
9
[UREA], M
FIG. 2. Relaxation times as a function of urea concentration for the wild-type DHFR in 10 mM phosphate, 0.2 mM EDTA, 1 mM 2-mercaptoethanol, pH 7.8, and 15°. 15Circles and squares correspond to manual mixing data; all other symbols correspond to stopped-flow measurements. Lines are drawn to aid the eye.
FIG. 1. Reaction coordinate diagrams for folding reactions which involve a native conformation, N, a transient intermediate, I, and an unfolded form, U. TS~ is the transition state for the rate-limiting interconversion of N and I; TS2 is that for the rapid interconversion of I and U. (A) The reaction coordinate diagram under strongly refolding conditions. (B) The reaction coordinate diagram at the midpoint of the transition. (C) The reaction coordinate diagram under strongly unfolding conditions.
120
PROTEINS AND PEPTIDES" PRINCIPLES AND METHODS
[7]
of four species which are stable under unfolding conditions and which interconvert slowly. These later two forms are proposed to give rise to the '/'3 and ¢4 refolding phases. According to this model, folding through these channels is faster than interconversion between the channels. Proline isomerization is a potential explanation for the parallel folding pathways. 16-18 When we apply the above analysis to the data for the Zl and T2 slow folding phases in DHFR, we can obtain estimates of the free energies of the intermediates and transition states relative to the stable species, namely, the native and unfolded forms in our model. These results can be combined with the equilibrium data and displayed in a reaction coordinate diagram (Fig. 3). The significance of this diagram is that, in addition to being a summary of the properties of the folding reaction for the wild-type protein, it also serves as a basis for comparison of the effects of amino acid replacements on the folding reaction. By noting the species whose energies are altered when the mutation is introduced, one can determine the point in the folding reaction where that particular side chain becomes involved. In other words, one can probe the involvement of the relevant side chain in the tertiary structure of various intermediates and transition states along the folding pathway. Reaction Coordinate Diagram for L28R Mutation in Helix B To demonstrate both the power and the limitations of the mutagenic analysis, the effects of an amino acid replacement at position 28 on the equilibrium and kinetic folding data for D H F R are shown in Fig. 4. The
16 T. Kiefhaber, H.-P. Grunert, U. Hahn, and F. X. Schmid, Biochemistry 29, 6475 (1990). i7 T. B. White, P. B. Berget, and B. T. Nail, Biochemistry 26, 4358 (1987). is Frieden has proposed an alternative, sequential folding model in which the ~'s, z4, and ~'3 reactions precede, in that order, the T2 reaction. In this model, the rate-limiting step in folding still involves the formation of the native conformation from a transient intermediate via the z 2 reaction. Presumably the slowest reaction, ~-i, was not observed because its long relaxation time and small amplitude make it difficult to detect by stopped-flow techniques. Although this study did not examine the unfolding reaction, our results clearly show ¢2 reaction as a rate-limiting step in unfolding as well. In this case, the microscopic rate constants required for the free energy analysis can still be obtained from the inverse of the ¢2 relaxation time in the native and unfolded baseline regions. The faster folding reactions cannot be treated in the simple fashion described above. See C. Frieden, Proc. Natl. Acad. Sci. U.S.A. 87, 4413 (1990). 19 p. A. Jennings, S. M. Saalau-Bethell, J. J. Onuffer, K. M. Perry, E. E. Howell, and C. R . . Matthews, manuscript in preparation.
[7]
MUTATIONAL
ANALYSIS
15
OF PROTEIN
FOLDING
TSI ( N I / I 1) // i I I /
10
o
'
i~ TSI ( N 2 / 1 2 } t
~1 tl
0
it II
H
H
ii
,',
~t
'ti II
o It
~
it
','
f
;I
5;
I
I I
I l
I 1 ~I
N,
i
I
, :
)r
/,
-5
I I It I tl
P" if
H
~t t TS2 ( U 2 / I 2) )~ , t ~t
i t I I' i J i
,,~
/;
121
aroma,,t TS 2 ( U I / I 1) a ,J ;' , t
t. II
H //
MECHANISMS
:
u2
f
2
N2
-10 REACTION COORDINATE FIG. 3. Reaction coordinate diagram for the species involved in the ~'l and ¢2 channels of the folding model proposed for wild-type DHFR. The rate-limiting step in both channels is the interconversion of the N and I forms.
replacement of leucine by arginine in an active-site helix causes an increase in the midpoint and slope of the equilibrium unfolding transition (Fig. 4A). These changes correspond to an increase in stability of 1.7 --- 0.4 kcal/ mol. Kinetic studies reveal that both the ~'1 and z2 relaxation times for the unfolding of L28R increase by a factor of 3 and have nearly the same urea dependence (slopes) as those for wild-type DHFR. In refolding, the 71 relaxation time is virtually identical with that for wild-type protein. The r2 refolding relaxation time has a steeper slope; however, the magnitude of the relaxation time in the native baseline region (0 to 2 M urea) is nearly identical to that for the wild-type protein. The fit of these data in the native and unfolded baseline regions with Eqs. (2) and (3) yields estimates of the activation free energies for the rate-limiting steps in the absence of denaturant. The corresponding reaction coordinate diagram is shown in
122
PROTEINS
1.1
I
AND PEPTIDES"
I
I
I
I
PRINCIPLES
I
!
I
AND METHODS
I
I
I
[7]
I
I
A
I
I
o
0.9
~
O
O
o
/ , 0.7
~
g
_
o.
u.~0.5
-
0.3
~
-
0.1
_
-0.1
,
0
I
,
1
I
2
i
I
3
i
I
4
=
I
=
5
I
6
,
I
7
,
8
[UREA],M Fxc.4. (A)Apparentfractionofunfoldedprotein,Fapp, asafunctionofureaconcentration for the wild-type and L28R DHFR proteins at pH 7.8 and 15°. 20 Lines represent computer fits to the data using a two-state model as described previously. 15(B) rl and r2 relaxation times as a function of urea concentration for the L28R mutant protein. Filled symbols correspond to unfolding relaxation times and open symbols to refolding relaxation times. The dashed lines are linear fits to the unfolding and refolding baseline region data as described in the text. The solid lines are fits to the wild-type data. Fig. 5. F o r clarity, o n l y the d i a g r a m s for the m a j o r ~'z reactions in the wildt y p e and m u t a n t proteins are s h o w n . T h e results f o r the ~'1 p h a s e are similar. B y aligning the e n e r g y o f the u n f o l d e d f o r m s for the wild-type and m u t a n t proteins, it c a n be seen that the native, transition state, and interm e d i a t e f o r m s o f the L 2 8 R m u t a n t a p p e a r to be stabilized with r e s p e c t to the u n f o l d e d protein. T h e increases in stability c a n be calculated with the following equations: A A t,-2H20
H20 • H20 ,~,-,NU = AGNu (wild type) - AGNu (mutant)
AAc~o
(8)
-20 (wild • type) - AGTsl -2o (mutant) = AGTsl - - h e ° -- A G~H2°](wild type) = "t~(-/NU rA~n2 ° AG~n2°](mutant) - - tt,,AVN U - -
(9)
[7]
MUTATIONAL
I
B
ANALYSIS
I
OF PROTEIN
I
FOLDING
I
I
~e
123
MECHANISMS
I
I
I
~O
1000 "1
Fi[] A 0
G)
E
'= e-
100
.o_
f
O::
-
H
10 0
-
L28R
--
m W l "
I 1
I 2
I 3
I 4
I 5
I 6
I 7
I 8
[UREA] U F I G . 4. AA ~H20
~(JIU
H20
(continued)
"
H20
=
AGIu (wild type) - AGIo (mutant)
=
rA~H20 L~uNU
--
FA/'~H20
(AG~ H2°
A G*_Hl:°)](wild type)
- L,~'JN, -- (AG~ - AG~_0](mutant)
(10)
Insertion o f the appropriate values for the wild type and the L28R mutant • H20 H20 gives AAGN, -- - 1.6 -+ 0.4 kcal/mol, AAGTs I = - 1.3 +- 0.4 kcal/mol, and A A G ~ ° = - 1.0 +- 0.4 kcal/mol for the z2 channel. In the convention adopted here, a negative value for AAG corresponds to an increase in stability due to the mutation. Clearly, the side chain at position 28 is involved in structure formation as early as the intermediate in the folding pathway. Fitting all the relaxation time data to Eq. (6) yields the same values for the change in free energy for the native, transition state, and intermediate within experimental error. The question o f differential effects of the mutation on the native,
124
PROTEINS A N D PEPTIDES: PRINCIPLES AND METHODS
[7]
20
15
WT WT J , ~
i
t i
L28R
x
o E
h
10
¢
~
5
t
l
tI
ii
i s
r
t 0 11 s 0 L L
Ii
ar
r
i, b
~e
' #
~ l
,, II II ii
i
if
ti I
I ;
II #i
iI i I
I I
lI
iI
l + +
l# ]J
0 0
+
...,,--.r
!7
i
t
i i i , i a ,
II
h rs
-5
t
i
~t o
,,I I ';
0
i
e o
i
b b ij
Z
,
i i
r
~e
o,
h i
~iim
,WT .x..--. L28R
L2SR
L28R
-10 N
TS1 REACTION
I
TS2
U
COORDINATE
FIG. 5. Reaction coordinate diagram for the major ¢2 folding phase of wild-type and L 2 8 R m u t a n t D H F R . The relative energy of TS 2 must be determined from stopped-flow measurements and is not available for the L28R mutant protein.
transition state, and intermediate energies is obscured by the magnitude of the errors. To address this issue, the unfolding and refolding relaxation times for the wild-type and mutant proteins can be compared at urea concentrations in the appropriate baseline regions. This procedure avoids the errors introduced by extrapolation. For example, the unfolding activation energy of the 'rE phase for the L28R mutant protein is 0.5 -+ 0.3 kcal/mol greater than wild-type DHFR at 6 M urea. For refolding to 1 M urea the activation energies are not significantly perturbed; AG ~_I ( W T ) = 19.2 -+ 0.2 and AG~_l(L28R) = 19.0 -+ 0.2. Therefore, the replacement of Leu-28 by arginine stabilizes the native conformation by 0.5 kcal/mol relative to the transition state and the intermediate. The structural changes which give rise to this differential increase in stability must occur after the transition state is passed in refolding. Similar results for the D27N and F31V mutant DHFR proteins suggest that the rearrangement of the segment containing these residues
17]
MUTATIONAL ANALYSIS OF PROTEIN FOLDING MECHANISMS
125
and perhaps the entire B helix (residues 24-35) on the fl sheet is one of the last events in folding. 2° Comparison of the activation energies in the native and unfolded baseline regions seems reasonable in cases where the urea dependences of the relaxation times are similar, namely, where the slopes of the log ~- versus [denaturant] plots are parallel. If the slopes are not parallel one should use this alternative approach with caution. Large changes in slope can result in either increases or decreases in the rate constant depending on the urea concentration selected as a reference state. In such cases, the best reference state is the absence of denaturant. Following this prescription for the ~'2 refolding relaxation time for the L28R mutant protein, where an increase in slope is observed (Fig. 4B), one would still conclude that refolding is not significantly perturbed. Another concern with the mutagenic analysis is whether the observed effects are replacement specific. The best way to answer this question is to make additional replacements at the same site and then to look for common effects on the folding reaction. As an example, consider the results of a series of replacements at position 75 in DHFR. 6 The isopropyl side chain of Val-75 in the wild-type protein is buried in a large hydrophobic cluster. Replacement with smaller neutral residues (Ala or Cys) had no effect on either equilibrium or kinetic properties. Mutations involving a larger side chain (Ile), a charge (Arg), or which have polar substituents (Ser, His, and Tyr) all lowered the stability of the mutant protein with respect to wild-type D H F R by a similar amount, 1.9-2.8 kcal/mol. Kinetic studies of the V75H, V75I, and V75Y mutants showed a decrease in the unfolding relaxation time and an increase in the refolding relaxation time of the major z2 phase. These qualitatively similar, but quantitatively different, results imply that the side chain at position 75 can participate in the rate-limiting step of the folding reaction. If only the V75A or V75C mutants had been examined, one would have concluded that this position does not play a role in the rate-limiting step in folding. Thus, it seems prudent to make at least three or four replacements at an individual site to obtain a reliable interpretation. Conclusion The current results from our laboratory and others on the mutagenic analysis of protein folding reactions provide strong support for the view 2o K. M. Perry, J. J. Onuffer, N. J. Touchette, C. S. Herndon, M. S. Gittelman, C. R. Matthews, J. T. Chen, R. J. Mayer, K. Taira, S. J. Benkovic, E. E. Howell, and J. Kraut, Biochemistry 26, 2674 (1987).
126
PROTEINS AND PEPTIDES: PRINCIPLES AND METHODS
[8]
that this approach will dramatically increase our understanding of the development of tertiary structure during folding.l-3'5'2°-23 When this information is combined with high-resolution information on the formation of secondary structure from NMR spectroscopy, 24-26 significant advances will be made in deciphering the "folding c o d e . " 21D. P. Goldenberg,Annu. Rev. Biophys. Biophys. Chem. 17, 481 (1988). 22T. Alber, Annu. Rev. Biochem. 58, 765 (1989). 23D. P. Goldenberg,R. W. Frieden, J. A. Haak, and T. B. Morrison,Nature (London) 338, 127 (1989). 24H. Roder, G. A. Elove, and S. W. Englander, Nature (London) 335, 700 (1988). 25j. B. Udgaonkar and R. L. Baldwin, Nature (London) 335, 694 (1988). 26M. Bycroft,A. Matouschek, J. T. Kellis, L. Serrano, and A. R. Fersht, Nature (London) 346, 488 (1990).
[8] C l e f t s a n d B i n d i n g S i t e s in P r o t e i n R e c e p t o r s B y RICHARD A. LEWIS
Introduction An enzyme can increase the rate of a biochemical reaction by bringing the reactants together and holding them in the correct orientation. This will increase the probability of a successful collision between the reacting groups. A membrane-bound receptor can promote interaction between its own functional groups and the ligand in a similar way. The host protein should make multiple contacts with the substrate in order to maximize efficiency and specificity. X-Ray studies of the structures of proteins have shown that most binding sites are found in clefts, holes, or in the boundaries between protein domains, where the ligand can be held firmly: studies of receptor-ligand complexes show that, after binding, the ligand is often completely surrounded by the receptor and hence immobilized for reaction. There is a price to be paid for this increase in the reaction rate. The freezing of a mobile molecule into a semicrystalline state inside the receptor binding site requires the loss of entropy; this must be balanced by the utilization of binding energy between the ligand and the receptor.~ A ligand can gain more binding energy from a complex with its host when it is surrounded in space by receptor atom groups that display complementary w. P. Jenks, in "Chemical Recognition in Biology" (F. Chapeville and A.-L. Haenni, eds.), p. 3. Springer-Vedag, Berlin, 1980. METHODS IN ENZYMOLOGY, VOL. 202
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
MUTATIONAL
ANALYSIS
OF PROTEIN
FOLDING
MECHANISMS
113
[7] M u t a t i o n a l A n a l y s i s o f P r o t e i n F o l d i n g M e c h a n i s m s By PATRICIA A. JENNINGS, SUSANNE M. SAALAU-BETHELL, BRYAN E . FINN, XIAOWU CHEN, a n d C. ROBERT MATTHEWS Introduction
Site-directed mutagenesis has proved to be a powerful technique for probing the relationship between the amino acid sequence of a protein and its folding, stability, structure, and function.l-4 In a previous contribution to this series, we discussed the application of mutagenesis to the investigation of the mechanism by which the sequence directs the rapid and efficient folding to the unique native conformation, that is, the mechanism of protein folding. 5 Although the data available at that time were certainly encouraging, it was left for future studies to determine the ultimate utility of the mutagenic approach to the solution of one of the most vexing problems in biochemistry. An increasing number of contributions to the literature has indicated that the initial promise was real. There are now a number of examples in which amino acid replacements in globular proteins have provided new insight into the molecular events involved in rate-limiting steps in folding reactions. 6-8 The purpose of this chapter is (1) to extend the approach to a three-state folding model involving a transient intermediate, (2) to demonstrate how the integration of kinetic and equilibrium folding data can provide a more comprehensive understanding of the effects of mutations on the folding mechanism, and (3) to summarize some of the recent findings from our laboratory on the folding of dihydrofolate reductase using this approach. 1 L. M. Gierash and J. King (eds.), "Protein Folding: Deciphering the Second Half of the Genetic Code." AAAS Press, Washington, D.C., 1990. 2 D. L. Oxender (ed.), "Protein Structure, Folding and Design." Alan R. Liss, New York, 1986. 3 C. S. Craik, R. Fletterick, C. R. Matthews, and J. Wells (eds.), "Protein and Pharmaceutical Engineering." Wiley-Liss, New York, 1990. 4 j. j. Villafranca (ed.), "Current Research in Protein Chemistry: Techniques, Structure, and Function." Academic Press, New York, 1990. 5 C. R. Matthews, this series, Vol. 154, p. 498. 6 E. P. Garvey and C. R. Matthews, Biochemistry 28, 2083 (1989). 7 A. Matouscheck, J. T. Kellis, L. Serrano, M. Bycroft, and A. R. Fersht, Nature (London) 346, 440 (1990). 8 N. B. Tweedy, M. R. Hurle, B. A. Chrunyk, and C. R. Matthews, Biochemistry 29, 1539 (1990).
METHODS IN ENZYMOLOGY, VOL. 202
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
114
PROTEINS AND PEPTIDES: PRINCIPLES AND METHODS
[7]
Methods A detailed description of the experimental techniques used to obtain both equilibrium and kinetic data on the reversible folding reaction can be found in our previous chapter. 5 To summarize briefly, one can determine the free energy difference, AGNu ,9a between the native and unfolded states in a two-state equilibrium reaction from chemical denaturation experiments. Formally, AGNu = - R T In KNU, where KNU = [U]/[N]. As the concentration of denaturant increases, the equilibrium shifts to favor the unfolded form. Consequently, the value of KNU increases while that for AGNu decreases. The value of AGNu at any denaturant concentration can be calculated from A ~H20
AGNu = ,-,t-/su + ANu[denaturant]
(1)
A ~H20
where ,~t/NU is the free energy difference in the absence of denaturant and ANU is a parameter which reflects the cooperativity of the transition (Asu < 0 in this formalism). From the kinetic data, one can calculate the activation free energy, AG t, which represents the difference in free energy between one of the species in the kinetic mechanism and the transition state separating it from other species in the reaction. The relevant equation is AG* = - R T ln[kh/kBT]
(2)
where k is the microscopic rate constant, h, kB, and R are the Planck, Boltzmann, and universal gas constants, respectively, and Tis the absolute temperature. The activation free energies for folding reactions have been observed to depend on the denaturant concentration according to Eq. (3)5,9 AG* = AG*H2° + A*[denaturant]
(3)
where AG*H2° and A* have interpretations similar to their equilibrium counterparts. For unfolding, A*u < 0 and for refolding A*R > 0. Analysis The free energy data from both equilibrium and kinetic studies of the folding reactions of mutant and wild-type proteins can be summarized in a reaction coordinate diagram. In this diagram, the free energies of the various species along the folding pathway are plotted as a function of the reaction coordinate. This coordinate does not correspond to any particular 9 B. Chen, W. A. Baase, and J. A. Schellman, Biochemistry 28, 691 (1989). 9~ The free energy difference is that between the native and unfolded forms of the protein at standard state conditions. This stipulation is typically denoted by a superscript zero which has been deleted in this and other equations to simplify the notation.
[7]
MUTATIONAL ANALYSIS OF PROTEIN FOLDING MECHANISMS
115
molecular dimension but rather reflects the progress of the folding reaction between the two end states, the native and the unfolded conformations. In our previous chapter, 5 we addressed the case of a two-state folding mechanism which involved a native, N, and an unfolded, U, conformation. We now extend the discussion to the case in which a transient intermediate, I, is also present: N.
kl " k_ I slow
I
k2 "U "k_2 fast
kl and k z represent microscopic rate constants for the unfolding reactions, and k_l and k_ z are rate constants for the refolding reactions. Let us further stipulate that the interconversion of the native and intermediate forms is rate limiting: k2, k_2 >>k~, k_ 1. This situation has been observed for a number of proteins and appears to be a common (if somewhat simplified) folding mechanism. Note that the identification of I as a transient intermediate means that it is not measurably populated at equilibrium; the equilibrium unfolding process would still follow the behavior expected for a simple two-state model. 5 Perturbations of this system from equilibrium result in two relaxation times.l° With the above restrictions on the rate constants, these relaxation times can be expressed in terms of the microscopic rate constants as follows: q'falt = k2 + k_2 (4) 'rsl-olw = kl + k_l[(kE/(k_2 + k2)] (5) alternatively, 7"sl-olw = kl +
k-l[(1/(1
+ KIU)]
(6)
where Kiu = k2/k-2 = [ U ] / [ I ] is the equilibrium constant for the fast reaction between I and U. If the folding intermediate, I, has spectroscopic properties which differ from N and U, the slow relaxation time will be observed in both unfolding and refolding experiments. It is coupled to the fast reaction by the 1/(1 + Kiu) term. Under strongly unfolding conditions where kl >> k_ 1 and Kiu >> 1, ~-~olw~ kl. A similar simplification for the refolding reaction under strongly folding conditions where k_ 1 >> kl and Kiu < 1 shows that Cslow1 k_ 1. These equations provide a means of determining the microscopic rate constants, kl and k_ l, in the native and unfolded baseline regions, respectively. These data can be converted to activation free energies using Eq. (2) and the activation energy at any denaturant concentration calculated with Eq. (3). l0 C. F. Bernasconi, "Relaxation Kinetics." Academic Press, New York, 1976.
116
PROTEINS AND PEPTIDES: PRINCIPLES AND METHODS
[7]
One can also fit the slow relaxation time data in the transition region to Eq. (6). In this case, the equilibrium constant Kio should also reflect the urea dependence of the I ,~ U reaction: . • ~H20
Kit; = exp[-[/xL/itj
+ Aio[denaturant])/RT]
(7)
For the two-state equilibrium model, Kio is greater than unity in the transition region, that is, [U] >> [I]. The contribution of this term to the observed relaxation time is relatively small, and one obtains rather poor estimates of its value from the nonlinear least-squares fits. This procedure does have the advantage of using a greater portion of the data set for fitting and of avoiding the possibility of a change in the nature of the rate-limiting step from folding to isomerization in the native baseline region• For a number of proteins, the dependence of the slow relaxation time on the denaturant concentration under strongly folding conditions changes markedly from its dependence in the transition zone. n-13 The denaturantindependent relaxation times observed for folding under these conditions have been suggested to reflect proline isomerization.14 With the rate constants for the interconversion of N and I in hand, the related equilibrium constant, KNI = [I]/[N], at any urea concentration can be calculated from KNI = kl/k_l. The free energy difference between the native and intermediate conformations is then determined from AGNI = - R T In KNI. Equivalently, A G N I = AGI - AG*_1. One can also position the transition state energy for the N ~ I reaction on the reaction coordinate diagram relative to the energy of the native conformation from the value of the activation free energy of the unfolding reaction, namely, AGI. Thus, kinetic studies on the rate-limiting step in this three-state folding reaction permit the measurement of the free energy of the transition state and intermediate relative to that of the native conformation. The fast relaxation time can only be detected in refolding experiments if the free energy of the intermediate is less than that of the unfolded form under these conditions. If the free energy of the intermediate was always higher than that of the unfolded form, the intermediate would never become sufficiently populated to contribute to the observed signal. For the case in which the intermediate is not significantly populated in the transition zone (two-state equilibrium model), one would expect to detect the fast phase only under strongly refolding conditions. In this instance, 'rf~slt = k _ 2 . The activation free energy between the unfolded form and the transition state leading to the intermediate can be obtained at any urea n C. R. M a t t h e w s a n d M. M. Crisanti, Biochemistry 20, 784 (1981). 12 M. R. Hurle, C. B. M a r k s , P. A. K o s e n , S. A n d e r s o n , and I. D. K u n t z , Biochemistry 29, 4410 (1990). 13 R. F. Kelley and E. Stellwagen, Biochemistry 23, 5095 (5095). 14 j. F. Brandts, H. R. H a l v o r s e n , and M. B r e n n a n , Biochemistry 14, 4953 (1975).
[7l
MUTATIONAL ANALYSIS OF PROTEIN FOLDING MECHANISMS
117
concentration as described above for the slow phase. In this case, the energy of the transition state is measured relative to that of the unfolded form. The combination of the equilibrium and kinetic data allows one to calculate the relative energies of all these species in the above threestate mechanism. Hypothetical reaction coordinate diagrams under three different conditions are shown in Fig. 1. Figure 1A reflects the relative energies of these species in the absence of the perturbation required to shift the equilibrium between these species, that is, chemical denaturant. The reaction coordinate diagram at the midpoint of the transition is shown in Fig. lB. The energies of the native and unfolded forms are equal whereas that of the intermediate must be higher to satisfy the requirements of the two-state equilibrium model. Figure 1C summarizes the conditions which favor the unfolded form. Applications
Reaction Coordinate Diagram for Wild-Type Dihydrofolate Reductase As an example, this analysis can be applied to the urea-induced unfolding of wild-type dihydrofolate reductase (DHFR) from Escherichia coli. The equilibrium unfolding reaction follows a two-state model in which the free energy of the native conformation is 5.9 kcal/mol lower than that of the unfolded form at pH 7.8, 15°) 5 Kinetic studies show that the reaction is actually more complex; multiple phases are observed in both unfolding and refolding. The two phases detected in unfolding have counterparts which are also seen in refolding studies. Additional faster phases are also detected in refolding. A semilog plot of the dependence of these relaxation times on the final urea concentration is shown in Fig. 2. The inverted V or chevron shape observed for the two slowest reactions, z i and T2, is characteristic of folding reactions and reflects the kinetic consequences of varying the denaturant concentration. 5The fact that there are two such reactions for D~£FR has been interpreted in terms of a folding model in which there are two native conformations in slow equilibrium that unfold and refold by parallel pathways. 15 The TI and z2 phases are presumed to reflect the interconversion of these native forms with a pair of unstable intermediates. These intermediates are then linked to a corresponding pair of unfolded forms by a pair of much faster reactions, which have similar relaxation times. These later reactions are designated by their relaxation times as the z 5 folding phases (Fig. 2). The unfolded forms equilibrate with two additional unfolded forms, resulting in a manifold 15 N. A. T o u c h e t t e , K. M. Perry, and C. R. M a t t h e w s ,
Biochemistry 25,
5445 (1986).
118
PROTEINS
AND
PEPTIDES:
PRINCIPLES
AND
TS 1 I-'N I t
TS 2 I I
I I I I f I I I I I I
t I I t I t I I I
m
Z m m m
I t t t I I t t
t m
U
!
m
N
TS 1 I
t
I
t
I # I
~e
B
I I
Z
T S2
t i t
r"N J t
I t I I
I f I f
I
..~
t
t J I J
I
t t
J
I I t
][
t
N
U
TS r...~ 1 r i
~e
|
i t
i i
i
t
I f
| t
I r I
Z I
m
N
TS 2 r-'t I t I I I
! I t t t
I I I I
I
1 t L./ !
I I I t I I I t 1
i ! !
!-_ u
REACTION
COORDINATE
METHODS
[7]
[7]
MUTATIONAL ANALYSIS OF PROTEIN FOLDING MECHANISMS I
I
I
I 2
I 3
119
I
I
I
I
I
I 4
I 5
I 6
I 7
I 8
1000
100
g
u~ v ¢J
10
n-
Y _.....I ~5
0.1 0
I 1
9
[UREA], M
FIG. 2. Relaxation times as a function of urea concentration for the wild-type DHFR in 10 mM phosphate, 0.2 mM EDTA, 1 mM 2-mercaptoethanol, pH 7.8, and 15°. 15Circles and squares correspond to manual mixing data; all other symbols correspond to stopped-flow measurements. Lines are drawn to aid the eye.
FIG. 1. Reaction coordinate diagrams for folding reactions which involve a native conformation, N, a transient intermediate, I, and an unfolded form, U. TS~ is the transition state for the rate-limiting interconversion of N and I; TS2 is that for the rapid interconversion of I and U. (A) The reaction coordinate diagram under strongly refolding conditions. (B) The reaction coordinate diagram at the midpoint of the transition. (C) The reaction coordinate diagram under strongly unfolding conditions.
120
PROTEINS AND PEPTIDES" PRINCIPLES AND METHODS
[7]
of four species which are stable under unfolding conditions and which interconvert slowly. These later two forms are proposed to give rise to the '/'3 and ¢4 refolding phases. According to this model, folding through these channels is faster than interconversion between the channels. Proline isomerization is a potential explanation for the parallel folding pathways. 16-18 When we apply the above analysis to the data for the Zl and T2 slow folding phases in DHFR, we can obtain estimates of the free energies of the intermediates and transition states relative to the stable species, namely, the native and unfolded forms in our model. These results can be combined with the equilibrium data and displayed in a reaction coordinate diagram (Fig. 3). The significance of this diagram is that, in addition to being a summary of the properties of the folding reaction for the wild-type protein, it also serves as a basis for comparison of the effects of amino acid replacements on the folding reaction. By noting the species whose energies are altered when the mutation is introduced, one can determine the point in the folding reaction where that particular side chain becomes involved. In other words, one can probe the involvement of the relevant side chain in the tertiary structure of various intermediates and transition states along the folding pathway. Reaction Coordinate Diagram for L28R Mutation in Helix B To demonstrate both the power and the limitations of the mutagenic analysis, the effects of an amino acid replacement at position 28 on the equilibrium and kinetic folding data for D H F R are shown in Fig. 4. The
16 T. Kiefhaber, H.-P. Grunert, U. Hahn, and F. X. Schmid, Biochemistry 29, 6475 (1990). i7 T. B. White, P. B. Berget, and B. T. Nail, Biochemistry 26, 4358 (1987). is Frieden has proposed an alternative, sequential folding model in which the ~'s, z4, and ~'3 reactions precede, in that order, the T2 reaction. In this model, the rate-limiting step in folding still involves the formation of the native conformation from a transient intermediate via the z 2 reaction. Presumably the slowest reaction, ~-i, was not observed because its long relaxation time and small amplitude make it difficult to detect by stopped-flow techniques. Although this study did not examine the unfolding reaction, our results clearly show ¢2 reaction as a rate-limiting step in unfolding as well. In this case, the microscopic rate constants required for the free energy analysis can still be obtained from the inverse of the ¢2 relaxation time in the native and unfolded baseline regions. The faster folding reactions cannot be treated in the simple fashion described above. See C. Frieden, Proc. Natl. Acad. Sci. U.S.A. 87, 4413 (1990). 19 p. A. Jennings, S. M. Saalau-Bethell, J. J. Onuffer, K. M. Perry, E. E. Howell, and C. R . . Matthews, manuscript in preparation.
[7]
MUTATIONAL
ANALYSIS
15
OF PROTEIN
FOLDING
TSI ( N I / I 1) // i I I /
10
o
'
i~ TSI ( N 2 / 1 2 } t
~1 tl
0
it II
H
H
ii
,',
~t
'ti II
o It
~
it
','
f
;I
5;
I
I I
I l
I 1 ~I
N,
i
I
, :
)r
/,
-5
I I It I tl
P" if
H
~t t TS2 ( U 2 / I 2) )~ , t ~t
i t I I' i J i
,,~
/;
121
aroma,,t TS 2 ( U I / I 1) a ,J ;' , t
t. II
H //
MECHANISMS
:
u2
f
2
N2
-10 REACTION COORDINATE FIG. 3. Reaction coordinate diagram for the species involved in the ~'l and ¢2 channels of the folding model proposed for wild-type DHFR. The rate-limiting step in both channels is the interconversion of the N and I forms.
replacement of leucine by arginine in an active-site helix causes an increase in the midpoint and slope of the equilibrium unfolding transition (Fig. 4A). These changes correspond to an increase in stability of 1.7 --- 0.4 kcal/ mol. Kinetic studies reveal that both the ~'1 and z2 relaxation times for the unfolding of L28R increase by a factor of 3 and have nearly the same urea dependence (slopes) as those for wild-type DHFR. In refolding, the 71 relaxation time is virtually identical with that for wild-type protein. The r2 refolding relaxation time has a steeper slope; however, the magnitude of the relaxation time in the native baseline region (0 to 2 M urea) is nearly identical to that for the wild-type protein. The fit of these data in the native and unfolded baseline regions with Eqs. (2) and (3) yields estimates of the activation free energies for the rate-limiting steps in the absence of denaturant. The corresponding reaction coordinate diagram is shown in
122
PROTEINS
1.1
I
AND PEPTIDES"
I
I
I
I
PRINCIPLES
I
!
I
AND METHODS
I
I
I
[7]
I
I
A
I
I
o
0.9
~
O
O
o
/ , 0.7
~
g
_
o.
u.~0.5
-
0.3
~
-
0.1
_
-0.1
,
0
I
,
1
I
2
i
I
3
i
I
4
=
I
=
5
I
6
,
I
7
,
8
[UREA],M Fxc.4. (A)Apparentfractionofunfoldedprotein,Fapp, asafunctionofureaconcentration for the wild-type and L28R DHFR proteins at pH 7.8 and 15°. 20 Lines represent computer fits to the data using a two-state model as described previously. 15(B) rl and r2 relaxation times as a function of urea concentration for the L28R mutant protein. Filled symbols correspond to unfolding relaxation times and open symbols to refolding relaxation times. The dashed lines are linear fits to the unfolding and refolding baseline region data as described in the text. The solid lines are fits to the wild-type data. Fig. 5. F o r clarity, o n l y the d i a g r a m s for the m a j o r ~'z reactions in the wildt y p e and m u t a n t proteins are s h o w n . T h e results f o r the ~'1 p h a s e are similar. B y aligning the e n e r g y o f the u n f o l d e d f o r m s for the wild-type and m u t a n t proteins, it c a n be seen that the native, transition state, and interm e d i a t e f o r m s o f the L 2 8 R m u t a n t a p p e a r to be stabilized with r e s p e c t to the u n f o l d e d protein. T h e increases in stability c a n be calculated with the following equations: A A t,-2H20
H20 • H20 ,~,-,NU = AGNu (wild type) - AGNu (mutant)
AAc~o
(8)
-20 (wild • type) - AGTsl -2o (mutant) = AGTsl - - h e ° -- A G~H2°](wild type) = "t~(-/NU rA~n2 ° AG~n2°](mutant) - - tt,,AVN U - -
(9)
[7]
MUTATIONAL
I
B
ANALYSIS
I
OF PROTEIN
I
FOLDING
I
I
~e
123
MECHANISMS
I
I
I
~O
1000 "1
Fi[] A 0
G)
E
'= e-
100
.o_
f
O::
-
H
10 0
-
L28R
--
m W l "
I 1
I 2
I 3
I 4
I 5
I 6
I 7
I 8
[UREA] U F I G . 4. AA ~H20
~(JIU
H20
(continued)
"
H20
=
AGIu (wild type) - AGIo (mutant)
=
rA~H20 L~uNU
--
FA/'~H20
(AG~ H2°
A G*_Hl:°)](wild type)
- L,~'JN, -- (AG~ - AG~_0](mutant)
(10)
Insertion o f the appropriate values for the wild type and the L28R mutant • H20 H20 gives AAGN, -- - 1.6 -+ 0.4 kcal/mol, AAGTs I = - 1.3 +- 0.4 kcal/mol, and A A G ~ ° = - 1.0 +- 0.4 kcal/mol for the z2 channel. In the convention adopted here, a negative value for AAG corresponds to an increase in stability due to the mutation. Clearly, the side chain at position 28 is involved in structure formation as early as the intermediate in the folding pathway. Fitting all the relaxation time data to Eq. (6) yields the same values for the change in free energy for the native, transition state, and intermediate within experimental error. The question o f differential effects of the mutation on the native,
124
PROTEINS A N D PEPTIDES: PRINCIPLES AND METHODS
[7]
20
15
WT WT J , ~
i
t i
L28R
x
o E
h
10
¢
~
5
t
l
tI
ii
i s
r
t 0 11 s 0 L L
Ii
ar
r
i, b
~e
' #
~ l
,, II II ii
i
if
ti I
I ;
II #i
iI i I
I I
lI
iI
l + +
l# ]J
0 0
+
...,,--.r
!7
i
t
i i i , i a ,
II
h rs
-5
t
i
~t o
,,I I ';
0
i
e o
i
b b ij
Z
,
i i
r
~e
o,
h i
~iim
,WT .x..--. L28R
L2SR
L28R
-10 N
TS1 REACTION
I
TS2
U
COORDINATE
FIG. 5. Reaction coordinate diagram for the major ¢2 folding phase of wild-type and L 2 8 R m u t a n t D H F R . The relative energy of TS 2 must be determined from stopped-flow measurements and is not available for the L28R mutant protein.
transition state, and intermediate energies is obscured by the magnitude of the errors. To address this issue, the unfolding and refolding relaxation times for the wild-type and mutant proteins can be compared at urea concentrations in the appropriate baseline regions. This procedure avoids the errors introduced by extrapolation. For example, the unfolding activation energy of the 'rE phase for the L28R mutant protein is 0.5 -+ 0.3 kcal/mol greater than wild-type DHFR at 6 M urea. For refolding to 1 M urea the activation energies are not significantly perturbed; AG ~_I ( W T ) = 19.2 -+ 0.2 and AG~_l(L28R) = 19.0 -+ 0.2. Therefore, the replacement of Leu-28 by arginine stabilizes the native conformation by 0.5 kcal/mol relative to the transition state and the intermediate. The structural changes which give rise to this differential increase in stability must occur after the transition state is passed in refolding. Similar results for the D27N and F31V mutant DHFR proteins suggest that the rearrangement of the segment containing these residues
17]
MUTATIONAL ANALYSIS OF PROTEIN FOLDING MECHANISMS
125
and perhaps the entire B helix (residues 24-35) on the fl sheet is one of the last events in folding. 2° Comparison of the activation energies in the native and unfolded baseline regions seems reasonable in cases where the urea dependences of the relaxation times are similar, namely, where the slopes of the log ~- versus [denaturant] plots are parallel. If the slopes are not parallel one should use this alternative approach with caution. Large changes in slope can result in either increases or decreases in the rate constant depending on the urea concentration selected as a reference state. In such cases, the best reference state is the absence of denaturant. Following this prescription for the ~'2 refolding relaxation time for the L28R mutant protein, where an increase in slope is observed (Fig. 4B), one would still conclude that refolding is not significantly perturbed. Another concern with the mutagenic analysis is whether the observed effects are replacement specific. The best way to answer this question is to make additional replacements at the same site and then to look for common effects on the folding reaction. As an example, consider the results of a series of replacements at position 75 in DHFR. 6 The isopropyl side chain of Val-75 in the wild-type protein is buried in a large hydrophobic cluster. Replacement with smaller neutral residues (Ala or Cys) had no effect on either equilibrium or kinetic properties. Mutations involving a larger side chain (Ile), a charge (Arg), or which have polar substituents (Ser, His, and Tyr) all lowered the stability of the mutant protein with respect to wild-type D H F R by a similar amount, 1.9-2.8 kcal/mol. Kinetic studies of the V75H, V75I, and V75Y mutants showed a decrease in the unfolding relaxation time and an increase in the refolding relaxation time of the major z2 phase. These qualitatively similar, but quantitatively different, results imply that the side chain at position 75 can participate in the rate-limiting step of the folding reaction. If only the V75A or V75C mutants had been examined, one would have concluded that this position does not play a role in the rate-limiting step in folding. Thus, it seems prudent to make at least three or four replacements at an individual site to obtain a reliable interpretation. Conclusion The current results from our laboratory and others on the mutagenic analysis of protein folding reactions provide strong support for the view 2o K. M. Perry, J. J. Onuffer, N. J. Touchette, C. S. Herndon, M. S. Gittelman, C. R. Matthews, J. T. Chen, R. J. Mayer, K. Taira, S. J. Benkovic, E. E. Howell, and J. Kraut, Biochemistry 26, 2674 (1987).
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PROTEINS AND PEPTIDES: PRINCIPLES AND METHODS
[8]
that this approach will dramatically increase our understanding of the development of tertiary structure during folding.l-3'5'2°-23 When this information is combined with high-resolution information on the formation of secondary structure from NMR spectroscopy, 24-26 significant advances will be made in deciphering the "folding c o d e . " 21D. P. Goldenberg,Annu. Rev. Biophys. Biophys. Chem. 17, 481 (1988). 22T. Alber, Annu. Rev. Biochem. 58, 765 (1989). 23D. P. Goldenberg,R. W. Frieden, J. A. Haak, and T. B. Morrison,Nature (London) 338, 127 (1989). 24H. Roder, G. A. Elove, and S. W. Englander, Nature (London) 335, 700 (1988). 25j. B. Udgaonkar and R. L. Baldwin, Nature (London) 335, 694 (1988). 26M. Bycroft,A. Matouschek, J. T. Kellis, L. Serrano, and A. R. Fersht, Nature (London) 346, 488 (1990).
[8] C l e f t s a n d B i n d i n g S i t e s in P r o t e i n R e c e p t o r s B y RICHARD A. LEWIS
Introduction An enzyme can increase the rate of a biochemical reaction by bringing the reactants together and holding them in the correct orientation. This will increase the probability of a successful collision between the reacting groups. A membrane-bound receptor can promote interaction between its own functional groups and the ligand in a similar way. The host protein should make multiple contacts with the substrate in order to maximize efficiency and specificity. X-Ray studies of the structures of proteins have shown that most binding sites are found in clefts, holes, or in the boundaries between protein domains, where the ligand can be held firmly: studies of receptor-ligand complexes show that, after binding, the ligand is often completely surrounded by the receptor and hence immobilized for reaction. There is a price to be paid for this increase in the reaction rate. The freezing of a mobile molecule into a semicrystalline state inside the receptor binding site requires the loss of entropy; this must be balanced by the utilization of binding energy between the ligand and the receptor.~ A ligand can gain more binding energy from a complex with its host when it is surrounded in space by receptor atom groups that display complementary w. P. Jenks, in "Chemical Recognition in Biology" (F. Chapeville and A.-L. Haenni, eds.), p. 3. Springer-Vedag, Berlin, 1980. METHODS IN ENZYMOLOGY, VOL. 202
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