Langmuir 1993,9, 3166-3173
3166
Cooperativity in the Binding of Avidin to Biotin-Lipid-Doped Langmuir-Blodgett Films Shulei Zhao,t D. S. Walker, and W. M. Reichert' Department of Biomedical Engineering and the Center for Emerging Cardiovascular Technologies, Duke University, Durhum, North Carolina 27708-0281 Received April 13, 1993. In Final Form: July 2 2 , 1 9 9 9
Monolayers of arachidicacid (AA) doped with either biotinylated DPPE (B-DPPE)or a chain extended biotinylated DPPE (B-x-DPPE)were deposited onto alkylsilane treated surfaces of quartz evanescent fiber optic sensors (EFO) by the Langmuir-Blodgett (LB) technique. The surfacemodified EFOs were usedto obtainbindingisothermsof fluorescein-labeledavidintothe biotin-lipid-dopedLB f i i . Hyperbolic binding isotherms were observed for all B-DPPE doped LB films and for B-x-DPPE doped f i i s with 0.5 for slope, p < 0.05 for intercept). Thus, a fourth observation is that q increases with increasing receptor density for 50.63 mol % B-x-DPPE. Of all binding experiments, five of them yielded q estimates of less than unity (Figure 5 and Table I),which is indicative of negative cooperativity. In four of these instances q is within a standard deviation (0.37)centered at 1.0and, therefore, probablyrepresents noncooperativity.
Zhao et al.
3170 Langmuir, Vol. 9,No. 11, 1993
/A
I
I
adsorbed native non-specifically LE-bound , specifically
LB-bound adsorbed denatured
ii -t
1650 1600 1550 Wavenumber (cm-1)
1750 1700
10
106
100
Blotin surface density (mol%)
Figure 6. Intrinsic association constante (KO) of avidin binding to B-DPPE (open circles) or B-x-DPPE (filled circles) in LB monolayers. The vertical line indicates a biotin surface density
1500
Figure 7. Amide I and I1 bands of background-subtractedATRFTZR spectra of native avidin and thermally denatured avidin adsorbedto bare silicon ATR crystals,nonspecificallyLB-bound avidin (AA f i i with 0 mol % biotin lipid),and specifically LBbound avidin (AA with 0.5 mol % B-DPPE).
of 0.63 mol %. Table 111. Estimated Apparent Association Constant of Avidin Binding to LB Monolayers Containing B-DPPE or B-X-DPPE biotin density (mol %) 0.17 0.28 0.40 0.50 0.63 0.76 0.99 1.41 1.79 2.17 3.23 6.25 16.7 100 mean Kapp
K~~~(107~ - 1 ) B-DPPE B-X-DPPE 6.5 33 7.9 5.8 8.2 14 22 16 5.1 8.8 11 25 5.2 11
1l.f 8.1
96 118 34 45 62 44 80 80 84
52 59 48 74 78 68 f 23
Only once was 7 significantly less than 1.0 (0.56f 0.241, suggesting a fairly strong negative cooperativity. It has been shown that Scatchard plots of binding isotherms can appear to be negatively cooperative if the "large-ligand effect" is p r e ~ e n t . ' ~ 'This ~ * ~effect ~ can be observed when a large protein ligand binds noncooperativity to one or two receptors and at the same time inactivates (or covers) other neighboring receptors. Therefore, this effect is also called "pseudocooperativity". However, it is more plausible that this low cooperativity coefficient resulted from experimental error rather than an isolated occurrence of the large-ligand effect. Figure 6 is a log-log plot of the intrinsic association constant KOagainst biotin surface density. KOvalues for the entire range of B-DPPE surface densities showed no correlation to the biotin receptor density in the LB monolayer and had an approximate average of (7 f 5) X lo7 M-l. On the other hand, KOfor B-x-DPPE dopant decreased quickly with increasing B-x-DPPE surface density and leveled off to an approximate average of (2.0 f 0.6)X lo7 M-' for B-x-DPPE densities of 20.63 mol % (the vertical dashed line). The apparent association constant (Kapp) is the association constant observed at equilibrium. If virtually all accessible biotin groups are bound at equilibrium, then Kapp can be approximated from eq 5, i.e. Kapp= K0v4. Table I11containsthe estimated values of Kapp for B-DPPE and B-x-DPPE. The average values of Kappfor B-DPPE (20) McGhee, J. D.; von Hippel, P. H. J . Mol. Biol. 1974,86,469-489.
and B-x-DPPE are (11f 8)X lo7M-l and (68f 23)X lo7 M-l, respectively. Kapp appears to be independent of receptor doping density, but the average value of Kapp for B-x-DPPE receptor is approximately6times greater than that for B-DPPE. ATR-FTIR Study. Figure 7 compares backgroundsubtracted,unmassaged, ATR-FTIR spectra of (1)native avidin and (2)thermally denatured avidin adsorbed to bare silicon ATR crystals, (3) avidin nonspecifically adsorbedto an AA film without biotin lipid, and (4)avidin specifically bound to a biotin-lipid-doped AA f i b (0.5 mol % B-DPPE). The amide I and amide I1 bands of avidin are evident in Figure 7 at 1640 and 1540 cm-', respectively. It is apparent from Figure 7 that the ATR spectrum of specifically LB-bound avidin differs significantly from the spectra of adsorbed native avidin and nonspecifically LB-bound avidin and more closely resembles the spectrum of adsorbed denatured avidin. Table IV lists the wavenumber locations of the amide I and amide I1 band maxima, and the ratio of the amide I1 to amide I band intensities, for avidin bound to LB films doped from 0 to 5 mol % biotin lipid and for native and thermally denatured avidin adsorbed to bare ATR crystals. The amide I and I1 band maxima, respectively, appeared as follows: 1536 and 1648 cm-' for adsorbed native avidin; 1531 and 1635cm-l for adsorbed denatured avidin; and 1538-1542cm-' and 1638-1637 cm-l for both nonspefically and specifically LB-bound avidin. Therefore, the amide I and I1 band maxima of denatured avidin were shifted to lower wavenumbers than that observed for all of the LB-bound avidin spectra,while only the amide I1 maxima of native avidin was shifted significantly from that observed with LB-bound avidin. The intensity of the amide I band for LB-bound avidin increased and then leveled off with increasing mol ?6 biotin lipid, whereas the amide I1 band intensity initially decreased with increasing mol % biotin lipid and then appeared to vanish from the ATR spectrum before returning at higher doping densities. When viewed as the ratio of the amide I1 and amide I band intensities (Table IV), the ATR data seem to represent a loss of and then an apparent return to a native conformation, where the maximum conformational change occurred at 0.25,0.50,and 0.75 mol % for B-x-DPPE and at 0.75,1.0, and 2.5 mol % for B-DPPE. However, it seems unlikely that surface bound avidin would become more conformationally altered than thermally denatured avidin, and the curious disappearance of the amide I1 band may be an artifact of the background subtraction process. For example, biotin has a strong absorption at 1550 cm-' Io that may have
Binding of Avidin to Biotin Lipids
Langmuir, Vol. 9, No. 11,1993 3171
Table IV. Amide I and Amide I1 Band Locations and Ratio of Band Intensities of Adsorbed and LB-Bound Avidin amide IUamide I amide IIIamide I surface of ATR crystal band locations (cm-1) intensity ratio bare surface, native avidin 164811536 0.72 bare surface, denatured avidin 163511531 0.15 biotin-free LB film,native avidin 163811542 0.78 biotin-containing LB fiis, native avidin mol % biotin lipid 0.10 0.25
0.50 0.75 1.00 2.50
5.00 a
B-DPPE
B-X-DPPE
B-DPPE
B-X-DPPE
163811542 163811541 163711539 a11537 a/1536 411538 1638/1538
163811538 all538 411539 411538 163711540 163811540 163811540
0.49 0.35 0.15
0.19
4 4 4
4
a 4
0.08
0.09 0.57 0.83
Discernible absorption peak not identified.
obscured a weak avidin amide I1 band when background subtracted, while the spectrum of adsorbed denatured avidin was collected on a bare ATR crystal. Nonetheless, one can conclude from the ATR measurementsthat biotin lipid bound avidin becomes conformationally altered, and the greater extent of conformational change occurs at a lower receptor density for B-x-DPPE than for B-DPPE.
et al. also showed that the lack of a spacer between the hydrophobic tails and the biotin head group in the lipid prevents streptavidin binding to biotin. However, when they substituted avidin for streptavidin, no protein domains were observed through light microscopy. Ku et al. recently used electron microscopy to show that avidin also forms domains on biotin lipid monolayers at the airwater interface, albeit very small ones.26 The difference Discussion in domain size suggeststhat the interaction between biotinbound avidin molecules is more combersome than that Positive cooperativity refers to activity that facilitates between the biotin-bound streptavidin. ligand binding by the presence of other ligand participants, In the current paper, our estimates of the cooperativity while negative cooperativity concerns activity that hinders I and 11, Figure 5) indicate that two coefficient (Tables ligand binding. Noncooperativity corresponds to the conditions were necessary for the occurrence of positive binding where all participants act independently. Theories (1) asufficiently high biotin surface density; cooperativity have been developed that ascribe cooperativity to intra(2) good access of the biotin group in the LB film to the protein and interprotein interactions that require the avidin molecules. participating moieties to be in proper j u x t a p ~ s i t i o n . ~ ~ - ~binding ~ Clearly, if individual receptors are too far apart, proteinConformationalchanges of the protein or its subunits are protein interactions do not occur. This point is supported considered to be essentialin inducing cooperativityin these by considering a receptor density of 0.63 mol % biotin theories. Chay and ChoZ4proposed that cooperativitycan lipid in the LB monolayer; which is close to the approxalso arise in biological membranes as a consequence of imate minimum receptor density of 0.6 mol % necessary microenvironmentalchange. Specifically, if a ligand has to bind a contiguous monolayer of avidin. [(3025-47)/18 preferential binding to either a protonated or an unpro= 165 AA molecules per single biotin lipid per 3025 A2, tonated state of the protein and if the net charge of the where 3025 A2 (55A X 55 A),1s 47 A2, and 18 A2 are the liganded state is different from unliganded state, the molecular areas of biotin lipid-bound avidin, a biotin lipid, existence of two environmentsmay induce cooperativity. and an AA fatty acid, respectively." (1/165)X 100 = 0.6 Streptavidin is a protein of bacterial origin that shares mol % biotin lipid in the LB film therefore corresponds the same high binding affinity for biotin as egg white to the minimum receptor density needed to bind a avidin.6 Blankenburg et al.25observed apparent proteincontiguousmonolayer of avidin.] Thus, it is not surprising protein interaction in their study of streptavidin binding that noticeably sigmoid binding curves were observed only to biotinylated phospholipid monolayers at the air-water for experiments with 10.63 mol % B-x-DPPE (Figure 3 interface. Streptavidin was observed to form highly and Table 11)where the avidin molecules are sufficiently ordered domainsat the air-water interface, but only when close to interact. However, q appears to increase up to 1 biotin lipid receptors were present in the monolayer, mol % B-x-DPPE (Figure 51,suggesting that the level of suggesting that binding with biotin was necessary for the cooperativity continuesto increase until most of the avidin streptavidin-streptavidin interaction to occur. Interestmolecules are bound by two B-x-DPPEreceptors (e.g. >1.2 ingly, they demonstrated that the presence of a highly mol %). organized monolayer, i.e. the solid-condensed state, is not The explanation for the second condition is somewhat necessary for the formation streptavidin domains. These more speculative and may be related to the conformation domains were observed even when the biotin lipid monoof the surface-bound avidin. That is, since cooperativity layer was in the uncompressed state. This observation was observed only with the more accessible B-x-DPPE indicates that streptavidin is driven to aggregation, i.e. biotin lipid, we hypothesize that avidin bound to B-x"swim around", after they bind with biotin. Blankenburg DPPE was conformationally altered in a manner that contributed to the cooperativity observed in our study. (21) Monod, J.; Wyman,J.; Changeux, J.-P. J. Mol. Biol. 1965, 12, Indirect support for this hypothesis can be inferred again 88-118. from streptavidin. (22) Koshland, D. E.; Nemethy, G.; Filmer, D. Biochemistry 1966,5, 365-385. _.. Streptavidin, like avidin, consists of four identical (23) Changeux,J.-P.; Thiery, J.;Tung, Y.;Kittel, C. R o c . Natl. Acad. subunits, each with a single high affinity biotin binding Sci. U.S.A. 1967,57,335-341. ~~
~~~
(24) Chay, T. R.; Cho, S.-H. Biophys. Chem. 1982,15,271-275. (25) Blankenburg,R.; Meller,P.;Rmgsdorf,H.; Salesee,C. Biochemistry 1989,243, 8214-8221.
~~
(26) Ku, A.
~
C.; Damt, S. A.; Kornberg, R. D.; Robertson, C. R.; Gast,
A. P . Langmuir 1992,8, 2357-2360.
Zhao et al.
3172 Langmuir, Vol. 9, No. 11,1993 site. Weber et al.27 used X-ray crystallography to show that the biotin binding pockets of streptavidin lie at the end of /3 barrels, and biotin binding involves'displacement of bound water, formation of multiple interactionsbetween biotin heteroatoms and the binding site residues, and the burial of biotin through ordering of a surface loop. Since streptavidinshares many similaritieswithavidin,including 38% sequence homology," it is reasonable that biotinbound avidin undergoes a similar a conformational change and that this change is greatest when the biotin head group is accommodated deeply in the binding pocket. Unfortunately, analogous crystallographic data are not yet available for avidin, and one is tempted to use what is known definitively about streptavidin to infer information about avidin. However, such extrapolations should be made with caution. Raman and Fourier transform infrared (FTIR) are complementary spectroscopy techniques commonly used to determine the secondary structure of macromole~ u l e s . ~ 8The 9 ~ ~main vibrational absorptions of proteins are the N-H stretch ( 3300 cm-9, amideI (C=Ostretch, 1640cm-l), amide 11(N-H bend, C-N stretch, 1550 cm-l) and amide I11 (mixed N-H and C-N vibrations, 1250 cm-l) absorptions. Honzatkoand Williams30used Raman spectroscopy to determine that avidin consisted of 55% &sheet and 10% a-helix conformation, but observed virtually no difference in the amide I and amide I1 regions in solution phase Raman spectra of avidin and the avidin biotin complex. Barbucci et al.'O used ATRFTIR to examine the amide bands of avidin and the avidinbiotin complex, assigning a structure to absorptions at 1668,1307,and 1275 cm-l and j3 structure to absorptions at 1630 and 1250 cm-l, but did not assign structure to the 1540-cm-l amide I1 absorption band. Barbucci et al. also observed only minor differences in the ATR-FTIR spectrum of the avidin-biotin complex when compared to a summed spectrum of avidin and biotin. Note that both Honzatko and Williams and Barbucci et al. collected spectra from extremely concentrated (i.e. 100 mg/mL) avidin solutions. In order to investigate the possibility of a conformational change in surface-bound avidin, we performed a series of ATR-FTIRmeasurementsincluding native and denatured avidin adsorbed to bare ATR crystals, avidin nonspecifically bound to biotin-freeLB films, and avidin specifically bound to biotin-lipid-doped LB films. Contrary to the solution phase work of Honzatko and Williamsm and Barbucci et al.,l0 we observed significant spectral differences between the adsorbed, nonspecifically LB-bound, and specifically LB-bound cases (Figure 8). However, unlike the above reporta, there were no bulk contributions to any of our spectra, and all of our data were derived strictlyfrom monolayer and submonolayer films of avidin. Using the intensity ratio technique of Kato et al.,I1 it also appears that specifically LB-bound avidin experienced conformational changes that were effected by both the biotin lipid surface density and the biotin lipid accessibility (Table IV). Unfortunately, signal averaging 1024 scans at 8 cm-l resolution smoothed out the fine structure in the data, making it difficult to resolve the primary amide I and amide I1absorbancesinto the subpeaksand shoulders observed by Barbucci et al.'O The lower absorbances also
-
-
-
(27) Weber, P. C.; Ohlendorf, D. H.; Wendoloski, J. J.; Salemme, F. R. Science 1989,243,8548. (28) Colthup, N. B.Zntroduction toZnfraredandRamnnSpectroscopy, 3rd 4. Academic ; Press: Boston, MA, 1990. (29) Parker, F. S. Applications of Infrared Spectroscopy in Biochemistry, Biology, and Medicine; Plenum Press: New York, 1971. (30) Honzatko, R. B.; Williams, R. W. Biochemistry 1982,21, 62016205.
led to difficulty in discerning the amide I1 peak when it fell into noise (see Results). Therefore, our ATR results are by no means definitive but do provide evidence that a conformational change occurs when avidin binds a biotin lipid at a surface and that the conformational change is qualitativelydifferent for avidin bound to B-x-DPPE than to B-DPPE. Finally, the intrinsic association constantKO determined for LB films doped with B-DPPE did not show a clear trend (Figure 6),suggesting that B-DPPE surface density did not significantly affect the affinity of avidin for the biotin group in the LB film. However, KO decreased linearly with increasing B-x-DPPE surface density until approximately0.63 mol 7% ,after which is leveled off. In a previously published, independent experiment we similarly observed that the kinetic rate of avidin binding to B-x-DPPE doped LB films decreased rapidly with increased receptor density and then leveled off at a steady value.8 In that study it was reasoned that a drop in binding affmity resulted from a decreased accessibility of avidin to the individual B-x-DPPE receptors as the receptor density increased. When doping density was 0.63 mol 9% or higher, it is not clear why KO values for B-x-DPPE(&,am = (2.0f 0.6) X lo7M-l) were on the average slightlysmaller than those for B-DPPE (&,a", = (7 f 5) X lo7 M-l). A possible explanationmight be that greater steric hindrance arose from neighboring B-x-DPPEthan from neighboring B-DPPEq8However, the estimated apparent association constant Kapp for B-x-DPPE was approximately6 times greater than the corresponding Kapp for B-DPPE (Table TTT\
111).
Summary
An expression for the isotherm of cooperative protein binding to an array of receptors was developed and tested experimentally using the avidin-biotin lipid ligandreceptor system. Quantitative information on the intrinsic affinity of avidin for biotin in LB monolayers and the level of cooperativity was obtained by fitting experimental data to this model through nonlinear regression. Cooperative binding was observed only for the more accessible B-x-DPPE ligand and only at receptor densities of 10.63 mol %, which is sufficient to bind a near monolayer of avidin. From these results we conclude that the avidinavidin interactions necessary for cooperative binding occurred when two conditionswere met: (1)biotin surface density was high enough to provide adequate proximity of binding sites; (2) the biotin groups were extended sufficiently above the LB monolayer so they would fit with facility into the binding pocket of avidin. Based upon available data for streptavidin, we postulate that facile binding of the B-x-DPPE ligand by avidin resulted in a conformation change on the protein that promoted cooperative activity. ATR-FTIR studies provided evidence that a conformational change occurred in avidin when it bound biotin lipid in the LB film and that this conformational change was at least qualitatively different for avidin bound to B-DPPE and to B-x-DPPE. Acknowledgment. This work was funded by a biomedical research grant from the Whitaker Foundation, NIH Grant HL 32132, and a graduate fellowship from the NSF/ERC Center for Emerging Cardiovascular Technologies at Duke University. We thank Dr. G. A. Truskey of Duke University for his keen advice and helpful discussion. An anonymous reviewer is thanked for providing the analysis presented in Appendix B.
Langmuir, Vol. 9, No. 11,1993 3173
Binding of Avidin to Biotin Lipids
Appendix A Association Constant as a Function of Free Energy Change The binding of a protein ligand (A) to its receptor (B) on a surface can be treated as a bimolecular reaction
COlUmn
A+B+AB (AI) The equilibrium constant K , for this reaction is defied as Ke = am/ ( U A ~ B ) ,where a ~aB, , and am are the activities In of species A, B, and AB a t equilibrium, re~pectively.~~ a dilute solution of nonelectrolytesthe activity of a species is equal to its mole fraction in the solution.31 Thus the equilibrium constant can be expressed as
where XA,X B , and XABare the mole fractions of species A, B, and AB at equilibrium, respectively. Since mole fraction of a species is proportional to its concentration, XA,XB,and XABcan be written as XA= CYA[A],XB = crg[B], and XAB= CYAB[ABI, where [AI, [Bl, and [AB1 are the concentrationsof speciesA, B, and AB at equilibrium, respectively, and CYA, CYB,and CYABare proportionality constants which convertthe units of concentrationto mole fractions. Because the units for [Bl and [AB] are the same (e.g., moles per unit area), CYBequals CYAB,yielding
Ke = KJK,
(A31
where
For any chemical reaction, the equilibrium association constant Ke is related to the standard free energy change AG by AG = -RT In Ke, where R is the universal gas constant and T is temperature in Kelvin.31 Using this relation and eq A3, we obtain the expression of K as a function of AG
K = K , exp(-AGIRT)
1
2
3
" '
1
...
1 0
0
0
0
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0
2 0
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: 0
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. ...
. ...
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0
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..
..
..
(31)Klotz, I. M.; Rosenberg, R. M. Chemical Thermodynamics, 4th ed.; Benjamin/Cummings, Menlo Park, CA, 1986; pp 354.
..
N
0
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Figure 8. Matrix representation of an ideal square lattice of protein receptors. Each open circle represents a receptor. Each receptor has four nearest neighbors.
number of protein-bound nearest neighboring receptors (n). This in turn requires assuminga receptor distribution. The simplest receptor distribution is a square lattice represented by the matrix (Figure 8)
s,
=
Sjj = 1 if receptor i j is occupied
= 0 if receptor i j is vacant
]
031)
The number of occupied sites neighboring a given receptor Sij is given by Sj-1: + sj+lj + si j+l + si j-1. Therefore, the averagenumber of nearest neighboringreceptors occupied by protein ( n ) is given by
where N is the totalnumber of receptors. Assuming array is infiiite (Nis very large), the four terms in eq B2 are equivalent and eq B2 becomes
(A51
Appendix B: Average Number of Protein-Bound Neighboring Receptors To model protein binding to the biotin-lipid receptors, it is necessary to determine the statistical average of the
..
N-1
"'."
4$$sij
n=
N
The dual summation in the numerator in eq B3 is simply the number of protein-bound receptors,which divided by the total receptors N,is the fraction of bound receptors X I . Therefore, eq B3 becomes n = 4x1
034)
