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Insights into the Conformational Equilibria of Maltose-binding Protein by Analysis of High Affinity Mutants

2003; Elsevier BV; Volume: 278; Issue: 36 Linguagem: Inglês

10.1074/jbc.m301004200

1083-351X

Patrick G. Telmer, Brian H. Shilton,

Protein purification and stability

The affinity of maltose-binding protein (MBP) for maltose and related carbohydrates was greatly increased by removal of groups in the interface opposite the ligand binding cleft. The wild-type protein has a KD of 1200 nm for maltose; mutation of residues Met-321 and Gln-325, both to alanine, resulted in a KD for maltose of 70 nm; deletion of 4 residues, Glu-172, Asn-173, Lys-175, and Tyr-176, which are part of a poorly ordered loop, results in a KD for maltose of 110 nm. Combining the mutations yields an increased affinity for maltodextrins and a KD of 6 nm for maltotriose. Comparison of ligand binding by the mutants, using surface plasmon resonance spectroscopy, indicates that decreases in the off-rate are responsible for the increased affinity. Small-angle x-ray scattering was used to demonstrate that the mutations do not significantly affect the solution conformation of MBP in either the presence or absence of maltose. The crystal structures of selected mutants showed that the mutations do not cause significant structural changes in either the closed or open conformation of MBP. These studies show that interactions in the interface opposite the ligand binding cleft, which we term the “balancing interface,” are responsible for modulating the affinity of MBP for its ligand. Our results are consistent with a model in which the ligand-bound protein alternates between the closed and open conformations, and removal of interactions in the balancing interface decreases the stability of the open conformation, without affecting the closed conformation. The affinity of maltose-binding protein (MBP) for maltose and related carbohydrates was greatly increased by removal of groups in the interface opposite the ligand binding cleft. The wild-type protein has a KD of 1200 nm for maltose; mutation of residues Met-321 and Gln-325, both to alanine, resulted in a KD for maltose of 70 nm; deletion of 4 residues, Glu-172, Asn-173, Lys-175, and Tyr-176, which are part of a poorly ordered loop, results in a KD for maltose of 110 nm. Combining the mutations yields an increased affinity for maltodextrins and a KD of 6 nm for maltotriose. Comparison of ligand binding by the mutants, using surface plasmon resonance spectroscopy, indicates that decreases in the off-rate are responsible for the increased affinity. Small-angle x-ray scattering was used to demonstrate that the mutations do not significantly affect the solution conformation of MBP in either the presence or absence of maltose. The crystal structures of selected mutants showed that the mutations do not cause significant structural changes in either the closed or open conformation of MBP. These studies show that interactions in the interface opposite the ligand binding cleft, which we term the “balancing interface,” are responsible for modulating the affinity of MBP for its ligand. Our results are consistent with a model in which the ligand-bound protein alternates between the closed and open conformations, and removal of interactions in the balancing interface decreases the stability of the open conformation, without affecting the closed conformation. ATP binding cassette transporters couple ATP hydrolysis to the transmembrane transport of a diverse range of compounds. Members of the ATP binding cassette transporter superfamily are characterized by two membrane-integral domains that each contain 6 or more membrane spanning helices, but are otherwise poorly conserved, and two peripheral ATP binding cassette domains that display sequence conservation across the entire superfamily (1Hyde S.C. Emsley P. Hartshorn M.J. Mimmack M.M. Gileadi U. Pearce S.R. Gallagher M.P. Gill D.R. Hubbard R.E. Higgins C.F. Nature. 1990; 346: 362-365Crossref PubMed Scopus (949) Google Scholar). In addition to the membrane complex, ATP binding cassette systems that catalyze nutrient uptake have primary receptors (binding proteins) that serve two functions: they provide a high affinity binding site for the transported molecule and they regulate the ATPase activity of the integral membrane complex. We are interested in the function of the primary receptors in the transport process. As a group, these proteins have been intensively studied by x-ray crystallography and other biophysical techniques (for a review, see Ref. 2Quiocho F.A. Ledvina P.S. Mol. Microbiol. 1996; 20: 17-25Crossref PubMed Scopus (450) Google Scholar). They typically contain two domains separated by a hinge region; the substrate binds in the cleft between the two domains, and the protein undergoes a large conformational change, leading to closure of the cleft. With respect to the maltose transport system, domain closure in the binding protein is thought to be the first step toward molecular shape recognition by the membrane complex (3Davidson A.L. Shuman H.A. Nikaido H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2360-2364Crossref PubMed Scopus (231) Google Scholar, 4Hor L. Shuman H.A. J. Mol. Biol. 1993; 233: 659-670Crossref PubMed Scopus (71) Google Scholar), although it has been shown that both substrate-loaded and substrate-free binding proteins have a role in the transport cycle (5Bohl E. Shuman H.A. Boos W. J. Theor. Biol. 1995; 172: 83-94Crossref PubMed Scopus (44) Google Scholar, 6Shilton B.H. Mowbray S.L. Protein Sci. 1995; 4: 1346-1355Crossref PubMed Scopus (21) Google Scholar, 7Merino G. Boos W. Shuman H. Bohl E. J. Theor. Biol. 1995; 177: 171-179Crossref PubMed Scopus (51) Google Scholar, 8Ames G.F. Liu C.E. Joshi A.K. Nikaido K. J. Biol. Chem. 1996; 271: 14264-14270Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). The association and dissociation of the substrate, and attendant conformational changes in the binding protein (MBP), 1The abbreviations used are: MBP, maltose-binding protein; SAXS, small-angle X-ray scattering; APS, advanced photon source; SPR, surface plasmon resonance; MBP-WT, wild-type maltose-binding protein; MBP-Del, MBP with a deletion of residues 172, 173, 175, and 176; MBP-Ala, maltose-binding protein with M321A and Q325A mutations; MBP-DM, maltose-binding protein harboring the mutations of both MBP-Del and MBP-Ala; RU, response units; MalFGK2, the membrane complex consisting of integral membrane subunits MalF and MalG, and the peripheral ATP binding cassette subunits MalK.1The abbreviations used are: MBP, maltose-binding protein; SAXS, small-angle X-ray scattering; APS, advanced photon source; SPR, surface plasmon resonance; MBP-WT, wild-type maltose-binding protein; MBP-Del, MBP with a deletion of residues 172, 173, 175, and 176; MBP-Ala, maltose-binding protein with M321A and Q325A mutations; MBP-DM, maltose-binding protein harboring the mutations of both MBP-Del and MBP-Ala; RU, response units; MalFGK2, the membrane complex consisting of integral membrane subunits MalF and MalG, and the peripheral ATP binding cassette subunits MalK. may have direct effects on transport kinetics and regulation of ATP hydrolysis by MalFGK2. To investigate the role of binding protein affinity on the transport process, our goal was to engineer MBP molecules with greater affinity for maltose, without changing residues in either the maltose binding site or in regions thought to interact with MalFGK2. Crystal structures of MBP in both the closed and open conformations have been solved (9Spurlino J.C. Lu G.-Y. Quiocho F.A. J. Biol. Chem. 1991; 266: 5202-5219Abstract Full Text PDF PubMed Google Scholar, 10Sharff A.J. Rodseth L.E. Spurlino J.C. Quiocho F.A. Biochemistry. 1992; 31: 10657-10663Crossref PubMed Scopus (427) Google Scholar), and they show that binding of maltose results in a large conformational change of the protein, bringing the two domains together such that the substrate is buried inside the cleft. In solution, unliganded MBP is in the open conformation (11Shilton B.H. Flocco M.M. Nilsson M. Mowbray S.L. J. Mol. Biol. 1996; 264: 350-363Crossref PubMed Scopus (110) Google Scholar); however, there is no obvious energetic barrier to closure of the ligand binding cleft, either in the hinge or in the interface surrounding the ligand binding site. Rather, an interface on the opposite side of the hinge from the ligand binding site appears to maintain the protein in an open conformation (Fig. 1): in the closed conformation, this interface is broken and becomes solvent exposed. Changes to this interface should affect the open-closed equilibrium, and consequently the maltose affinity of the protein. This idea is supported by a study by Marvin and Hellinga (12Marvin J.S. Hellinga H.W. Nat. Struct. Biol. 2001; 8: 795-798Crossref PubMed Scopus (129) Google Scholar), in which they demonstrated that introduction of large, branched groups into the interface (either as natural amino acids or by site-specific chemical modification) increased the affinity of the protein for maltose. We have found that by simply removing certain interactions in this interface, we could increase the affinity for maltose by approximately 2 orders of magnitude. On this basis, the interface is postulated to play an active role in maintaining the open conformation of MBP, and we have therefore called this region the “balancing interface” (Fig. 1). In an effort to understand how removal of interactions in the balancing interface effects such a pronounced change in ligand affinity, we have characterized the binding properties, solution conformations, and crystal structures of MBP molecules harboring mutations in the balancing interface. The results from these studies are consistent with the idea that the mutations have increased ligand affinity by destabilizing the open conformation. Site-directed Mutagenesis—Mutant MBP molecules MBP-Ala (M321A/Q325A) and MBP-Del (Δ172–173 and Δ175–176) were produced using oligonucleotide-directed mutagenesis. This was carried out by two rounds of PCR, following standard methods (13Cormack B. Chanda V.B. Series ed A usubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Greene Publishing Associates and Wiley-Interscience, New York1987Google Scholar). The first round of PCR consisted of two reactions, each including either a 5′ or 3′ mutagenic primer and a corresponding non-mutagenic flanking primer. The PCR products obtained from this first round were pooled together and used as template for the second round to obtain the full-length mutagenized product. PCR products were then subcloned back into the parent plasmid pLH1 (4Hor L. Shuman H.A. J. Mol. Biol. 1993; 233: 659-670Crossref PubMed Scopus (71) Google Scholar) to yield the mutant plasmids as follows: pLH1-Ala was made by insertion of a BglII/NdeI fragment from the PCR product into pLH1, whereas pLH1-Del was made by insertion of a BglII/NcoI fragment from the PCR product into pLH1. To make plasmid pLH1-DM, which harbors the combined mutations, a BglII/Eco47III fragment from pLH1-Del was subcloned into pLH1-Ala. All mutations were confirmed by automated DNA sequencing. Expression and Purification of MBP—All chromatographic media were purchased from Amersham Biosciences. Plasmids containing wild-type or mutant MalE coding regions were transformed into Escherichia coli strain HS3309, which does not produce MBP (14Treptow N.A. Shuman H.A. J. Mol. Biol. 1988; 202: 809-822Crossref PubMed Scopus (66) Google Scholar). Cultures were grown with vigorous shaking at 37 °C in LB broth containing 100 μg/ml ampicillin for 16–18 h. Periplasmic proteins were extracted by osmotic shock (15Neu H.C. Heppel L.A. J. Biol. Chem. 1965; 240: 3685-3692Abstract Full Text PDF PubMed Google Scholar) and dialyzed against 50 mm Tris-HCl, pH 8.5, prior to ion exchange chromatography. The extract was applied onto a 2.6 × 15-cm column packed with Q-Sepharose Fast Flow and eluted with a linear gradient from 0 to 1 m NaCl. Fractions containing MBP were pooled, dialyzed against 50 mm Tris-HCl, pH 8.5, and further purified on DEAE-Sephacel resin, 2.6 × 30 cm, using a linear gradient from 0 to 1 m NaCl. The DEAE-Sephacel step was not effective for purification of MBP-DM. In this case, fractions from the Q-Sepharose column were applied to a 1.6 × 15-cm column of amylose affinity resin (New England Biolabs) and eluted with 10 mm maltose. Preparation of Maltose-free Protein—To prepare maltose-free binding protein, the MBP was concentrated by ion-exchange chromatography using a 1-ml HiTrap Q column (Amersham Biosciences) and then denatured by adding guanidine HCl to a concentration of 6 m. The concentrated and denatured protein was loaded onto a 2.6 × 60-cm column of Superdex 200 Prep Grade gel filtration resin, which had been previously equilibrated with 6 m guanidine HCl. This column was developed with 6 m guanidine HCl at a flow rate of 1 ml/min; fractions containing denatured protein were pooled and dialyzed exhaustively against 50 mm Tris-HCl, pH 8.5. To remove aggregates, the refolded MBP was again concentrated and applied to a 2.6 × 60-cm column of Superdex 200 Prep Grade gel filtration resin, previously equilibrated with 100 mm KCl, 20 mm Tris-HCl, pH 8.5, 1 mm EDTA. Fluorescence Titrations—Fluorescence titrations were carried out at protein concentrations of 10–100 nm, in 3 ml of 50 mm Hepes, 100 mm NaCl, 5 mm NaN3, pH 7.5, using a Flourolog 3 spectrofluorimeter (ISA Instruments). The excitation and emission wavelengths were 280 and 349 nm, respectively, with 0.25 nm excitation and 15 nm emission slit widths. The value for relative minimum fluorescence (F min) was obtained using Equation 1 (16Lakiwicz J.R. Principles of Fluorescence Spectroscopy. Plenum Press, New York1983Crossref Google Scholar). F0-FF0=Fmin[ligand][ligand+c(Eq. 1) Where F 0 is the starting fluorescence, F is the fluorescence after addition(s) of ligand (maltose or maltotriose, both from Sigma), and c is a constant. The KD was then calculated using the following equation (17Miller D.M. Olson J.S. Pflugrath J.W. Quiocho F.A. J. Biol. Chem. 1983; 258: 13665-13672Abstract Full Text PDF PubMed Google Scholar), with the KD as the single variable. F0-FF0-Fmin=KD+[ligand]+[MBP]-KD+[ligand]+[MBP])2-4[ligand][MBP]2[MBP](Eq. 2) The “solver” function in Microsoft Excel was used to fit the data to the equations and obtain values for the relevant parameters. Surface Plasmon Resonance Analysis of Maltodextrin Binding—Surface plasmon resonance analyses were performed using a BIAcore X instrument. A CM5 sensor chip (BIAcore AB) was derivatized with either amylose or pullulan as follows. Amylose (Sigma) was dissolved in 1 m NaOH to yield a concentration of 50 mg/ml, and then diluted with 0.1 m NaOH, and the pH adjusted to 5.5 with acetic acid to yield a final amylose concentration of 1 mg/ml. 1 ml of the 1 mg/ml amylose solution was treated with NaIO4 at a concentration of 0.2 mm. The solution was incubated for 2 h at 20 °C to effect periodate cleavage of the amylose, and then 400 μl of the amylose solution was desalted on a 2-ml column of Sephadex G-25, equilibrated, and developed with 0.1 m sodium acetate, pH 4.5. Prior to derivatization, the CM5 chip was equilibrated with 10 mm Hepes, 0.15 m NaCl, 3 mm EDTA, 0.005% polysorbate 20, pH 7.4. The periodate-oxidized amylose was coupled to the surface in flow cell 2 by sequential injection of the following solutions at a flow rate of 5 μl/min: 28 μl of a solution of 200 mm N-hydroxysuccinimide and 50 mm N-ethyl-N′-(dimethylaminopropyl)carbodiimide, 35 μl of 5 mm hydrazine, 2× 100-μl injections of the oxidized amylose solution, 35 μl of 1 m ethanolamine, pH 8.5. Finally, the chip was stabilized with 40 μl of 0.1 m NaCNBH3 in 0.1 m sodium acetate, pH 4.5, at a flow rate of 2 μl/min. A total of 1300 response units (RU) of amylose was immobilized to flow cell 2, whereas flow cell 1 was left unmodified for use as a reference cell. Derivatization of a CM5 chip with pullulan followed essentially the same procedure, except that the pullulan, which is highly soluble, could be dissolved directly in 0.1 m sodium acetate, pH 5.5. Samples of wild-type and engineered MBP molecules were dialyzed against 50 mm Hepes, 150 mm NaCl, 5 mm NaN3, pH 7.4, and serially diluted to yield concentrations ranging from 4 nm to 4 μm. To minimize changes in bulk refractive index, the running buffer for the SPR experiments was exactly the same as the dialysis buffer, with the exception that for some experiments the SPR running buffer contained 100 μm maltose to abrogate rebinding effects. For experiments with the amylose-coupled chip, the flow rate was 50 μl/min, and the injection volumes were 100 μl; for the pullulan-coupled chip, the flow rate was 25 μl/min and the injection volume was 40 μl. For both the amylose and pullulan chips, the response from flow cell 1 (the flow cell with a non-derivatized surface) was subtracted from the response from flow cell 2. Small Angle x-ray Scattering—To remove aggregates prior to SAXS analysis, concentrated protein samples were purified using a 2.6 × 65-cm Superdex 200 Prep Grade gel filtration column (Amersham Biosciences), equilibrated and developed with 100 mm KCl, 20 mm Tris-HCl, 1 mm EDTA, pH 8.5. Samples for SAXS were dialyzed against 10 mm Tris-HCl, 100 mm NaCl, 5 mm NaN3, pH 8.0. The dialysis buffers were reserved for background measurements. Measurements of MBP-WT, MBP-Ala, and MBP-Del were made at the European Molecular Biology Laboratory Outstation at the Deutsches Elektronen Synchrotron (Hamburg, Germany), beamline X33 (18Koch M.H.J. Bordas J. Nucl. Instr. Methods. 1983; 208: 461-469Crossref Scopus (287) Google Scholar). Measurements of MBP-WT and MBP-DM proteins were made at BioCAT (beamline 18ID) of the Advanced Photon Source. Details of SAXS measurements and data processing have been described previously (19Del Rizzo P.A. Bi Y. Dunn S.D. Shilton B.H. Biochemistry. 2002; 41: 6875-6884Crossref PubMed Scopus (87) Google Scholar, 20Dempsey B.R. Economou A. Dunn S.D. Shilton B.H. J. Mol. Biol. 2002; 315: 831-843Crossref PubMed Scopus (26) Google Scholar). Crystallization and Crystal Structure Determination—MBP was crystallized by vapor diffusion using the hanging drop method; equal volumes of protein and reservoir solutions were mixed to yield drop sizes of 2 to 4 μl. For maltose-bound MBP-DM, the reservoir solution was comprised of 18–24% PEG 5K monomethyl ether (Hampton Research), 0.1 m cacodylate, pH 6.2, 200 mm sodium acetate, 10 mm zinc acetate, 5 mm NaN3, and 1 mm maltose. For unliganded and liganded MBP-Del, the reservoir contained 10 mm cacodylate, pH 6.5, 200 mm NaCl, 18 mm zinc acetate, and either 20% PEG 8K (Fluka) or 24% PEG 4K, and 1 mm maltose. All data were collected using a rotating anode source (Rigaku RUH3R) with graded-multilayer optics (Osmic; λ = 1.5418 Å), and a MAR345 image plate detector (MAR Research). Data for the maltose-bound conformations of MBP-Del and MBP-DM were collected at 100 K; the crystals were soaked briefly in 30% PEG 400, 200 mm sodium acetate, 0.1 m cacodylate, 1 mm maltose, pH 6.2, and then frozen in a stream of N2 gas at 100 K. Data processing and reduction were carried out with DENZO and Scalepack (21Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref Scopus (38253) Google Scholar). Molecular replacement solutions were found using the open and closed conformations of wild-type MBP (9Spurlino J.C. Lu G.-Y. Quiocho F.A. J. Biol. Chem. 1991; 266: 5202-5219Abstract Full Text PDF PubMed Google Scholar, 10Sharff A.J. Rodseth L.E. Spurlino J.C. Quiocho F.A. Biochemistry. 1992; 31: 10657-10663Crossref PubMed Scopus (427) Google Scholar) as search models, with the program Amore in the CCP4 suite (22CCP4Acta Crystallogr. Sect. D. 1994; 50: 760-763Crossref PubMed Scopus (19668) Google Scholar, 23Navaza J. Acta Crystallogr. Sect. A. 1994; 50: 157-163Crossref Scopus (5026) Google Scholar). The crystallographic model was manipulated using the program O (24Jones T.A. Bergdoll M. Kjeldgaard M. Bugg C.E. Ealick S.E. Crystallographic and Modeling Methods in Molecular Design. Springer-Verlag, New York1990: 189-195Crossref Google Scholar), and refinement and map calculations were carried out with CNS (25Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16919) Google Scholar). Rationale for Mutations—As discussed in the Introduction, we believe that interactions between the two domains of MBP, on the side of the hinge opposite the ligand binding cleft, provide an energetic barrier to closure of the ligand binding cleft, and thereby stabilize the open conformation (Fig. 1A). This region of domain contacts will be termed the balancing interface because the present work, and that of another group (12Marvin J.S. Hellinga H.W. Nat. Struct. Biol. 2001; 8: 795-798Crossref PubMed Scopus (129) Google Scholar), demonstrates that it plays a role in modulating the maltose binding affinity. It has been demonstrated that introduction of larger groups into the balancing interface, by either mutagenesis or chemical modification, results in an increase in maltose affinity (12Marvin J.S. Hellinga H.W. Nat. Struct. Biol. 2001; 8: 795-798Crossref PubMed Scopus (129) Google Scholar). The presence of bulky groups in the balancing interface may affect the overall structure of the open conformation of the protein, and therefore our strategy to increase the affinity of MBP for maltose was to remove important contacts in the balancing interface (Fig. 1B). The assumption here was that the interface has an active role in stabilizing the open conformation. We located two potentially important interactions in the balancing interface; in both cases, these interactions only take place when the protein is in the open conformation. The first interaction occurs between the side chains of Met-321 on the C-terminal domain, and Tyr-90, Phe-92, Glu-308, and Ala-301 on the N-terminal domain: in the ligand-free conformation, the Met-321 SD and CE atoms are projected into a pocket formed by the hydrophobic parts of the other four side chains, resembling a ball-and-socket joint. In addition, the side chain of Gln-325 sits over the side chain of Met-321, protecting the assembly from solvent. In the ligand-bound conformation, this interaction is disrupted: the Met-321 side chain, the “ball,” moves almost 4 Å of the “socket” formed by Tyr-90, Phe-92, Glu-308, and Ala-301, and the hydrophobic surfaces that were buried become solvent exposed. Further evidence for important structural changes in this region comes from an analysis of the temperature factors, which provide an indication of atomic mobility. Temperature factors for the side chain atoms of Met-321 and Gln-325 are greater in the ligand-bound conformation (Table I), indicating an increase in mobility for these atoms when the protein binds maltose. These observations suggested that Met-321 and Gln-325 mediate important stabilizing interactions in the ligand-free, but not the ligand-bound, conformation. We mutated both residues to alanine to abrogate these interactions and selectively destabilize the open conformation. The M321A/Q325A mutant will be referred to as MBP-Ala.Table ITemperature factor analysis of ligand-free and ligand-bound MBPGroupAverage B-factorLigand-freeMaltose-boundMet-321 side chain atoms16.726.3Gln-325 side chain atoms33.760.6Residues 171-17743.459.2Whole protein25.922.1 Open table in a new tab The loop containing residues 171 to 177 was also identified as a potential stabilizing factor for the open conformation. In various crystal structures of MBP, this loop appears in different conformations with relatively high temperature factors, indicating that it is probably not important for the overall structure or fold of MBP. Nevertheless, this loop is retained with moderate sequence conservation in MBP molecules from various organisms, suggesting that it may have an important function. This loop is part of the C-terminal domain, but in the ligand-free conformation it makes a number of contacts with the N-terminal domain; these contacts are broken when the protein binds ligand. As with the side chains of Met-321 and Gln-325, the temperature factors of residues 171–177 are greater for the ligand-bound conformation (Table I), indicating that the loop becomes more mobile when the protein binds maltose. Note that the loop does not participate in crystal contacts in the structures analyzed. Based on reasoning similar to that given for the MBP-Ala mutant, we decided to shorten this loop to selectively destabilize the ligand-free, open conformation of MBP. Residue 174, a glycine, is located in the middle of the β-turn of the loop, and given its unique properties, we decided to shorten the loop by removing two residues on either side of Glu-174, namely Glu-172, Asn-173, Lys-175, and Tyr-176. This deletion mutant will be referred to as MBP-Del. The mutations in MBP-Ala and MBP-Del caused an increase in affinity for maltose (see below). Because the mutations in MBP-Ala and MBP-Del are completely independent, we decided to combine them in an attempt to further increase the affinity for maltose. The MBP molecule produced by combining the mutations in MBP-Ala with the deletions in MBP-Del will be referred to as MBP-DM (for “double mutant”). Functional Characteristics of MBP and Engineered Mutants—MBP molecules that cannot bind maltose, or interact productively with MalFGK2, will not function in transport, and we first tested the mutants for their ability to participate in maltose transport. The MBP mutants were put into a background, E. coli HS3309 (14Treptow N.A. Shuman H.A. J. Mol. Biol. 1988; 202: 809-822Crossref PubMed Scopus (66) Google Scholar), which has a disruption of the maltose operon such that there is no chromosomally encoded MBP present. In this background, all of the mutants were able to support growth on minimal maltose media, indicating that they are fully functional. We monitored binding of maltose to both MBP-WT and the engineered mutants MBP-Ala and MBP-Del by changes in intrinsic fluorescence. To ensure that the proteins were absolutely free from maltose, they were unfolded in 6 m guanidine HCl, purified by gel filtration chromatography, and refolded (see “Experimental Procedures”). The mutations attenuated the fluorescence quenching normally caused by maltose binding: upon addition of a saturating concentration of maltose, wild-type MBP exhibited a decrease in fluorescence of 14%, whereas the decrease for MBP-Ala and MBP-Del, under exactly the same conditions, was 8% (Table II). The change in fluorescence for MBP-Ala and MBP-Del, although modest, was still sufficient to carry out fluorescence titrations to determine the KD for maltose binding (Fig. 2). The KD values obtained from these experiments are listed in Table II.Table IIAffinity of MBP molecules for maltose and maltotrioseProteinMaltoseMaltotrioseQuenchingaAverage ± S.D. for three determinations.KD aAverage ± S.D. for three determinations.QuenchingaAverage ± S.D. for three determinations.KD aAverage ± S.D. for three determinations.%nm%nmMBP-WT14 ± 11200 ± 20022 ± 2660 ± 60MBP-Ala8 ± 270 ± 1016 ± 2120 ± 10MBP-Del8 ± 3110 ± 2018 ± 2100 ± 10MBP-DM<2ND12 ± 36 ± 1a Average ± S.D. for three determinations. Open table in a new tab The MBP-DM showed no significant change in fluorescence upon addition of maltose, but there was an 11–14% decrease in fluorescence upon addition of a saturating concentration of maltotriose (Table II). This change in fluorescence allowed us to carry out titrations with maltotriose and determine that the KD for this sugar is 6 ± 1 nm, approximately 2 orders of magnitude lower than the KD for the wild-type protein (Table II). The mutations produced an additive attenuation in the ligand-induced quenching of tryptophan fluorescence (Table II). None of the mutations produced an observable change in structure in the region of any tryptophan residue (see crystal structure analysis, below), and therefore it would appear that the strategy of destabilizing the open conformation of the protein has also had a general effect on ligand-induced fluorescence quenching. Of the eight tryptophan residues in MBP, three residues, Trp-10, Trp-94, and Trp-129, are located away from both the balancing interface and the ligand binding cleft, and show no change in structure or environment in the open and closed forms of the protein. Four residues are located in the ligand binding cleft; three of these, Trp-62, Trp-230, and Trp-340, make direct interactions with bound maltose, whereas the fourth, Trp-232, does not, but all experience a change in environment as a result of ligand binding. The eighth tryptophan residue, Trp-158, is part of the C-terminal domain, but is located close to the balancing interface. The contributions of the various tryptophan residues to maltose-induced fluorescence quenching were assessed previously in a site-directed mutagenesis study, where each tryptophan was mutated to an alanine and the binding and fluorescence properties of the resulting proteins were measured (26Martineau P. Szmelcman S. Spurlino J.C. Quiocho F.A. Hofnung M. J. Mol. Biol. 1990; 214: 337-352Crossref PubMed Scopus (58) Google Scholar). In that study, the two most striking changes in tryptophan fluorescence were produced by the W230A and W158A mutants: the W230A mutation caused an increase in maltose-induced quenching to 30%, whereas the W158A mutation resulted in a complete loss of maltose-induced quenching. Given the location of Trp-158 near to the balancing interface, and the fact that mutation to alanine results in a loss of maltose-induced quenching (similar to what we have observed for MBP-DM) Trp-158 is most likely the source of the attenuated lig