[5] Avidin and streptavidin

[5] Avidin and streptavidin

[5] AVIDIN AND STREPTAVIDIN 51 3.5.1.12) may eventually be extensively exploited in avidin-biotin technology, as a replacement for avidin, for biot...

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[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).

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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

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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).

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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

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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 (<20%), there are m a n y possible alignments which give almost equivalent scores and that the highest score does not necessarily c o r r e s p o n d to the structurally most significant alignment. The alignments 42 H. A. Elo, M. S. Kulomaa, and A. O. Niemela, Comp. Biochem. Physiol. 68A, 323 (1981). 43 R. B. Honzatko and R. W. Williams, Biochemistry 21, 6201 (1982). N. M. Green and M. D. Melamed, Biochem. J. 100, 614 (1966).

45j. S. Richardson, this series, Vol. 115, p. 341. 46 M. E. Newcomer, T. A. Jones, J. Aquist, J. Sundelin, U. Eriksson, L. Rask, and P. A. Peterson, E M B O J. 3, 1451 (1984). 47M. Z. Papiz, L. Sawyer, E. Eliopoulos, A. C. T. North, J. B. C. Findlay, R. Sivraprasadarao, T. A. Jones, M. E. Newcomer, and P. J. Kraulis, N a t u r e (London) 324, 383 (1986). 48j. Godovac-Zimmerman, Trends Biochem. Sci. 13, 64 (1988). 49 R. Huber, M. Schneider, O. Epp, I. Mayr, A. Messerschmidt, J. Pflugrath, and H. Kayser, J. Mol. Biol. 195, 423 (1987).

[5]

AVIDIN AND STREPTAVIDIN

Streptavidin

59

30

12

12

9

Avidin

18

9

8

28

7

9

8

4

Fatty acid ( r a t )

II

Retinol ( c e l l u l a r ) Retinol

(serum)

21

Lactoglobulin FIG. 2. Identity matrix between orthogonal B-barrel proteins arranged in three subclasses. The percent identities were calculated for the alignments shown in Fig. 1. The percent identity for the lactoglobulin subclass was taken from a structurally based alignment, 49 which was then matched with Fig. 1. The greatest resemblances outside the subclasses are between avidin and the fatty acid-binding protein. Structures of the underlined proteins have been determined. 4°,46,47

in Fig. 1 were obtained by matching the N-terminal motifs and as far as possible matching predicted strands and bends with corresponding features of the known structure. Several lysine and tryptophan residues marked with an asterisk in Fig. 1 contribute to the biotin-binding site 37,38 and are mostly located in regions homologous to those in the fatty acidbinding protein which make contact with ligand. One feature of the structure of the fatty acid-binding protein which is not present in the predicted secondary structures of the avidins is the pair of short a helices between the A and B strands. These are also absent from the lactoglobulin subclass, where they are replaced by a short /3 hairpin which extends from the A and B strands. Although uncertainties of both the alignment and the secondary structure prediction prevent detailed structural conclusions, it can be said that avidins are probably orthogonal /3 barrels with binding sites enclosed between/3 sheets. Such a deep binding site is consistent both with the very high affinity and with the deep burial of the binding sites, implied by the results with bifunctional ligands. 5° However, deep burial of a site does not necessarily correlate with a very high affinity. The dissociation constants for fatty acids and retinol, for example, are between 10 -6 and 10 -8 M. 50 N. M. Green, L. Konieczny, E. J. Toms, and R. C. Valentine, Biochem. J. 125, 781 (1971).

60

BIOTIN-BINDING PROTEINS

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Dissociation Constants for Biotin

The rate constant for biotin binding has been measured only once (7 × sec -I for avidin and biotin at pH 5, 25°), so that values quoted for dissociation constants of other ligands or other conditions, which are based on this value, are only approximate. Direct measurements of the much higher dissociation constants of the D and I_ isomers of hexylimidazolidone have been compared with those calculated from the rate constants 6 and were found to be higher by a factor of about 4. This could result partly from the rate of binding for the uncharged ligand being lower than that for biotin, since avidin and biotin carry opposite charges at pH 5. Other factors are also involved so that absolute values of most binding constants estimated from rate constants could be in error by an order of magnitude. Since no measurements of rate constants for either binding or dissociation for the widely used biotin-streptavidin system have been published, some results of recent measurements of the exchange rate are given here (Table II). 51,52 ;(hey are compared with previous results for avidin52 and show a considerably faster release of biotin, in agreement with an earlier comment referring to unpublished work. 53The rate constants increased as the pH rose, opposite to the behavior of avidin. This could be a consequence of the different isoelectric points. Streptavidin would be expected to show maximum stability near pH 5 and to release its ligand more readily at extremes of pH, where the net charge on the protein increases. In contrast, the net charge on avidin would fall with rising pH up to pH 10.5. The results in Table II are consistent with this hypothesis. 10 7 M -1

Avidin Polymers Current knowledge of the spatial relations between the four binding sites of avidin comes from an electron microscopic study of polymers made with bifunctional biotinyldiamines.5° Now that the structure of streptavidin is known at high resolution it is worth comparing its behavior with these reagents, since this could reveal differences in subunit relationships. In the reaction with avidin, at least 9 methylene groups between biotinamides are required to produce polymers, and 12 are required to make them sufficiently stable to resist depolymerization by biotin, implying binding sites of considerable depth (15 A). A further increase in length s~ R. K. Garlick and R. W. Giese, J. Biol. Chem. 263, 210 (1988). 52 N. M. Green and E. J. Toms, Biochem. J. 133, 687 (1973). 53 F. M. Finn, G. Titus, and K. Hofmann, Biochemistry 23, 2554 (1984).

[5]

AVIDIN AND STREPTAVIDIN

61

T A B L E II DISSOCIATION RATES OF AVIDIN-BIOTIN COMPLEXESa pH

Rate and half-life Avidin k (sec -~ × 107) t~/: (days) Streptavidin k (sec -j × 107) tl/2 (days)

1.7

2.0

3.0

5.0

7.0

9.2

10.5

---

200 0.4

9 9

0.9 90

0.4 200

---

---

35 2.3

---

19 4.2

8.7 9.2

28 2.9

64 1.25

100 0.8

Exchange rates for streptavidin (Celltech, Slough, U.K.) were measured at 25 ° using [~4C]biotin that had been purified by HPLC. 51 The exchange followed a simple firstorder course apart from the first 10% which, as with avidin, exchanged more rapidly.

The methods and the results for avidin are taken from Ref. 52.

of linking chain (19-22 atoms) greatly decreases the length of the polymers, showing that intramolecular bridging occurs and that the shortest path between biotin carboxyls (not necessarily linear) is about 25 .~ (Fig. 3 and Ref. 50). Pairs of sites are positioned at opposite ends of the short (40 ,~) axis of the avidin molecule, each pair being located in a depression in the protein surface. Similarly situated sites have recently been observed in a tetrameric biliverdin-binding protein, 49 a member of the lactoglobulin family. The presence of a depression in the protein surface could account for the observation that the length of linking chain required to produce an effective heterobifunctional reagent, such as those introduced by Hof-

a

Avidin

b

Streptavidin

B25

FIG. 3. Spatial relations between neighboring binding sites in avidin and streptavidin. Avidin polymers form when there are more than 8 methylene groups between biotins. Intramolecular bifunctional binding of bisbiotinyl compounds requires a linking chain of at least 19 atoms for avidin (a), whereas a chain of 12 is sufficient for streptavidin (b).

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BIOTIN-BINDING PROTEINS

[5]

m a n n f o r i s o l a t i n g h o r m o n e r e c e p t o r s , 27 c a n b e s h o r t e r t h a n t h a t r e q u i r e d to g i v e a v i d i n h o m o p o l y m e r s . It s u g g e s t s t h a t a s m a l l o r f l e x i b l e s e c o n d p r o t e i n c a n a p p r o a c h m o r e c l o s e l y to t h e b i o t i n site t h a n c a n a s e c o n d a v i d i n m o l e c u l e . A f u r t h e r e x a m p l e o f this is p r o v i d e d by t h e D N P g r o u p o f D N P b i o t i n h y d r a z i d e , w h i c h w h e n b o u n d to a v i d i n is a c c e s s i b l e to

FIG. 4. Avidin polymers. Electron micrographs of streptavidin combined with (a) bis(biotinamido)dodecane (B ~5) and (b) bis-[biotinamidobutyramido]decane (B25). In both cases 2 tool of the reagent is bound to each tetramer. The sparsity of polymeric species in (a) implies that most of the bifunctional ligand molecules are bridging two sites within a tetramer (Fig. 3). Only the long-chain reagent gave significant amounts of polymer (b). The repeat distance for this polymer was 46 ~,, identical to that observed with avidin and the same B2.~ reagent. 5° The results shown were obtained with streptavidin from Celltech. A second set of experiments with unprocessed streptavidin gave similar results. Bar, 4 nm.

[5]

AVIDIN AND STREPTAVIDIN

63

anti-DNP antibody, as measured by fluorescence quenching, although there are only two nitrogen atoms between the DNP group and the carboxyl group of biotin (N. M. Green, unpublished). Experiments on the reactions between streptavidin and the same series of bisbiotinyl compounds showed interesting differences. Titrations in the presence of the dye 2-(4'-hydroxyazobenzene)benzoic acid (HABA) showed some bifunctional behavior when there were 8 methylene groups on the linking chain (compound BI 0, and this increased gradually as the chain length was increased to 12 methylene groups (B~5). This is similar to previous observations with avidin, except that the change from monofunctional to bifunctional behavior is not so sharply defined. Electron microscopy of the products from B12 to B15 showed them to be 90% monomeric (Fig. 4a) with a few dimers and trimers. This differs markedly from avidin, which gave long p o l y m e r s 6'44 (n = 10-20). The streptavidin products resembled those given by avidin with the long-chain B25 reagent, suggesting that binding sites of streptavidin are much closer together and that in this case intramolecular bifunctional binding is strongly preferred over polymer formation, even at short chain lengths. Again, in contrast to avidin, the long-chain B25 reagent gave the longest polymers (n = 7, Fig. 4b), probably reflecting differences in local topography of the sites, which made it difficult for biotin at the end of a long chain to find its way back to the neighboring site (Fig. 3b). We conclude that while the individual subunits and binding sites of avidin and streptavidin have similar structures, based on sequence similarity and conservation of tryptophan involvement, the angular relations between subunits in the tetramer is such as to bring the openings into the binding clefts of streptavidin closer together by about 10 ,&. The very high affinity and the 2-fold symmetry of the tetramer form the basis of one of the most useful attributes of the avidin-biotin system. The initial binding is almost irreversible, provided that the biotinyl residue is accessible, and the 2-fold symmetry ensures that if the avidin is binding to a surface molecule many outwardly directed biotin sites will remain vacant and can be saturated with a second biotinyl ligand without displacement of the avidin from the labeled site. This provides the basis for the variety of sandwich and amplification techniques. The polymers themselves can be prepared with their terminal binding sites vacant, and these have been used as markers for both nucleotide sites and reactive thiol groups on myosin. 54 The polymers can be extensively labeled with concanavalin A (Con A) (Fig. 5), showing that the carbohydrate is not located on the surfaces which carry the binding sites, 54 K. Sutoh, K. Y a m a m o t o , and T. Wakabayashi, Proc. Nat. Acad. Sci. U.S.A. 83, 212 (1986).

64

BIOTIN-BINDING PROTEINS

[5]

FIG. 5. Avidin polymers (BI5), labeled with concanavalin A. Avidin polymers (15 k~g/ml) were adsorbed on carbon film. The film was transferred to a solution of Con A (20 p~g/ml, 0.15 M NaC1, pH 7.5) and left for 0.5-5 min to adsorb. The field shown was labeled for 2 min. Con A tetramers (MW 100,000) are larger than avidin, and their profiles are sometimes triangular. They can be seen in rows, sometimes on both sides of an avidin polymer. Bar, 4nm.

since these would be inaccessible. Linear polymers have also been obs e r v e d w h e n a v i d i n b i n d s to o l i g o m e r i c b i o t i n y l e n z y m e s , s u c h as p y r u v a t e c a r b o x y l a s e , 55,56 f r o m w h i c h c o n c l u s i o n s c o u l d b e d r a w n a b o u t t h e l o c a t i o n s o f t h e c a t a l y t i c sites.

K i n e t i c s of D i s p l a c e m e n t of B i o t i n y l L i g a n d s T h e r e h a v e b e e n a n u m b e r o f s t u d i e s o f the effects o f a v a r i e t y o f s u b s t i t u e n t s at t h e n i t r o g e n o f b i o t i n a m i d e on t h e r a t e o f d i s p l a c e m e n t b y b i o t i n u n d e r d i f f e r e n t c o n d i t i o n s ( T a b l e III). T h e r a t e s i n c r e a s e r a p i d l y w i t h r i s e in t e m p e r a t u r e ( × 2 f o r 5°), w h i c h is c o n s i s t e n t w i t h the high ~ H f o r b i o t i n b i n d i n g . 6 T h e y a l s o i n c r e a s e w h e n a b u l k y s u b s t i t u e n t is p r e s e n t , a n d this e f f e c t c a n b e e l i m i n a t e d b y i n c o r p o r a t i n g a s i x - c a r b o n s p a c e r a r m . z6,27 S m a l l h y d r o p h o b i c g r o u p s s u c h as i o d o p h e n o l c a n d e c r e a s e t h e d i s s o c i a t i o n r a t e . E f f e c t s o f p H a r e v a r i a b l e (see a l s o T a b l e II) a n d a r e e s p e c i a l l y m a r k e d w h e n c h a r g e d g r o u p s a r e i n t r o d u c e d into b i o t i n itself. 6 S o m e o f t h e s e e f f e c t s a r e d i s c u s s e d in d e t a i l in an e a r l i e r r e p o r t on 55 W. J o h a n n s s e n , P. V. Attwood, J. C. Wallace, and D. B. Keech, Eur. J. Biochem. 133, 201 (1983).

~6M. Rohde, F. Lira, and J. C. Wallace, Eur. J. Biochern. 156, 15 (1986).

[5]

AVIDIN AND STREPTAVIDIN

65

o~ ~ . ~~

*

~m

.~-~ ~ea •

~

~"NE + + + +

~

+

-~

a~~ ~

0 Z 0

>

0

z

h 0 ~L

d. 0

h

o

~

o.~.~ ~ ~ ~ - ~ = ~ ~.~

~-~-

~.~

Z'~Z,

"~. z z ' ~ z

66

BIOTIN-BINDING PROTEINS Cis

B_S

[5]

Trans

L-B B-£

L-B

FIG. 6. Interpretation of slow and rapid stages of the exchange process for bulky biotinyl ligands. When bulky ligands occupy neighboring sites, one is displaced relatively rapidly by biotin, leaving firmly bound ligands in the trans position that do not interfere and exchange more slowly.

the use of iodinated biotin derivatives. 52 Succinylation of the avidin has relatively little effect on the exchange rate. 5~ A surprising observation of slow- and fast-exchanging ligands has been made in several sets of measurements, 52,56-59 in apparent conflict with earlier conclusions (from titrations of weakly binding analogs) that the four biotin-binding sites are equivalent and noninteracting. 6 The effect is apparent only in complexes with bulky substituents on the biotin carboxyl and is most marked with fully saturated AB4 complexes. AB complexes show only a single, slow exchange rate. The effect can be interpreted in terms of the 2-fold symmetry of the subunit arrangements. The binding sites are organized in pairs on opposite faces of the avidin tetramer, with the biotin carboxyls separated by 15-25 A (Fig. 6). After saturation of the first site, the second mole of ligand can enter either a cis or a trans position relative to an occupied site. If the biotin carries a bulky substituent, then the cis arrangement will be less stable, and in a fully saturated avidin (AB4), one-half of the ligands will exchange relatively rapidly. These conclusions were originally drawn by Sinha and ChignelW from a study of the binding of the spin-labeled derivatives such as biotin amido tetramethylpiperidine N-oxide. The ESR spectrum of the complex with two sites occupied changed slowly from one which included a major component characteristic of interacting nitroxides (implying a separation of <15 A) to one in which there was no detectable interaction. The rate constant was the same as the rate of displacement of the first two biotinyl 57 H. Romovacek, F. M. Finn, and K. Hofmann, Biochemistry 22, 904 (1983). 58 S. Lavielle, G. Chassaing, and A. Marquet, Biochem. Biophys. Acta 759, 270 (1983). 59 B. K. Sinha and C. F. Chignell, this series, Vol. 62, p. 295.

[5]

AVIDIN AND STREPTAVIDIN

67

nitroxides from a 4 : 1 complex (Table III). This interesting observation is consistent with random initial binding followed by dissociation of nitroxide from the less stable cis sites and recombination at trans sites (Fig. 6). Conclusions

This chapter provides a summary of recent advances in understanding the interactions between avidin and biotin derivatives, some of which may prove useful for the development of new techniques. Undoubtedly many novel approaches will emerge, stimulated by knowledge of the detailed structure of streptavidin and by the cloning of the avidin genes. Combined with new methods for making biotinyl nucleotides, 6° a range of applications in molecular genetics can be foreseen. The system could also be expanded by employing a hapten such as DNP, which induces antibodies of very high affinity, to provide a second type of noncovalent linker. This would allow controlled coupling of different macromolecules and could serve to simulate mechanisms or test hypotheses of transmembrane signaling. Another area yet to be developed is the use of avidin monolayers as a basis for biosensor technology. An important new biological role for avidin appears likely to emerge from a recent observation of an avidinlike domain (30% identity) in a gene expressed in the developing ectoderm of the sea urchin embryos. 61,62 The gene product also includes multiple repeats of an epidermal growth factorlike sequence, and it seems likely that it has some biotin-dependent control function. Acknowledgments I thank Dr. N. G. Wrigley for electron micrographs, Dr. Meir Wilchek and Dr. R. M. Buckland (Celltech) for samples of streptavidin, and Mr. B. J. Trinnaman for skilled assistance.

60 j. M. Rothenberg and M. Wilchek, Nucleic Acids Res. 16, 7197 (1988). 6t D. A. Hursh, M. E. Andrews, and R. A. Raft, Science 237, 1487 (1987). 62 W. C. Barker, L. T. Hunt, and D. G. George, Protein Sequence Data Anal. 1, 363 (1988).