Probing Denaturation by Simultaneously Monitoring Residual Enzyme Activity and Intrinsic Fluorescence An Undergraduate Biochemistry Experiment Todd P. Silverstein and Lars E. Blomberg Willamette University, Salem, OR 97301 All undergraduate biochemistry courses stress the relationship between molecular structure and function. Biological macromolecules (e.g., proteins, DNA, RNA, lipids, etc.) must be folded or packed in a specific native conformation for full activity and function. For instance, enzvmes that are easilv unfolded bv various denaturants tend to lose activity ~recipitonslya s the concentration of denaturant increases above a certain threshold value. I n snite of the imnortance of this concent. it is difficult to find undergraduate biochemistry labor&o& pmjerrs that exnlore the efyerts of foldine and unfoldine. Over the vears thk Journal has attemptegto fill this gapby presenting a few experiments that monitor the effects of denaturants on conformation ( 1 , Z ) . Although these projects allow students to monitor the process of unfolding, they do not actually follow the specific activity of the protein during the denaturation process. Accordingly, we propose here a simple experiment that uses a W-vis snectronhotometer to monitor enzvme activity and a fluorimeter to monitor enzyme intrinsic fluorescence. As the enzyme unfolds, we generally observe a close correlation between residual enzyme activity and intrinsic fluorescence
Conformation Proteins consist of individual amino acids that are covalently attached in a specific sequence that accounts for the motein's nrimarv structure. T h i s covalent oolvnentide chain folds into various tightly packed conformations that involving interactions that stabilize specific protein-folding patterns. These important intramolecular interactions include the following.
.
"A
.
disulfide bridges salt bridges hydrogen bonds hydrophobic farces Such interactions account for the levels of higher-order structure. secondary structure, which is based on local, small-scalein-
teractions tertiary strueturn, which is based on domainwide larger-
scale interactions quarternary structure, whieh is based on interactions he-
tween separate polypeptide chains Disulfide bridges, which are covalent bonds, are clearly the stroneest interaction. However.. hvdroeen bonds and " hydrophobic forces are typically the most common and the most critical in stabilizing the native conformation of a protein.
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Denaturation Recause these intramolecular interactmns are reaponslble for stabilinng a specifir native conformation of the pmtein, reagents that weaken the interactions allow the prot e i n to unfold t o a more random, high-entropy conformation. This process, which is called denaturation, is invariably associated with a loss of specific activity. One or m i r e of the stabilizing inter&tions listed above can be targeted bv various denaturing - conditions, such as those listed below. high ionic strength high temperature pH extremes added solutes (detergents,amides, alcohols, etc.)
.
Fluorescence While monitoring activity loss is oRen fairly straightforward, sophisticated spectroscopic techniques (e.g., 2-D NMR, Raman IR, Circular Dichroism) are usually chosen to follow the three-dimensional process of unfolding. However, the intrinsic fluorescence due to tryptophan (and tyrosine) aromatic side chains is easy to measure. The flnorescence of aromatic chromophores is highly sensitive to solvent polarity. Thus, changes in protein structure that alter the local dielectric surrounding the chromophore will cause a corresponding change in fluorescence intensity. This laboratory project follows changes in chymotrypsin esterase activity and intrinsic flnorescence upon denaturation in guanidine hydrochloride (CIHZN=C(NH~)~) solutions. Experimental Materials All reagents were purchased from S i m a or Aldrich and used witI;out f~rthe;~urificatitm.u-Chymotrypsin I bU mg) was dissolved in 100 mL of 10 m.M H K l ' l . 3 buffer. oH 6.5, and used fresh daily. The substrate pnitropheny1 icetatd (PNPA)was dissolved in buffer (10 mM HEPES.. . pH 6.5) to make a 3.0 mM stock solution. Procedure p-Nitrophenolate ( p N P ) production was measured a t 405 nm usin a Beckman UV 5240. Its molar absorptivity is 18,800 M-'cm-'. The increase which was linear with time. was followed for 1-2 min to determine the s l o ~ e MAt. Guanidine hydrochloride (GnHCl) was dissolved in buffer to give a highly concentrated stock solution (9.5 M). Samples containing 5.0 mL of enzyme stock solution and variable amounts of GuHCl and buffer were mixed in 10.00-mLvolumetric flasks and incubated for 3&60 min to establish equilibrium.
Measurements to Detect Coupling
GuHCI-Induced Unfolding of Chyrnotrypsin [GuHCI] M
Enzyme Hydrolysis Act~vity (MIS)
IN
Ku
Enzyme Intrinsic Fluorescence
-RTlnKu
(a.u.)
f~
Ku
RTinKu
1.00 0.00 0.00 undef. 114. undef. 113. 0.98 0.022 +2.28 0.0091 +2.80 0.96 0.044 +1.86 0.00 undef. 112. 104. 0.79 0.270 +0.78 0.283 +0.75 0,45 , 1.27 4.14 3.32 4.72 81. 0.30 2.36 4.51 69. 0.04 22.5 -1.86 12.1 -1.48 -2.28 68. 0.02 46.0 16.1 -1.66 110. -2.80 67.5 0.01 93.0 -2.70 All measurement$made at 26.8 'C in buffer (10 mM HEPES. pH 6.5).FluoresQnce readings in arbitrary units, excitation at 290 nm, emission at 335 nm. Act~vityvalues in moles per liter of pNP- produced per second,as measured at 405nm. ~ 4 0 5= 18,800M'cm-'. f~ = (X -Xo) I (XN-XO); Ku = (XN-X) I (X - XD); for activity. aw = 25.2 MIS, ao = 3.0 M h . while for activity, FIN= 114.. Flo = 67. 0.00 0.75 1.00 1.35 1.70 2.00 2.35 2.70 3.00
25.2 25.0 25.2 20.3 12.8 8.14 4.7 4.3 3.2
1.00 0.99 1.00 0.78 0.44 0.23 0.08 0.06 0.01
1
Sample cells were prepared by mixing 1.0 mL of pNPA stock with 2.0 mL of enzyme-GuHC1 stock. Chymotrypsin intrinsic fluorescence from tryptophan side chains was measured with a Varian SF330 with excitation a t 290 nm and emission a t 335 nm. Results The table shows results for a typical experiment at room temperature (26.8 usinp0-3 M GuHCi. Fluorescence is in arbitrary units. ~ i t i v igiven t ~ is given in units of concentration per second, [pNPlIs, though it could just a s easily be given as below in units of absorbance per time: AAadAt. To compare the two processes, it is easiest to convert them both to fractions of remaining native enzyme ( f ~ ) .
x-x, fN== where N and D represent the completely native or denatured state, respectively; and X is either a fluorescence or activity value. From the plot (Fig. 1) off^ vs. [GuHCIl we see that the decrease in intrinsic fluorescence closely follows the loss in activity.' This is not surprising because GuHCl, along with other amides (urea) and alcohols, is believed to disrupt both hydrogen bonds and hydrophobic interactions (3-5). Thus, the resultant protein unfolding would be quite general and widespread, equally affecting all protein domains (e.g., active site and distant tryptophan).
Mozhaev e t al. (4) have tested other amide and alcohol denaturants, finding a similar close relationship between changes in fluorescence and activity loss. However, we have tested both thermal and salt-induced denaturation, finding t h a t under these conditions changes in fluorescence are significantly uncoupled from activity loss. Thus, the conformational coupling between tryptophan fluorescence and activesite interactions depends on the type of denaturant used. Discussion Equilibrium
Threshold dose-dependence effects are fairly common in biochemistry Sigmoidal binding curves are tvnicallv analvzed usine a Hill ;lot, which g i k a s b p e oin. This the Hill coefficient, which describes the degree of cooperativity of the binding interaction. To determine the level of cooperativity involved in the denaturant-induced unfolding of a protein, a formalism was developed by Knapp and Pace (6,7).This simplified theory posits that native protein (PN)unfolds to denatured protein (P,) in a single denaturant-dependent step.
+m GuHCl
p~
P~
C
-m GuHCl
Assuming that we can follow this process by monitoring the change in some parameter X (e.g., activitiy or fluorescence), we get an equilibrium constant for the unfolding and an equation of the following form.
where XN and Xo represent the values of X for the fully native and denatured states, respectively. Chymotrypsin/GuHCl D e n a t u r a t i o n at 26.8%
Half-Maximal Denaturation
Half-maximal denaturation (see Fig. 1) is achieved a t 1.7 M GuHCl, which is denoted as Cm.When [GuHClI = C,,q the concentrations of native and denatured chymotrypsin are equal. If the class is large enough. each student (or air of students, could be asslfgned dFfferent temperature;i to te%t,or d~fferentdenaturanrs to use. We h a w found that C , , decreases significantly a t higher temperatures, for example, C m = 1.1M a t 37 'C. Thus, heat makes the enzyme more susceptible to guanidine-induced unfolding. 'We have compared the fitted values for C,,2, m, or A q , H 2 0 that were calculated separately from the activity and the fluorescence data. We find that the differences are well within the standard deviation calculated for that particular value.
Native
Fraction
- -
- - - - - - . ..
0.4
Figure 1. Fraction of native chymotrypsin (fN) plotted as a function of the concentration of guanidine hydrochloride. Data were taken from the table for activity (+) and fluorescence (4.The solid line is a sigmoidal fit for the data: n = 6.8 and C,,= 1.65 M. Volume 69 Number 10 October 1992
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Free Energy of Chymotrypsin/GuHC1 Denaturation at 26.8%
Calculations When data from the table are plotted as -RT In K, vs. [GuHCII in Figure 2, the result is indeed linear with R = 0.9885. This again shows the close agreement between changes in activity (+I and fluorescence (X).' From the slope and intercept of this plot, we calculated the following.
Figure 2. Free energy of unfolding (-RTin K,) plotted as afunctionof guanidine hydrochloride wncentration. Data were taken from the table for activity (+) and fluorescence (X). The solid line is a linear-regression fit for the data: intercept = 4.0 i 0.2; slope = -2.3 f 0.1; correlation coefficient = 0.9885.
These values are quite reasonable when compared with those obtained for other proteins (5-7). As a further comparison, a t 37 'C we calculated the folowing values.
Thermodynamic Theory The free energy of unfolding can be given by the following equation.
Because this reaction is directly dependent on denaturant concentration, the following equationZis valid.
where m is an index of the sensitivity of the protein to the denaturant; and
The Effect of Temperature The free energy of unfolding changed very little over the 2 0 3 7 'C temperature range. This is not surprising because chymotrypsin normally functions a t 37 *C, and we can expect the protein to be stable a t that temperature. Because the unfolding process is generally (8, 9) endothermic
and disordering ILS:>o
higher temperatures can be expected to decrease is the free energy of unfolding in absence of denaturant. The factor m describes how steeply the free energy of unfolding decreases as [GuHClI increases. Thus, m gives a measure of the increase in the extent to which the protein is exposed to solvent upon denaturation (5, 6). Note that a plot of -RT In K, vs. [GuHCII should yield a straight line with slope = -m and intercept = AG:,
H,O
Furthermore, at C m we get
Summary This laboratory project has several distinct heuristic benefits. Data collection is easily accomplished in a single afternoon. 'Students are introduced to spectrophotometric monitoring of enzyme activity as well as fluorescence monitoring of protein dynamics. .Correlation between enzyme activity and conformation is demonstrated. Thermodynamic data analysis stresses the use of free energy and leads to a dynamic interpretation of the effects of denaturant and temperature.
and
Thus,
This panicular relationship was carefully verified for urea and GuHCl by Alonso and Dill in a recent paper (3. 854
until it becomes exergonic. At this point the protein will unfold spontaneously. In fact signifcant decreases in specific activity are observed in buffer above 37 'C (data not shown). The lower value of Cvz a t 37 'C, suggesting an increased susceptibility to guanidine, is due mainly to a substantial increase in m. Since protein motionincreases with temperature, internal protein-protein stabilizing interactions become weaker. Thus, it is not surprising that m, the sensitivity of the protein to denaturant, increases a t higher temperatures.
Journal of Chemical Education
Acknowledgment We would like to thank the Willamette University Chemistry Department for supporting this project, and Frances H. Chapple and Frank Zizza for help with data analysis.
Literature Cited
6. K~.RPP, J. A ; pace, C. N . B ~ K 1914.13, ~ ~ ~ . 128S1294.
1. Parmy-M-ale.A.:Bamn,C. J C h . Educ 1986,63(111,1003-1004. 2. Sehuh,M. D. J. Cbm. Edur 1988,65(81,740-741. 3. Kamoun,P. P. h n d r Biocbm. Sci 1988.13, 424-425. 4. ~ ~ K~ h ~ L.~;Seqeeeev ~ ~ M, ",;~ ~ , A, B,; ~ mYYYhko,~N,; Levaahou,A.V.; Martinek, K.Eur J. Biochem. 1989,184,597602. 5. Alonao, D. A.V.: Dill, K. A. Blachem. 1991,30, 59745985.
7. Caffrey, M. 3.; Daldd, F ; Holden, H. M ;Cusanovlch, M A . Blocham. 1991,30,4119 4125.
v v.;
8. BrandesC.;Tooze, J . l n t d u c ~ i o n t o D o l e i n ~ t ~ c ~ u m ; c a 1~9~9d1 :; ~~ ~ ~ ~ ~ ~ k , ~ ~ ~ , 256.
~
9. Zubay, 0.Biocharnlstv, 2nd d.; Macmillan: New Yark;p 65.
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