circular dichroism analysis of the glucan binding domain of streptococcus mutans glucan binding...

Ž .Biochimica et Biophysica Acta 1384 1998 112–120

Circular dichroism analysis of the glucan binding domain ofStreptococcus mutans glucan binding protein-A

Wolfgang Haas a,), Robert MacColl b, Jeffrey A. Banas a

a Albany Medical College-A68, 47 New Scotland AÕe., Albany, NY 12208, USAb Wadsworth Center, New York State Department of Health, P.O. Box 509, Albany, NY 12201, USA

Received 15 September 1997; revised 8 December 1997; accepted 15 December 1997

Abstract

Ž . Ž .The glucan binding domain GBD of the glucan binding protein-A GBP-A from the cariogenic bacterium Streptococ-Ž .cus mutans was studied using circular dichroism CD analysis, Chou–Fasman–Rose secondary structure prediction, and

absorption and fluorescence spectroscopy. Our data show that the binding domain undergoes a conformational shift uponbinding to the ligand dextran. The CD spectrum shows two positive bands at 280 nm and 230 nm which were assigned toaromatic residues. The 230-nm band was seen at 208C and 308C, lost intensity at 408C, and was eliminated at 458Ccoinciding with complete denaturation. The protein was stable at physiological pH, but precipitated at pH 5. A pH of 10changed the secondary structure but had no effect on the 230-nm band. Analysis of the CD data in the far UV using theSELCON computer program revealed a high content of b-sheets and a lack of a-helical structures. Secondary structureprediction based on the amino acid sequence of GBD agreed with the CD analysis. The fluorescence emission maximum at339 nm suggested that the majority of the tryptophans were located in the interior of the protein. This maximum shifted tohigher energy upon binding to the ligand dextran. q 1998 Elsevier Science B.V.

Ž .Keywords: Glucan binding domain; Circular dichroism; Secondary structure; Streptococcus mutans

1. Introduction

Commensal and pathogenic bacteria generally re-quire some mechanism of adherence to the host inorder to be able to colonize their ecological niche.This is especially true for bacteria that colonize thesmooth surfaces of the teeth where the constant flowof saliva and the mechanical forces of the tongue and

w xfood movement make strong adhesion necessary 1 .Oral streptococci, such as Streptococcus mutans, se-

) Corresponding author. Fax: q1-518-262-5748; E-mail:[email protected]

crete glucosyltransferases which use dietary sucroseto produce extracellular glucan polymers. These glu-cans are very adhesive by nature and allow the

w xbacteria to form a biofilm on the tooth surface 1,2 .S. mutans was shown to possess at least three

Ž .distinct, nonenzymatic glucan binding proteins GBPw xthat are either secreted or cell-bound 3–5 . While the

cell-surface protein GBP-C clearly has a lectin-likew xfunction 5 , the role of the other two proteins re-

mains to be elucidated. GBP-A, formerly describedw xas the 74-kDa GBP, was cloned and sequenced 3

and shown to bind to dextran-like glucans containinga-1,6-glucosidic linkages. Binding is mediated by a

Ž .glucan binding domain GBD which is present at the

0167-4838r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved.Ž .PII S0167-4838 98 00005-3

( )W. Haas et al.rBiochimica et Biophysica Acta 1384 1998 112–120 113

carboxyl-terminus of the protein and consists of aw xnumber of repeating units 3,6,7 . This domain has

homologous counterparts in all glucosyltransferasesfrom oral streptococci, the Toxins A and B fromClostridium difficile, and several muramidases from

w xStreptococcus pneumoniae and its bacteriophages 6 .Determination of the structural basis underlying

the protein–polysaccharide interactions would notonly further our general understanding of the rela-tionship between protein structure and function, but itcould also lead to strategies to combat the infectiousdiseases caused by the organisms that express theseproteins. Protein crystals for X-ray crystallographyare not yet available whereas analysis by nuclear

Ž .magnetic resonance NMR is not possible due to thehigh molecular weight of the proteins. The circular

Ž .dichroism CD spectrum of a protein reveals infor-mation about the conformation of the peptide back-bone and allows calculation of the contribution ofa-helix, b-sheet, b-turn, and random coil to the

w xsecondary structure of the protein 8,9 . CD is veryeffective in monitoring changes in the secondary andtertiary structures in protein binding studies. Here wereport the use of CD and absorption and fluorescencespectroscopy to study the structure of the GBP-AGBD under various conditions.

2. Materials and methods

2.1. Construction of fusion protein B:GBD

The DNA region encoding the GBD beginningwith amino acid 158 and proceeding to the carboxyl-

w xterminus 3 of S. mutans GBP-A was amplified byŽ .polymerase chain reaction PCR , using the phospho-

rylated primers TCTCTCCAACCAATAGCTTCTTTand CCGCCATATTTACCGTTTTCAA. The PCRproduct was ligated into the EcoRV site of the

Ž .PinPoint Xa1 vector Promega , yielding the plasmidpDPL4. Cloning and plasmid transformation were

w xdone using standard procedures 10 . The plasmidexpressed, after induction with isopropyl-b-D-thioga-

Ž . Ž .lacto-pyranoside IPTG , a fusion protein B:GBDbetween the biotinylated 1.3 S transcarboxylase sub-unit from Propionibacterium shermanii and the GBD.B:GBD reacted with an anti-GBP-A antibody on aWestern immunoblot and had the same molecular

weight on an SDS-PAGE as deduced from the aminoŽ .acid sequence data not shown . The advantages of

the B:GBD fusion protein, compared to the nativeprotein, were that it could be expressed in highamounts in Escherichia coli cells, it could be easilypurified, and the GBD could be separated from thetranscarboxylase subunit by specific protease treat-ment. Additionally, it was ascertained that the B:GBDfusion had virtually identical glucan binding proper-ties as the native GBP-A.

2.2. Protein purification

E. coli JM109 cells containing pDPL4 were grownw xat 378C in 2xYT 10 medium containing 100 mgrml

ampicillin and 2 mM biotin. Overnight cultures werediluted 1:100 in fresh medium and allowed to growfor 1 h before expression of the fusion protein wasinduced by adding IPTG to a final concentration of100 mM. The cells were harvested after 4–5 h bycentrifugation at 3000=g for 20 min at 48C in aSorvall GSA rotor. Cells were resuspended in 10

Ž . Žvolumes wt.rvol. TEN3 buffer 20 mM TRIS–HCl,pH 7.2, 1 mM EDTA, 300 mM NaCl, 100 mM

.phenylmethylsulfonyl fluoride and disrupted byŽpassing twice through a French pressure cell 1 IN.

DIA., model 4-3339, American Instrument, Silver.Spring, MD at a flow rate of 2 mlrmin at 20,000

P.S.I. Cellular debris was removed by centrifugationfor 30 min at 10,000=g at 48C in a Sorvall SS34rotor. The supernatant was then incubated for 2 h at

Ž .48C with Sepharcyl S1000 Sigma which acts as anaffinity matrix for proteins containing a GBD. Thematrix was washed extensively with TEN3 buffer and0.5 M guanidinium hydrochloride. Bound protein waseluted with 6 M guanidinium hydrochloride and dia-lyzed at 48C overnight against 0.1 M phosphate

Ž .buffer pH 6.8 with multiple changes of buffer. Thisprotein preparation was subsequently incubated for 2

Ž .h at 48C with SoftLink resin Promega . This resinconsists of avidin, which specifically binds to bio-tinylated proteins such as B:GBD and therefore func-tions as a second affinity column. The matrix waswashed with 0.1 M phosphate buffer and B:GBD waseluted with buffer containing 5 mM biotin. The pro-tein was dialyzed overnight at 48C against 0.05 M

Ž .phosphate buffer pH 6.8 unless indicated otherwiseand used the following day for analysis.

( )W. Haas et al.rBiochimica et Biophysica Acta 1384 1998 112–120114

2.3. CleaÕage of B:GBD with factor Xa

The PinPoint vector was designed such that arecognition site for the protease factor Xa was placedat the carboxyl-terminus of the transcarboxylase sub-unit. This specific protease site allows separation ofthe two components of the fusion protein withoutfurther degrading the two resulting peptides. Diges-tion with factor Xa was performed as follows: 350mg protein was digested with 14 mg protease at pH7.4 in a total volume of 1.2 ml for 16 h at 258C. Thetranscarboxylase subunit was removed by passingover an avidin column and the remaining GBD-peptide was dialyzed against 0.05 M phosphate buffer.Around 6–8 mg of B:GBD or GBD were analyzedby SDS-PAGE gels, visualized using the Silver Stain

Ž .Kit from Boehringer Mannheim Indianapolis, INand found to be free of other proteins.

2.4. Circular dichroism analysis

A JASCO J720 spectropolarimeter was used forCD analysis. The light path for the near UV regionwas 10 mm, and for the far UV region 0.5 mm wasused. Measurements were taken in 0.05 M sodiumphosphate buffer, pH 6.8. Temperature was kept at

Ž .208C unless indicated otherwise with circulatingwater from a Neslab RTE III refrigerated circulator.Far-UV data were recorded within the wavelengthrange of 260 nm to 180 nm at 0.2-nm decrementswith a scan speed of 20 nmrs, a 1.0-nm band width,and an averaging time of 0.5 s. A base line was takenwith buffer or buffer containing dextran at the sameconditions as used for the sample and subtracted fromeach protein spectrum. Six scans in the far UV regionand four scans in the near UV-region were averagedon each sample to improve the signal-to-noise ratios.Noise was further reduced using a Savitzky–Golayfilter. For data in the far UV region, molar units werecalculated using a mean residue molecular weight of111.4.

2.5. Measurement of protein concentrations

Protein concentrations were determined either withŽ .the BCA Protein Assay Pierce, Rockford, IL using

bovine serum albumin as a standard or by measuringthe absorbance at 277 nm and dividing by the absorp-

tivity of 1.93 for a 1 mgrml solution in a 1-cm lightpath.

2.6. Prediction of protein secondary structure

The Self-Consistent Method of Protein SecondaryŽ .Structure Estimation SELCON by Sreerama and

w xWoody 11 was used to analyze the CD data. TheŽ . w xmethods of Kabsch and Sander KS 12 , Levitt and

Ž . w x Ž .Greer LG 13 and Hennessey and Johnson HJw x14 were used for the assignment of secondary struc-ture.

2.7. Absorption and fluorescence emission spectra

The UV absorption spectrum was determined forB:GBD at 208C using a Beckman DU640 spectro-photometer. The fluorescence emission spectrum ofB:GBD at 208C was obtained using a Perkin-Elmerfluorescence spectrophotometer LS50B. The excita-tion and emission slits were at 5 nm, and the excita-tion wavelength were 280 and 295 nm.

3. Results

3.1. CD analysis of the GBD in the presence andabsence of dextran

B:GBD was expressed and purified as describedabove. The GBD was released from the fusion pro-tein by proteolysis and subjected to far-UV CD anal-ysis. The corresponding CD spectrum revealed twopositive bands at 230 and 202 nm, a negative band at196 nm and a broad negative band between 210 and

Ž .215 nm Fig. 1 . Addition of dextran to yield a10-fold molar excess over the concentration of GBD

Ž .Fig. 1. Far-UV CD spectra of the GBD in the absence solid andŽ .presence of 30 mM dextran dotted .


resulted in an increase in ellipticity above 225 nmand a decreased ellipticity below 225 nm. The broadnegative band shifted to 208–214 nm while the posi-tion of the peaks at 202 and 196 nm did not change,although their ellipticity decreased drastically. SinceCD analysis in the far-UV spectrum yields informa-tion about the secondary structure of a protein, theobserved change in the spectrum of GBD indicatesthat the GBD undergoes a conformational shift uponbinding to dextran. The dextran-solution alone wasnot optically active and therefore not detected by CD.

3.2. CD analysis of the B:GBD fusion protein

The B:GBD fusion protein, with the transcarboxyl-ase subunit at the amino-terminus and the GBD at thecarboxyl-terminus, was analyzed in the presence andabsence of dextran. As seen with the GBD alone, thespectrum of B:GBD showed an intense positive bandat 230 nm and a change due to the addition ofdextran, indicating a change in protein conformation

Ždue to interactions with the polysaccharide data not.shown .

3.3. Secondary structure predictions

The computer program SELCON, using the as-signments of Kabsch and Sander, was used to predictthe secondary structures of GBD and B:GBD in thepresence and absence of dextran. As seen in Table 1,neither protein seemed to contain a-helical struc-tures, but consisted of at least 55% b-sheet. Usingthe assignments of Levitt and Greer, or Hennesseyand Johnson, the ratio of b-sheets to b-turn andother structures changed but did not result in the

Table 1Secondary structure of GBD and B:GBD in the presence andabsence of dextran as predicted by the SELCON computerprogram using the assignment of Kabsch and Sander

aa-Helix b-Sheet b-Turn Other

GBD y9.1 61.8 21.1 23.3GBDqdextran y0.5 55.3 23.0 22.0B:GBD y2.1 58.2 22.3 17.6B:GBDqdextran y1.4 55.1 24.7 22.0

Data are given in percent.aNegative values were introduced by the computer program formathematical reasons and should be regarded as ‘zero’.

prediction of any a-helices or a decrease of b-sheetsŽ . w xbelow 48% data not shown . Hennessey et al. 15

predicted 3% a-helices, 32% b-sheets and 18% b-turns for the 1.3 S transcarboxylase subunit at pH 5.8which changed only slightly at pH 9.0. Therefore, thepresence of the transcarboxylase subunit did not re-sult in drastic changes in secondary structure predic-tions from GBD to B:GBD. Our data indicate thatGBD contains only b-sheets and no a-helices whichhas been shown to be the case for other lectins such

w xas concanavalin A 16 . However, as will be dis-cussed below, the high amount of aromatic residuesin the two proteins and their contribution to thefar-UV spectrum may or may not have obscured the

w xsecondary structure analysis 9,17 . Secondary struc-ture predictions based on the amino acid sequence ofthe GBD using the methods of Chou and Fasmanw x w x Ž18,19 and Rose 20 MacDNASIS, Hitachi Soft-

.ware, San Bruno, CA also came to the conclusionŽ .that GBD is rich in b-sheet structures Fig. 2 ,

though 12–21% of the domain is predicted to havethe potential to form either a-helices or b-sheets.

3.4. Analysis of B:GBD from 230 to 350 nm

The CD spectrum of B:GBD was measured be-tween 230 and 350 nm to study the contributions ofthe aromatic residues to the near CD spectrum. Disul-fide bonds are known to contribute to the near-UVCD spectrum but were absent in the proteins studiedw x9,21 . As seen in Fig. 3, B:GBD gave rise to apositive band centered around 230 nm and a secondpositive band at 280 nm. Exciton splitting could notbe shown to be responsible for this unusual spectrumsince no negative band of equal intensity was ob-served in close proximity to either one of the positive

w xbands 9,22 . Exciton splitting occurs when two ormore chromophores are situated close together in theproper orientation, resulting in a CD spectrum thathas negative and positive bands of equal rotational

w xstrength. Previous reports 9,17,23,24 showed thataromatic amino acids, especially tyrosine residues,can create a positive band centered around 220–250nm that is not due to the folding of the peptide

Ž .backbone. The presence in GBD of 38 9.1% tyro-Ž . Ž .sine, 26 6.2% phenylalanine, and 11 2.6% trypto-

phan residues out of a total of 417 amino acids, andthe lack of cysteine residues, make it likely that these


Fig. 2. Secondary structure prediction for the GBD using the method of Chou, Fasman and Rose. Capital letters underneath an amino acidŽ . Ž . Ž . Ž .indicate that this residue is very likely to be present in an a-helix H , b-sheet S , b-turn T or random coil C . Lower case letters

indicate that the amino acid has the potential to be part of this secondary structure.

residues are responsible for the positive band at 230nm, as well as the band at 280 nm. Interestingly, 38of these aromatic residues are arranged in 12 clusters,each containing three or more aromatic amino acids.

Fig. 3. CD spectrum of B:GBD at 230–350 nm.

3.5. Protein conformation at Õarious pH

w xDay 23 showed for the G5 protein that a positiveband at 228 nm was due to tyrosine residues based onthe protein’s CD spectrum at various pH. We mea-sured the CD spectrum of B:GBD between pH 5.0

Ž .and 11.5 Fig. 4 . At pH 5.0, more than 75% of theprotein precipitated, which is not surprising since thepredicted p I for B:GBD is 5.0. The spectrum re-mained unchanged between pH 5.5 and 8.5, indicat-ing that the GBD is stable at physiological pH.Increasing the pH to 10.0 resulted in an alteration ofthe spectrum below 225 nm while the 230 nm bandremained unaffected. A pH of 11.5, which is wellabove the pK of 10.13 for the hydroxyl group ofa


Ž .Fig. 4. Far UV CD spectra of B:GBD at pH 6 light solid , pH 7Ž . Ž . Ž .solid , pH 8.5 dashed and pH 10 dotted .

w xtyrosine 25 , resulted in a total loss of the 230 nmŽ .band data not shown , which suggested that tyrosine

was the cause of the 230 nm band seen in GBD andB:GBD.

3.6. Effects of temperature on B:GBD’s conformation

To analyze the effects that elevated temperaturesmight have on protein folding, B:GBD was graduallyheated to the indicated temperature and allowed toremain at that temperature for 10 min before mea-

Ž .surements were taken Fig. 5 . The spectra remainedunchanged at 208 and 308C, but the intensity of the230-nm band decreased at 408C, while the spectrumbelow 220 nm changed only slightly. At 458C andabove, the 230-nm band had disappeared and thespectrum below 220 nm changed significantly fromthat seen at lower temperatures. Increasing the tem-perature up to 808C did not result in drastic changesin the CD spectrum, indicating that most of the

Ž .structural changes occurred at 458C data not shown .The protein did not regain its former conformationwhen the temperature was decreased from 408C or458C back to lower temperatures. These data indicatethat 408C marks a transition temperature where

Ž .Fig. 5. CD spectra of B:GBD at 208 and 308C solid , 408CŽ . Ž .dotted and 458C dashed . The sample was cooled down to

Ž .308C and the CD spectrum was measured again light solid .

B:GBD loses some of its tertiary structure whilemaintaining most of its secondary structure. Bindingstudies using B:GBD preincubated at 40 and 508Cshowed that the former protein bound to dextran aswell as the untreated B:GBD, while the latter did notbind at all. Furthermore, 40% of the protein heatedabove 458C precipitated, indicating that loss of nativeconformation results in loss of functionality and solu-

Ž .bility data not shown .

3.7. Absorption and fluorescence spectra

The absorption maximum of B:GBD was at 277Ž .nm Fig. 6A . The absorptivity for a 1 mgrml solu-

tion was determined to be 1.93 in a 1-cm light path.The fluorescence emission maximum was at 339 nmŽ .Fig. 6B . This maximum suggests that the majorityof the tryptophan residues were in the interior of the

w xprotein 26 . The emission spectra were analogous for280 and 295-nm excitations. The fluorescence emis-sion maximum was slightly blue shifted in the pres-

Ž .ence of dextran Fig. 6B . Heating B:GBD to 458Cfor 15 min before fluorescence analysis did not change

Ž . Ž .Fig. 6. Absorption A and fluorescence B spectra of B:GBD.The fluorescence emission spectra of B:GBD were obtained

Ž .using 295 nm excitation in the presence solid and absenceŽ .dotted of dextran.


the emission peak after excitation at 295 nm, indicat-ing that the environment of the tryptophane-residueswas unaltered.

4. Discussion

The presence of a GBD in virulence factors of oralstreptococci and homologous domains in several other

w xproteins from pathogenic bacteria 6 led us to studythe GBD from GBP-A. Rather than using the nativeprotein, we chose to use a fusion protein between the1.3 S transcarboxylase subunit of known CD spec-trum and the GBD. This fusion protein was equal tothe native protein in its ability to bind to dextran buthad the advantage that the GBD could be separatedfrom the transcarboxylase subunit by specific pro-tease digestion. The opportunity to use two indepen-dent affinity columns to obtain highly purified pro-tein preparations was a second advantage of thisapproach. The CD spectrum and proposed secondarystructure distributions of the 1.3 S transcarboxylase

w xsubunit were published by Hennessey et al. 15 . Theauthors describe a negative band from 220–240 nmand negative ellipticity starting at 200 nm.

The CD spectrum of B:GBD and GBD showed anunusual positive band at 230 nm that is believed to bedue to the contribution of aromatic side chains. Dayw x23 showed that a positive band at 228 nm in the CDspectrum of the G5 protein from bacteriophage fdwas due to phenolic transitions of tyrosine residues.Lytic amidase from S. pneumoniae and lysozyme

w xfrom phage Cp-1 27,28 contain C-terminal choline-binding domains that are homologous to the GBD ofGBP-A. The CD spectra of both proteins show astrong positive band around 230 nm that was at-tributed to the contribution of aromatic side chains.This was confirmed for B:GBD by measuring the CD

w xspectrum at various pH, since Day 23 and Sanz andw xGarcia 28 could show that this band is pH depen-

dent. Their results and our data demonstrate that the230 nm band is lost at a pH of 11.5 or higher, whichis sufficiently high to deprotonate the phenolic hy-droxyl group. The protein is stable between pH 5.5and 8.5, but precipitates at pH 5.0, which equals itspredicted p I.

The aromatic side chain contribution to the CDspectrum from 180 to 260 nm may or may not

obscure the secondary structure analysis as pointedw x w xout by Fasman 9 and Woody 17 . Multiple sets of

CD data obtained for B:GBD and GBD in the pres-ence or absence of dextran were analyzed using the

w xSELCON computer program 11 and the assign-ments of Kabsch and Sander, Levitt and Greer, andHennessey and Johnson. All assignments agreed thatthe GBD is made up of at least 48% b-sheets and

w xcontains no a-helices. Sanz and Garcia 28 usedseveral methods to assess the secondary structure ofthe choline binding domain of CPL-1 lysozyme, adomain homologous to GBD. They concluded thatthe C-terminal domain is made up of two sheet-bend-helix modules connected by five bend-sheetstructures and that the binding to choline results inthe redistribution of b-sheets and b-turns inlysozyme. However, definite structure assignmentswill have to await X-ray crystallography or NMRanalysis of the GBD or of a homologous structure.

Heat-denaturation experiments revealed that theGBD undergoes a transition at 408C. At this tempera-ture, the peak at 230 nm loses in intensity while theremainder of the spectrum undergoes little or nochange. This suggests that the changes affect only thetertiary structure that brings the aromatic residues inclose contact, but not the protein’s secondary struc-ture. If the temperature is raised to 458C, the CDspectrum loses the 230-nm band entirely, producing achange in the secondary structure of the protein aswell. A secondary structure prediction using themethod of Chou, Fasman and Rose revealed a largeamount of b-sheets and that at least 12% of the GBDhas the potential to form either a b-sheet or ana-helix. It is possible that all or some of these regionsare in a b-sheet conformation in the native state andat elevated temperatures undergo a shift to an a-heli-cal structure which is more stable, making the transi-tion irreversible. Although we could not predict thesecondary structure based on the CD spectrum ofheat-denatured B:GBD, this theory is supported bydata obtained for aged protein. The spectrum of6-week old B:GBD had a spectrum similar to that

Ž .obtained for heat-denatured protein data not shownin that it lacked a positive band at 230 nm and had abroad negative band between 205 and 225 nm. TheSELCON computer program predicted 16% a-helix,39% b-sheet, 24% b-turn and 23% other structuresfor this protein sample, which is in good agreement


with the Chou–Fasman–Rose data and would explainthe conformational changes seen at elevated tempera-tures.

The fluorescence emission maximum at 339 nm ischaracteristic of tryptophan residues found in theinterior of proteins. When Trp residues are on thesurface of proteins in the presence of water the Trpmaxima are at lower energies. The emission spectrawere similar using excitations at either 280 or 295nm. This result suggests that only Trp residues arefluorescing since excitation at 280 nm excites bothTrp and Tyr, whereas excitation at 295 nm excitesTrp only. Fluorescence of Tyr would be observed atabout 310 nm, and the absence of this band suggeststhat the Tyr emission is quenched by the Trp. TheTrp emission was slightly blue-shifted in the presenceof dextran. This suggests that some of the Trp residueswere now even more shielded from water. This non-polar environment could be caused by a conforma-

w xtional change 26 , which is in good agreement withthe CD data.

Much has been written about the GBD in GBP-Aand the GTFs and the repeating units present in theGBD. However, these publications were restricted todefining homologies or correlating the size of the

w xbinding domain to the affinity for a ligand 6,7,29,30 .With the combination of Chou–Fasman–Rose sec-ondary structure prediction and CD analysis we wereable to assign a secondary structure for the GBD. Ourdata show that the repeating units form b-sheetstructures that are interrupted by b-turn or randomcoil structures. The unusual positive band at 230 nmcould be contributed to the high amount of Tyrresidues in the protein. The contribution of certainhighly conserved amino acids in forming the sec-ondary structures and ligand binding is currentlyunder investigation. Binding to dextran induces aconformational shift in the GBD that might allow alarger portion of the protein to interact with theligand. More work is currently in progress in order toget a better understanding of how the GBD is struc-turally organized and binds to dextran.

Acknowledgements

Ž .The authors thank L.E. Eisele Biochemistry Corefor help with the SELCON computer analysis and

T.T. Andersen and L.A. Day for critical review of themanuscript. This research was funded by grantDE10058 from the National Institute of Dental Re-search.

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