[5]
AVIDIN AND STREPTAVIDIN
51
3.5.1.12) may eventually be extensively exploited in avidin-biotin technology, as a replacement for avidin, for biotinylation of binders, or as an analytical tool for releasing the biotin moiety from biotinylated material. The fact that biotin is a vitamin required in very small quantities for a variety of cellular processes reflects the very high affinity constants exhibited by the broad number of naturally occurring biotin-binding proteins which have been described. As time goes on, it would not be surprising if many more biotin-binding proteins will be described, many of which may prove suitable for application in avidin-biotin technology. In certain instances, some may even supercede the use of avidin, owing to improved molecular characteristics. The use of avidin-biotin technology would undoubtedly be increased enormously if other non-biotin-binding proteins which exhibit very high affinities for unrelated ligands would be described for complementary applications.
[5] A v i d i n a n d S t r e p t a v i d i n By N. MICHAEL Gl~EEN Introduction
The discovery of the protein avidin resulted from intensive nutritional investigations into the vitamin B complex.I Avidin proved to be a minor constituent of egg white that could induce a nutritional deficiency in rats by forming a very stable noncovalent complex with what was subsequently proved to be the B vitamin biotin. The biological role of avidin in egg white appeared to be that of a scavenger, inhibiting bacterial growth. A variant of lower affinity for biotin has been found in egg yolk, which may be important in regulating the supply of biotin during development. 2 Avidin is a highly specialized protein that is only rarely expressed. One might have expected that it was a vertebrate protein of fairly recent lineage were it not for the fact that a close relative, streptavidin, is expressed in a species of S t r e p t o m y c e s . 3 However, like proteins have not been found elsewhere in microorganisms, and the possibility remains that its occurrence in Streptomyces avidinii, a strain of Streptomyces lavendulae, reflects a chance transfection rather than an ancient lineage. The relation of the avidins to a recently discovered family of binding proteins is considered below. P. Gyorgy, in "The Vitamins" (W. H. Sebrell, Jr., and R. S. Harris, eds.), gol. 1, p. 527. Academic Press, New York, 1954. z H. W. Meslar, S. A. Camper, and H. B. White, J. Biol. Chem. 253, 6979 (1978). 3 L. Chaiet and F. J. Wolf, Arch. Biochem. Biophys. 106, 1 (1964).
METHODS IN ENZYMOLOGY, VOL. 184
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
52
BIOTIN-BINDING PROTEINS
[5]
Current interest in avidin derives mainly from its applications in biotechnology based on its rapid and almost irreversible binding of any molecule to which biotin can be linked. This has led to a proliferation of labeling and affinity purification methods, which are the subject of this volume. The biochemical basis for the very high affinity was first investigated by chemical modification.4 Interest was further stimulated by the discovery of the coenzyme function of biotin in CO2 transfer5 and by the subsequent use of avidin to identify biotinyl enzymes. More extensive characterization of the molecule and its binding sites followed, mainly from the author's laboratory, and the results of this were reviewed in 1975.6 At that time there was only one report of the use of avidin as a cytochemical label, 7 although from its general properties and its use as a high-resolution electron microscopic label for biotinyl enzymes, 8 it was clear that it had considerable potential in this field. Most work on avidin since the mid1970s has been on the development of such applications based on avidin labeled with gold, peroxidase, alkaline phosphatase, ferritin, or radioactive or fluorescent labels, using a variety of synthetic bridging agents to provide appropriate biotinyl sites for its attachment. 9,1° During this period there has been relatively little new biochemical work on the structure and binding properties of avidin or streptavidin. Considerable efforts have been devoted to an X-ray crystallographic approach to the structure, but it proved very difficult to grow acceptable crystals of sufficient size, J~ possibly because avidin is a glycoprotein and its carbohydrate is characteristically heterogeneous. ~2 In this respect, streptavidin, which is carbohydrate free, has proved advantageous. Its structure is now determined, but the results were not available for the preparation of this chapter. J3,J4 Another main advance has been the cloning and sequencing of the streptavidin gene.15 4 H. Fraenkel-Conrat, N. S. Snell, and E. D. Ducay, Arch. Biochem. Biophys. 39, 97 (1952). 5 S. J. Wakil, E. B. Titchener, and D. M. Gibson, Biochem. Biophys. Acta 29, 225 (1958). 6 N. M. Green, Adv. Protein Chem. 29, 85 (1975). 7 H. Heitzmann and F. M. Richards, Proc. Natl. Acad. Sci. U.S.A. 71, 3537 (1974). s N. M. Green, R. C. Valentine, N. G. Wrigley, F. Abroad, B. E. Jacobson, and H. G. Wood, J. Biol. Chem. 247, 6284 (1972). 9 E. A. Bayer and M. Wilchek, Methods Biochem. Anal. 26, 1 (1980). to M. Wilchek and E. A. Bayer, Anal. Biochem. 171, 1 (1988). ~i E. Pinn, A. Pahler, W. Saenger, G. A. Petsko, and N. M. Green, Eur. J. Biochem. 123, 545 (1982). t2 R. C. Bruch and H. B. White, Biochemistry 21, 5334 (1982). 13 p. C. Weber, D. H. Ohlendorf, J. J. Wendoloski, and F. R. Salemme, Science 243, 85 (1989). ~4 W. A. Hendrickson, A. P~hler, J. L. Smith, Y. Satow, E. A. Merritt, and R. P. Phizackerley, Proc. Natl. Acad. Sci. U.S.A. 86, 2190 (1989).
[5]
AVIDIN AND STREPTAVIDIN
53
In this chapter, I consider only fundamental molecular properties and those which are important for applications in biotechnology. Particular emphasis is placed on differences between avidin and streptavidin, since although streptavidin has become widely used as a label because of low nonspecific binding, its binding characteristics have not been studied quantitatively. The alternative use of succinylavidin ~6to diminish nonspecific avidin binding is less effective, probably because the carbohydrate of avidin also contributes to the nonspecific binding. ~7 Carbohydrate-free avidin can be obtained by use of deglycosylating enzymes, and it has been shown to be present in significant amounts in some commercial preparations from which it may be separated by use of lectin columns. ~sIts biotinbinding properties were unimpaired.
Affinity Methods for Purification of Avidins and Biotinyl Proteins Improvement on the original use of biotinyl cellulose ~9came with the introduction of iminobiotinyl derivatives of Sepharose 2°-23 which utilized the pH dependence of the binding 24 to achieve efficient elution. In iminobiotin, the ureido group becomes a guanidinium group, and only the form in which this is uncharged is strongly bound. The apparent affinity, therefore, should fall by a factor of 10 per pH unit provided that the protonated form does not bind [ K a p p : K B K / ( H + + K), where KB is the dissociation constant for uncharged iminobiotin (10 -13 M) and K is the acid dissociation constant of the guanidinium group (pK = 11.9)]. The dissociation constant of the avidin-iminobiotin complex increases from 0.03 /zM at pH 9 to 13 /zM at pH 6, by a factor of 430, rather than the theoretical factor of 10,000, the discrepancy being least above pH 8. It may be that the dissociation constant changes less at lower pH because of a contribution from weak binding of the protonated form of iminobiotin. 15 C. E. Argarana, I. D. Kuntz, S. Birken, R. Axel, and C. R. Cantor, Nucleic Acids Res. 14, 1871 (1986). t~ F. M. Finn. G. Titus, J. A. Montibeller, and K. Hofmann, J. Biol. Chem. 255, 5742 (1980). 17 T. V. Updyke and G. L. Nicolson, J. Immunol. Methods 73, 83 (1984). i8 y . Hiller, J. M. Gershoni, E. A. Bayer, and M. Wilchek, Biochem. J. 248, 167 (1987). J9 D. B. McCormick, Anal. Biochem. 13, 194 (1965). z0 K. Hofmann, S. W. Wood, C. C. Brinton, and F. M. Finn, Proc Natl. Acad. Sci. U.S.A. 77, 4666 (1980). 21 G. A. Orr, G. C. Heney, and R. Zeheb, this series, Vol. 122, p. 83. 22 G. A. Zeheb and G. A. Orr, this series, Vol. 122, p. 87. 23 E. A. Bayer, H. Ben Hur, G. Gitlin, and M. Wilchek, J.Biochem. Biophys. Methods 13, 103 (1986). 24 N. M. Green, Biochem. J. 101, 774 (1966).
54
BIOTIN-BINDING PROTEINS
[5]
The principle was extended by Hofmann's group, who made iminobiotinyl derivatives of hormones to allow efficient elution of hormone receptors,25,26 but unfortunately the affinities proved slightly too low to be useful. The dissociation constants for the aminohexanoate and lysine derivatives of iminobiotin at pH 6.8 (Kd = 10 5 M)25 were 5- to 10-fold greater than for the parent iminobiotin. The effects of using dethiobiotin and of incorporating spacer arms were also studied. A useful summary of the synthesis and properties of these derivatives is available. 27 The very high affinity of avidin for biotin, although ideal for specific labeling, is a disadvantage for affinity purifications of biotinyl enzymes, where it is not possible to substitute an iminobiotinyl residue. It is possible to weaken the binding by selective oxidation of avidin tryptophans with periodate (Kd = 10 -9 M ) , 6 but the affinity is still too high. A more successful approach has been the use of monomeric avidin-Sepharose, prepared by stripping off noncovalently bound subunits with guanidinium chloride. 6,28-3° The dissociation constant of the biotinyl complex of the monomer is about 10 7 M; however, other species with lower dissociation constants are present in these preparations, and these should be blocked with biotin before using the column. Lipoic acid has some stereochemical similarity to biotin, 6 and lipoyl Sepharose provides another matrix of moderate affinity which has been used for the purification of antibiotin antibodies. 31 It has also been used to purify complexes of myosin $1, with avidin, using the spare binding sites of an avidin molecule bound to biotinyl SI. 32 Conversely, avidinSepharose columns can, in principle, be used to purify lipoyl peptides. Streptavidin The gene for streptavidin has been cloned and sequenced with the ultimate objective of using it in general expression systems for detecting and isolating fusion proteins. ~5It coded for a sequence of 159 amino acids, some 30 residues longer than avidin and longer than expected from molec25 F. M. Finn and K. Hofmann, Proc. 7th Am. Peptide Symp. (Pierce Chemical Co., Rockford, ILL 1 (1981). 26 K. Hofmann, G. Titus, J. A. Montibeller, and F. M. Finn, Biochemistry 21, 978 (1982). 27 F. M. Finn and K. Hofmann, this series, Vol. 109, p. 418. 28 K. P. Henrikson, S. H. Allen, and W. L. Maloy, Anal. Biochem. 94, 366 (1979). 29 R. A. Kohanski and M. D. Lane, J. Biol. Chem. 260, 5014 (1985). 30 p. Dimroth, FEBS Lett. 141, 59 (1982). 31 F. Harmon, Anal. Biochem. 103, 58 (1980). n K. Yamamoto and T. Sekine, J. Biochem. 101, 519 (1987).
[5]
AV|DIN AND STREPTAVIDIN
55
TABLE I PROPERTIES OF AVIDINS Streptavidin Property Amino acid residues Subunit size F r o m sequence SDS gels Subunits Isoelectric pH e 28o
Ae233 (q- biotin) Fluorescence Xma×(nm) r (nsec) Binding of HABA Kd ( # M ) es0o Kd biotin (M) (pH 7, 25 °) [1/2 (days)
b c a e
Avidin egg white"
Unprocessed h
128
159
125-127
--
15,600 16,400 4 10 24,000 24,000
16,473 19,000 4 ----
13,400 14,500 4 5-6 34,000 8,000
-19,000 4 4.6 -7,000
338 (328) . 1.8 (0.8) . 6 35,000 0.6 x 10 15 200
. .
. . --
Processed C
Avidin, egg yolk J
. .
__
100 35,000 4 × 10 14
-7,000 1.7 × 10 12
--
2.9
0.07
-
-
F r o m Ref. 6. From Refs. 13 and 23. From Refs. 3 and 6. From Refs. 2 and 33. Fluorescence lifetimes were taken from Ref. 34. Numbers in parentheses refer to the avidin-biotin complex.
ular weight measurements. It was found that subunits of both low and high molecular weight were present and that the smaller one, the main constituent of most commercial preparations, was the result of processing at both the N and C termini to give " c o r e " streptavidin of 125-127 residues. 13,23 It had a much higher solubility in water than the unprocessed precursor. This core is identical to avidin at 33% of its residues, including the four tryptophan residues involved in the biotin-binding site. It also resembles avidin in its predicted secondary structure, predominantly/3 strands and bends, 15and in other features (Table I). 33,34 Some commercial preparations contain unprocessed streptavidin,13 and a recently published purification based on iminobiotin columns yields this as the main proda c t . 23
33 C. V. R. Murthy and P. R. Aliga, Biochem. Biophys. Acta 786, 222 (1984). 34 B. P. Maliwal and J. Lakowicz, Biophys. Chem. 19, 337 (1985).
56
BIOTIN-BINDING PROTEINS
[5]
Two differences from avidin are of some importance. Streptavidin contains no carbohydrate, the heterogeneity of which is the probable cause of the poor quality of avidin crystals,Jl and it has a slightly acid isoelectric point 3 (pH 5-6), which minimizes nonspecific adsorption to nucleic acids and negatively charged cell membranes. Quantitative data on its affinity for biotin and its analogs are sparse (see Table II below). General Properties of Avidins The main common features of biotin binding by both avidin and streptavidin (Table I) can be summarized briefly. The avidins are stable tetramers with 2-fold symmetry, the binding sites being arranged in two pairs on opposed faces of the molecule (see below). The stability is greatly enhanced by biotin binding, since the total free energy of binding is about 330 kJ/mol of tetramer. The dissociation constant for biotin is so low that it can be estimated only from the ratio of the rate constants for binding and exchange. The binding is accompanied by a red shift of the tryptophan spectrum and by a decrease in fluorescence, either of which can be used as the basis for quantitative assays. 35,36The spectral changes in the tryptophan residues are accompanied by a marked reduction in their accessibility to reagents such as a N-bromosuccinimide. In avidin, the four tryptophans of each subunit are protected when biotin is bound. In contrast, fluorescence quenching by oxygen is not diminished in the avidinbiotin complex; if anything, the rate constant for quenching is increased. 34 The binding of biotin can be blocked by oxidation of any of several tryptophan residues 6 or, in the case of avidin, by the dinitrophenylation of what appears to be a single lysine residue. 6 Isolation and sequencing of dinitrophenyl (DNP) peptides from avidin in which a single lysine is blocked showed that reaction at any of three lysines inactivates the avidin. 37 Similarly, two essential tryptophans were identified by isolation of peptides after labeling with hydroxynitrobenzyl bromide. 38 The location of these residues is shown in Fig. 1. These results show that inactivation by incorporation of 1 tool of reagent does not necessarily imply that a single specific group is involved. Reaction of any one of two or three different lysines or tryptophans led to inactivation and blocked further reaction. Furthermore, dinitrophenylation of the lysines blocked reaction of the tryptophans with the Koshland reagent, just as it had previously 35 N. 36 H. 37 G. 38 G.
M. Green, this series, Vol. 18A, p. 418. J. Lin and J. F. Kirsch, this series, Vol. 62, p. 287. Gitlin, E. A. Bayer, and M. Wilchek, Biochem. J. 242, 923 (1987). Gitlin, E. A. Bayer, and M. Wilchek, Biochem. J. 250, 291 (1988).
[5]
Streptav. 12 Avidin 1 B e t a pred. Conserved Fatty acid 1 Beta strands Ret.cell. 1
AVID[N AND STREPTAVIDIN i0 AAEAGITGTWYNQLGST ARKCSLTGKWTNDLGSN _
.
.
--
+
.
.
.+
.
20
.
:
.
.
57
30 FIVTAGAD MTIGAVNSR .
.
:*
. :
=
.
.
40 50 GALTGTYESAVGNAES GEFTGTYITAVT ATS
.
--=
.
.
.
.
:
.
.
.
: - -
MAFDGTWKVDRNENYEKFMEKMGINVVKRKLGAHDNLKLTITQEGNK . . . .
A . . . . . .
= : : : : : :
: : = = = : = =
. . . .
S .
.
.
.
.
.
.
PVDFNG~{KMLSNENFEEYLRALDVNVALRKIANLLKPDKEIVQDGDH
Streptav. 53 Avidin 42 B e t a pred. Conserved F a t t y a c i d 48 Beta strands Ret.cell. 49
RYVLTG NEIKES
60 70 80 90 RYDSAPATDGS GTALGWTVAWKNNYRNAHSATTWS PLHGTQNTINKRTQPTFGFTVNWKF SESTTVFT
_ _ _ *
.
Streptav. 97 Avidin 84 Beta pred. Conserved F a t t y a c i d 91 Beta strands Ret.cell. 94
VG FIDR
.
• ===
.
.
.
.
*
.
* *
100 QY GQC
. . . . . .
*.-
.=
= . - =
FTVKESSNFRNIDVVFEL GVDFAYSLAD GTELTGTWTMEGNKL -C . . . . . . . . D ........ E. . . . . . . F ....... MIIRTLSTFRNYIMDFQV GKEFEETGID DRKCMTTVSWDGDKL
. .--
.
Ii0 120 130 GAEARINTQWLL TSGTTEANAWKS NGKEVLKTMWLLRSSVNDIGDDWKA .
.
. ====.--
*----*---*
.--
140 150 TLVGHDTFTKVKPSAA TRVGINIFTRLRTQKE
* --=.--
*
. . . . . . . . . . . *
--
*=+
..
VGKFKRVDNGKELIAVREIS GNELIQTYTYEGVEAKRIFKKE G ......... H .......... I........ J--QCVQK GEKEGRGWTQWIE GDELHLEMRAEGVTCKQVFKKVH
FIG. 1. Alignment of avidin and streptavidin with fatty acid- and cellular retinol-binding proteins. The avidins t5 are aligned with the other two binding proteins, 39giving weight to the matching of predicted bends and strands ( - - - ) with the actual bends and strands ( - - X - - ) of the fatty acid-binding protein. 4° = = - indicates a helix. The serum retinol-binding protein and other members of the /3-1actoglobulin subclass are not shown because the percent identity is very low (Fig. 2) except for the N-terminal motif. Conservation is shown as follows: +, complete identity; =, identity of avidin and fatty acid-binding protein; - , conserved hydrophobic site;., other limited conservation. An asterisk indicates either a contact residue (bound fatty acid) or a site at which modification blocks biotin binding. 37-4°
been shown to protect them from periodate oxidation, 6 emphasizing the complex interactions which can occur in a restricted site. A gene for avidin has been cloned from chick oviduct. 4~ It corresponds to the variant with isoleucine at position 34. Its expression is induced in ~9 j. Sundelin, S. R. Das, U. Eriksson, L. Rask, and P. A. Peterson, J. Biol. Chem. 260, 6494 (1985). 40 j. C. Sacchetini, J. I. Gordon, and L. J. Banaszak, J. Biol. Chem 263, 5815 (1988). 41 M. t . Gope, R. A. Keinanen, P. A. Kristo, O. M. Conneely, W. G. Beattie, T. ZaruckiSchulz, B. W. O'Malley, and M. S. Kulomaa, Nucleic Acids Res. 15, 3595 (1987).
58
BIOTIN-BINDING PROTEINS
[5]
other tissues under inflammatory conditions, probably caused by a rise in progesterone levels. 42 Relation of Avidins to Proteins of Known Structure A search of the Protein Information Resource (PIR) database revealed no protein with significant similarity to avidin. A more detailed examination of potentially similar/3-structured proteins proved more informative. It is clear that avidin is c o m p o s e d almost entirely of/3 strands and bends, from analysis of its R a m a n spectrum, 43 from circular dichroism (CD) measurements, 6,44 and from the secondary structure predicted from the sequences. 15,43 Proteins with k n o w n antiparallel /3 structure fall into two main classes: (1) /3 sandwiches of 6-10 strands such as transthyretin (prealbumin) and immunoglobulins 45 and (2) orthogonal/3 barrels such as serum retinol-binding protein 46 and /3-1actoglobulin. 47 Comparison with the sequences of avidins showed no similarity to any of the first class but did reveal a short N-terminal motif (Fig. l) c o m m o n to all m e m b e r s of the second class. 39,48 The class of/3-barrel proteins has been extended by the discovery of several h o m o l o g o u s proteins ( - 4 0 % identity) which bind fatty acids, retinol, or steroids and share both the N-terminal motif and a /~-barrel structure 4° with the lactoglobulin subclass, although overall the sequences of the two subclasses show less than 20% identity. The alignments (Fig. l) and the derived matrix (Fig. 2) 49 show that avidin and streptavidin have greater sequence identity with this subclass of orthogonal/3-barrels than they have with the lactoglobulin subclass. The similarity is also greater than that b e t w e e n the two subclasses, although less than that between m e m b e r s of the same subclass. T a k e n in conjunction with the predicted secondary structure and the physical evidence for/3 strands, the evidence for a c o m m o n structure is good. N o t e that when the percent identity is as low as it is here (
AVIDIN AND STREPTAVIDIN
51
3.5.1.12) may eventually be extensively exploited in avidin-biotin technology, as a replacement for avidin, for biotinylation of binders, or as an analytical tool for releasing the biotin moiety from biotinylated material. The fact that biotin is a vitamin required in very small quantities for a variety of cellular processes reflects the very high affinity constants exhibited by the broad number of naturally occurring biotin-binding proteins which have been described. As time goes on, it would not be surprising if many more biotin-binding proteins will be described, many of which may prove suitable for application in avidin-biotin technology. In certain instances, some may even supercede the use of avidin, owing to improved molecular characteristics. The use of avidin-biotin technology would undoubtedly be increased enormously if other non-biotin-binding proteins which exhibit very high affinities for unrelated ligands would be described for complementary applications.
[5] A v i d i n a n d S t r e p t a v i d i n By N. MICHAEL Gl~EEN Introduction
The discovery of the protein avidin resulted from intensive nutritional investigations into the vitamin B complex.I Avidin proved to be a minor constituent of egg white that could induce a nutritional deficiency in rats by forming a very stable noncovalent complex with what was subsequently proved to be the B vitamin biotin. The biological role of avidin in egg white appeared to be that of a scavenger, inhibiting bacterial growth. A variant of lower affinity for biotin has been found in egg yolk, which may be important in regulating the supply of biotin during development. 2 Avidin is a highly specialized protein that is only rarely expressed. One might have expected that it was a vertebrate protein of fairly recent lineage were it not for the fact that a close relative, streptavidin, is expressed in a species of S t r e p t o m y c e s . 3 However, like proteins have not been found elsewhere in microorganisms, and the possibility remains that its occurrence in Streptomyces avidinii, a strain of Streptomyces lavendulae, reflects a chance transfection rather than an ancient lineage. The relation of the avidins to a recently discovered family of binding proteins is considered below. P. Gyorgy, in "The Vitamins" (W. H. Sebrell, Jr., and R. S. Harris, eds.), gol. 1, p. 527. Academic Press, New York, 1954. z H. W. Meslar, S. A. Camper, and H. B. White, J. Biol. Chem. 253, 6979 (1978). 3 L. Chaiet and F. J. Wolf, Arch. Biochem. Biophys. 106, 1 (1964).
METHODS IN ENZYMOLOGY, VOL. 184
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
52
BIOTIN-BINDING PROTEINS
[5]
Current interest in avidin derives mainly from its applications in biotechnology based on its rapid and almost irreversible binding of any molecule to which biotin can be linked. This has led to a proliferation of labeling and affinity purification methods, which are the subject of this volume. The biochemical basis for the very high affinity was first investigated by chemical modification.4 Interest was further stimulated by the discovery of the coenzyme function of biotin in CO2 transfer5 and by the subsequent use of avidin to identify biotinyl enzymes. More extensive characterization of the molecule and its binding sites followed, mainly from the author's laboratory, and the results of this were reviewed in 1975.6 At that time there was only one report of the use of avidin as a cytochemical label, 7 although from its general properties and its use as a high-resolution electron microscopic label for biotinyl enzymes, 8 it was clear that it had considerable potential in this field. Most work on avidin since the mid1970s has been on the development of such applications based on avidin labeled with gold, peroxidase, alkaline phosphatase, ferritin, or radioactive or fluorescent labels, using a variety of synthetic bridging agents to provide appropriate biotinyl sites for its attachment. 9,1° During this period there has been relatively little new biochemical work on the structure and binding properties of avidin or streptavidin. Considerable efforts have been devoted to an X-ray crystallographic approach to the structure, but it proved very difficult to grow acceptable crystals of sufficient size, J~ possibly because avidin is a glycoprotein and its carbohydrate is characteristically heterogeneous. ~2 In this respect, streptavidin, which is carbohydrate free, has proved advantageous. Its structure is now determined, but the results were not available for the preparation of this chapter. J3,J4 Another main advance has been the cloning and sequencing of the streptavidin gene.15 4 H. Fraenkel-Conrat, N. S. Snell, and E. D. Ducay, Arch. Biochem. Biophys. 39, 97 (1952). 5 S. J. Wakil, E. B. Titchener, and D. M. Gibson, Biochem. Biophys. Acta 29, 225 (1958). 6 N. M. Green, Adv. Protein Chem. 29, 85 (1975). 7 H. Heitzmann and F. M. Richards, Proc. Natl. Acad. Sci. U.S.A. 71, 3537 (1974). s N. M. Green, R. C. Valentine, N. G. Wrigley, F. Abroad, B. E. Jacobson, and H. G. Wood, J. Biol. Chem. 247, 6284 (1972). 9 E. A. Bayer and M. Wilchek, Methods Biochem. Anal. 26, 1 (1980). to M. Wilchek and E. A. Bayer, Anal. Biochem. 171, 1 (1988). ~i E. Pinn, A. Pahler, W. Saenger, G. A. Petsko, and N. M. Green, Eur. J. Biochem. 123, 545 (1982). t2 R. C. Bruch and H. B. White, Biochemistry 21, 5334 (1982). 13 p. C. Weber, D. H. Ohlendorf, J. J. Wendoloski, and F. R. Salemme, Science 243, 85 (1989). ~4 W. A. Hendrickson, A. P~hler, J. L. Smith, Y. Satow, E. A. Merritt, and R. P. Phizackerley, Proc. Natl. Acad. Sci. U.S.A. 86, 2190 (1989).
[5]
AVIDIN AND STREPTAVIDIN
53
In this chapter, I consider only fundamental molecular properties and those which are important for applications in biotechnology. Particular emphasis is placed on differences between avidin and streptavidin, since although streptavidin has become widely used as a label because of low nonspecific binding, its binding characteristics have not been studied quantitatively. The alternative use of succinylavidin ~6to diminish nonspecific avidin binding is less effective, probably because the carbohydrate of avidin also contributes to the nonspecific binding. ~7 Carbohydrate-free avidin can be obtained by use of deglycosylating enzymes, and it has been shown to be present in significant amounts in some commercial preparations from which it may be separated by use of lectin columns. ~sIts biotinbinding properties were unimpaired.
Affinity Methods for Purification of Avidins and Biotinyl Proteins Improvement on the original use of biotinyl cellulose ~9came with the introduction of iminobiotinyl derivatives of Sepharose 2°-23 which utilized the pH dependence of the binding 24 to achieve efficient elution. In iminobiotin, the ureido group becomes a guanidinium group, and only the form in which this is uncharged is strongly bound. The apparent affinity, therefore, should fall by a factor of 10 per pH unit provided that the protonated form does not bind [ K a p p : K B K / ( H + + K), where KB is the dissociation constant for uncharged iminobiotin (10 -13 M) and K is the acid dissociation constant of the guanidinium group (pK = 11.9)]. The dissociation constant of the avidin-iminobiotin complex increases from 0.03 /zM at pH 9 to 13 /zM at pH 6, by a factor of 430, rather than the theoretical factor of 10,000, the discrepancy being least above pH 8. It may be that the dissociation constant changes less at lower pH because of a contribution from weak binding of the protonated form of iminobiotin. 15 C. E. Argarana, I. D. Kuntz, S. Birken, R. Axel, and C. R. Cantor, Nucleic Acids Res. 14, 1871 (1986). t~ F. M. Finn. G. Titus, J. A. Montibeller, and K. Hofmann, J. Biol. Chem. 255, 5742 (1980). 17 T. V. Updyke and G. L. Nicolson, J. Immunol. Methods 73, 83 (1984). i8 y . Hiller, J. M. Gershoni, E. A. Bayer, and M. Wilchek, Biochem. J. 248, 167 (1987). J9 D. B. McCormick, Anal. Biochem. 13, 194 (1965). z0 K. Hofmann, S. W. Wood, C. C. Brinton, and F. M. Finn, Proc Natl. Acad. Sci. U.S.A. 77, 4666 (1980). 21 G. A. Orr, G. C. Heney, and R. Zeheb, this series, Vol. 122, p. 83. 22 G. A. Zeheb and G. A. Orr, this series, Vol. 122, p. 87. 23 E. A. Bayer, H. Ben Hur, G. Gitlin, and M. Wilchek, J.Biochem. Biophys. Methods 13, 103 (1986). 24 N. M. Green, Biochem. J. 101, 774 (1966).
54
BIOTIN-BINDING PROTEINS
[5]
The principle was extended by Hofmann's group, who made iminobiotinyl derivatives of hormones to allow efficient elution of hormone receptors,25,26 but unfortunately the affinities proved slightly too low to be useful. The dissociation constants for the aminohexanoate and lysine derivatives of iminobiotin at pH 6.8 (Kd = 10 5 M)25 were 5- to 10-fold greater than for the parent iminobiotin. The effects of using dethiobiotin and of incorporating spacer arms were also studied. A useful summary of the synthesis and properties of these derivatives is available. 27 The very high affinity of avidin for biotin, although ideal for specific labeling, is a disadvantage for affinity purifications of biotinyl enzymes, where it is not possible to substitute an iminobiotinyl residue. It is possible to weaken the binding by selective oxidation of avidin tryptophans with periodate (Kd = 10 -9 M ) , 6 but the affinity is still too high. A more successful approach has been the use of monomeric avidin-Sepharose, prepared by stripping off noncovalently bound subunits with guanidinium chloride. 6,28-3° The dissociation constant of the biotinyl complex of the monomer is about 10 7 M; however, other species with lower dissociation constants are present in these preparations, and these should be blocked with biotin before using the column. Lipoic acid has some stereochemical similarity to biotin, 6 and lipoyl Sepharose provides another matrix of moderate affinity which has been used for the purification of antibiotin antibodies. 31 It has also been used to purify complexes of myosin $1, with avidin, using the spare binding sites of an avidin molecule bound to biotinyl SI. 32 Conversely, avidinSepharose columns can, in principle, be used to purify lipoyl peptides. Streptavidin The gene for streptavidin has been cloned and sequenced with the ultimate objective of using it in general expression systems for detecting and isolating fusion proteins. ~5It coded for a sequence of 159 amino acids, some 30 residues longer than avidin and longer than expected from molec25 F. M. Finn and K. Hofmann, Proc. 7th Am. Peptide Symp. (Pierce Chemical Co., Rockford, ILL 1 (1981). 26 K. Hofmann, G. Titus, J. A. Montibeller, and F. M. Finn, Biochemistry 21, 978 (1982). 27 F. M. Finn and K. Hofmann, this series, Vol. 109, p. 418. 28 K. P. Henrikson, S. H. Allen, and W. L. Maloy, Anal. Biochem. 94, 366 (1979). 29 R. A. Kohanski and M. D. Lane, J. Biol. Chem. 260, 5014 (1985). 30 p. Dimroth, FEBS Lett. 141, 59 (1982). 31 F. Harmon, Anal. Biochem. 103, 58 (1980). n K. Yamamoto and T. Sekine, J. Biochem. 101, 519 (1987).
[5]
AV|DIN AND STREPTAVIDIN
55
TABLE I PROPERTIES OF AVIDINS Streptavidin Property Amino acid residues Subunit size F r o m sequence SDS gels Subunits Isoelectric pH e 28o
Ae233 (q- biotin) Fluorescence Xma×(nm) r (nsec) Binding of HABA Kd ( # M ) es0o Kd biotin (M) (pH 7, 25 °) [1/2 (days)
b c a e
Avidin egg white"
Unprocessed h
128
159
125-127
--
15,600 16,400 4 10 24,000 24,000
16,473 19,000 4 ----
13,400 14,500 4 5-6 34,000 8,000
-19,000 4 4.6 -7,000
338 (328) . 1.8 (0.8) . 6 35,000 0.6 x 10 15 200
. .
. . --
Processed C
Avidin, egg yolk J
. .
__
100 35,000 4 × 10 14
-7,000 1.7 × 10 12
--
2.9
0.07
-
-
F r o m Ref. 6. From Refs. 13 and 23. From Refs. 3 and 6. From Refs. 2 and 33. Fluorescence lifetimes were taken from Ref. 34. Numbers in parentheses refer to the avidin-biotin complex.
ular weight measurements. It was found that subunits of both low and high molecular weight were present and that the smaller one, the main constituent of most commercial preparations, was the result of processing at both the N and C termini to give " c o r e " streptavidin of 125-127 residues. 13,23 It had a much higher solubility in water than the unprocessed precursor. This core is identical to avidin at 33% of its residues, including the four tryptophan residues involved in the biotin-binding site. It also resembles avidin in its predicted secondary structure, predominantly/3 strands and bends, 15and in other features (Table I). 33,34 Some commercial preparations contain unprocessed streptavidin,13 and a recently published purification based on iminobiotin columns yields this as the main proda c t . 23
33 C. V. R. Murthy and P. R. Aliga, Biochem. Biophys. Acta 786, 222 (1984). 34 B. P. Maliwal and J. Lakowicz, Biophys. Chem. 19, 337 (1985).
56
BIOTIN-BINDING PROTEINS
[5]
Two differences from avidin are of some importance. Streptavidin contains no carbohydrate, the heterogeneity of which is the probable cause of the poor quality of avidin crystals,Jl and it has a slightly acid isoelectric point 3 (pH 5-6), which minimizes nonspecific adsorption to nucleic acids and negatively charged cell membranes. Quantitative data on its affinity for biotin and its analogs are sparse (see Table II below). General Properties of Avidins The main common features of biotin binding by both avidin and streptavidin (Table I) can be summarized briefly. The avidins are stable tetramers with 2-fold symmetry, the binding sites being arranged in two pairs on opposed faces of the molecule (see below). The stability is greatly enhanced by biotin binding, since the total free energy of binding is about 330 kJ/mol of tetramer. The dissociation constant for biotin is so low that it can be estimated only from the ratio of the rate constants for binding and exchange. The binding is accompanied by a red shift of the tryptophan spectrum and by a decrease in fluorescence, either of which can be used as the basis for quantitative assays. 35,36The spectral changes in the tryptophan residues are accompanied by a marked reduction in their accessibility to reagents such as a N-bromosuccinimide. In avidin, the four tryptophans of each subunit are protected when biotin is bound. In contrast, fluorescence quenching by oxygen is not diminished in the avidinbiotin complex; if anything, the rate constant for quenching is increased. 34 The binding of biotin can be blocked by oxidation of any of several tryptophan residues 6 or, in the case of avidin, by the dinitrophenylation of what appears to be a single lysine residue. 6 Isolation and sequencing of dinitrophenyl (DNP) peptides from avidin in which a single lysine is blocked showed that reaction at any of three lysines inactivates the avidin. 37 Similarly, two essential tryptophans were identified by isolation of peptides after labeling with hydroxynitrobenzyl bromide. 38 The location of these residues is shown in Fig. 1. These results show that inactivation by incorporation of 1 tool of reagent does not necessarily imply that a single specific group is involved. Reaction of any one of two or three different lysines or tryptophans led to inactivation and blocked further reaction. Furthermore, dinitrophenylation of the lysines blocked reaction of the tryptophans with the Koshland reagent, just as it had previously 35 N. 36 H. 37 G. 38 G.
M. Green, this series, Vol. 18A, p. 418. J. Lin and J. F. Kirsch, this series, Vol. 62, p. 287. Gitlin, E. A. Bayer, and M. Wilchek, Biochem. J. 242, 923 (1987). Gitlin, E. A. Bayer, and M. Wilchek, Biochem. J. 250, 291 (1988).
[5]
Streptav. 12 Avidin 1 B e t a pred. Conserved Fatty acid 1 Beta strands Ret.cell. 1
AVID[N AND STREPTAVIDIN i0 AAEAGITGTWYNQLGST ARKCSLTGKWTNDLGSN _
.
.
--
+
.
.
.+
.
20
.
:
.
.
57
30 FIVTAGAD MTIGAVNSR .
.
:*
. :
=
.
.
40 50 GALTGTYESAVGNAES GEFTGTYITAVT ATS
.
--=
.
.
.
.
:
.
.
.
: - -
MAFDGTWKVDRNENYEKFMEKMGINVVKRKLGAHDNLKLTITQEGNK . . . .
A . . . . . .
= : : : : : :
: : = = = : = =
. . . .
S .
.
.
.
.
.
.
PVDFNG~{KMLSNENFEEYLRALDVNVALRKIANLLKPDKEIVQDGDH
Streptav. 53 Avidin 42 B e t a pred. Conserved F a t t y a c i d 48 Beta strands Ret.cell. 49
RYVLTG NEIKES
60 70 80 90 RYDSAPATDGS GTALGWTVAWKNNYRNAHSATTWS PLHGTQNTINKRTQPTFGFTVNWKF SESTTVFT
_ _ _ *
.
Streptav. 97 Avidin 84 Beta pred. Conserved F a t t y a c i d 91 Beta strands Ret.cell. 94
VG FIDR
.
• ===
.
.
.
.
*
.
* *
100 QY GQC
. . . . . .
*.-
.=
= . - =
FTVKESSNFRNIDVVFEL GVDFAYSLAD GTELTGTWTMEGNKL -C . . . . . . . . D ........ E. . . . . . . F ....... MIIRTLSTFRNYIMDFQV GKEFEETGID DRKCMTTVSWDGDKL
. .--
.
Ii0 120 130 GAEARINTQWLL TSGTTEANAWKS NGKEVLKTMWLLRSSVNDIGDDWKA .
.
. ====.--
*----*---*
.--
140 150 TLVGHDTFTKVKPSAA TRVGINIFTRLRTQKE
* --=.--
*
. . . . . . . . . . . *
--
*=+
..
VGKFKRVDNGKELIAVREIS GNELIQTYTYEGVEAKRIFKKE G ......... H .......... I........ J--QCVQK GEKEGRGWTQWIE GDELHLEMRAEGVTCKQVFKKVH
FIG. 1. Alignment of avidin and streptavidin with fatty acid- and cellular retinol-binding proteins. The avidins t5 are aligned with the other two binding proteins, 39giving weight to the matching of predicted bends and strands ( - - - ) with the actual bends and strands ( - - X - - ) of the fatty acid-binding protein. 4° = = - indicates a helix. The serum retinol-binding protein and other members of the /3-1actoglobulin subclass are not shown because the percent identity is very low (Fig. 2) except for the N-terminal motif. Conservation is shown as follows: +, complete identity; =, identity of avidin and fatty acid-binding protein; - , conserved hydrophobic site;., other limited conservation. An asterisk indicates either a contact residue (bound fatty acid) or a site at which modification blocks biotin binding. 37-4°
been shown to protect them from periodate oxidation, 6 emphasizing the complex interactions which can occur in a restricted site. A gene for avidin has been cloned from chick oviduct. 4~ It corresponds to the variant with isoleucine at position 34. Its expression is induced in ~9 j. Sundelin, S. R. Das, U. Eriksson, L. Rask, and P. A. Peterson, J. Biol. Chem. 260, 6494 (1985). 40 j. C. Sacchetini, J. I. Gordon, and L. J. Banaszak, J. Biol. Chem 263, 5815 (1988). 41 M. t . Gope, R. A. Keinanen, P. A. Kristo, O. M. Conneely, W. G. Beattie, T. ZaruckiSchulz, B. W. O'Malley, and M. S. Kulomaa, Nucleic Acids Res. 15, 3595 (1987).
58
BIOTIN-BINDING PROTEINS
[5]
other tissues under inflammatory conditions, probably caused by a rise in progesterone levels. 42 Relation of Avidins to Proteins of Known Structure A search of the Protein Information Resource (PIR) database revealed no protein with significant similarity to avidin. A more detailed examination of potentially similar/3-structured proteins proved more informative. It is clear that avidin is c o m p o s e d almost entirely of/3 strands and bends, from analysis of its R a m a n spectrum, 43 from circular dichroism (CD) measurements, 6,44 and from the secondary structure predicted from the sequences. 15,43 Proteins with k n o w n antiparallel /3 structure fall into two main classes: (1) /3 sandwiches of 6-10 strands such as transthyretin (prealbumin) and immunoglobulins 45 and (2) orthogonal/3 barrels such as serum retinol-binding protein 46 and /3-1actoglobulin. 47 Comparison with the sequences of avidins showed no similarity to any of the first class but did reveal a short N-terminal motif (Fig. l) c o m m o n to all m e m b e r s of the second class. 39,48 The class of/3-barrel proteins has been extended by the discovery of several h o m o l o g o u s proteins ( - 4 0 % identity) which bind fatty acids, retinol, or steroids and share both the N-terminal motif and a /~-barrel structure 4° with the lactoglobulin subclass, although overall the sequences of the two subclasses show less than 20% identity. The alignments (Fig. l) and the derived matrix (Fig. 2) 49 show that avidin and streptavidin have greater sequence identity with this subclass of orthogonal/3-barrels than they have with the lactoglobulin subclass. The similarity is also greater than that b e t w e e n the two subclasses, although less than that between m e m b e r s of the same subclass. T a k e n in conjunction with the predicted secondary structure and the physical evidence for/3 strands, the evidence for a c o m m o n structure is good. N o t e that when the percent identity is as low as it is here (
