Desensitization of feedback inhibition of the Saccharomyces cerevisiae gamma-glutamyl kinase enhances proline accumulation and freezing tolerance

In response to osmotic stress, proline is accumulated in many bacterial and plant cells as an osmoprotectant. The yeast Saccharomyces cerevisiae induces trehalose or glycerol synthesis but does not increase intracellular proline levels during various stresses. Using a proline-accumulating mutant, we previously found that proline protects yeast cells from damage by freezing, oxidative, or ethanol stress. This mutant was recently shown to carry an allele of PRO1 which encodes the Asp154Asn mutant gamma-glutamyl kinase (GK), the first enzyme of the proline biosynthetic pathway. Here, enzymatic analysis of recombinant proteins revealed that the GK activity of S. cerevisiae is subject to feedback inhibition by proline. The Asp154Asn mutant was less sensitive to feedback inhibition than wild-type GK, leading to proline accumulation. To improve the enzymatic properties of GK, PCR random mutagenesis in PRO1 was employed. The mutagenized plasmid library was introduced into an S. cerevisiae non-proline-utilizing strain, and proline-overproducing mutants were selected on minimal medium containing the toxic proline analogue azetidine-2-carboxylic acid. We successfully isolated several mutant GKs that, due to extreme desensitization to inhibition, enhanced the ability to synthesize proline better than the Asp154Asn mutant. The amino acid changes were localized at the region between positions 142 and 154, probably on the molecular surface, suggesting that this region is involved in allosteric regulation. Furthermore, we found that yeast cells expressing Ile150Thr and Asn142Asp/Ile166Val mutant GKs were more tolerant to freezing stress than cells expressing the Asp154Asn mutant.     

Desensitization of Feedback Inhibition of the Saccharomyces cerevisiae γ-Glutamyl Kinase Enhances Proline Accumulation and Freezing Tolerance

In response to osmotic stress, proline is accumulated in many bacterial and plant cells as an osmoprotectant. The yeast Saccharomyces cerevisiae induces trehalose or glycerol synthesis but does not increase intracellular proline levels during various stresses. Using a proline-accumulating mutant, we previously found that proline protects yeast cells from damage by freezing, oxidative, or ethanol stress. This mutant was recently shown to carry an allele of PRO1 which encodes the Asp154Asn mutant γ-glutamyl kinase (GK), the first enzyme of the proline biosynthetic pathway. Here, enzymatic analysis of recombinant proteins revealed that the GK activity of S. cerevisiae is subject to feedback inhibition by proline. The Asp154Asn mutant was less sensitive to feedback inhibition than wild-type GK, leading to proline accumulation. To improve the enzymatic properties of GK, PCR random mutagenesis in PRO1 was employed. The mutagenized plasmid library was introduced into an S. cerevisiae non-proline-utilizing strain, and proline-overproducing mutants were selected on minimal medium containing the toxic proline analogue azetidine-2-carboxylic acid. We successfully isolated several mutant GKs that, due to extreme desensitization to inhibition, enhanced the ability to synthesize proline better than the Asp154Asn mutant. The amino acid changes were localized at the region between positions 142 and 154, probably on the molecular surface, suggesting that this region is involved in allosteric regulation. Furthermore, we found that yeast cells expressing Ile150Thr and Asn142Asp/Ile166Val mutant GKs were more tolerant to freezing stress than cells expressing the Asp154Asn mutant.     

Identification of a mutation that relieves gamma-glutamyl kinase from allosteric feedback inhibition by proline

A 1.75-kb DNA fragment containing the entire Escherichia coli proB+ gene has been sequenced. The proB locus encodes the structural gene for gamma-glutamyl kinase (GK), the enzyme responsible for the first step in proline biosynthesis, and the primary regulatory point of the pathway. We have previously reported the nucleotide (nt) sequence of a mutant proB gene isolated from an E. coli strain resistant to the toxic analog of proline, 3,4-dehydro-DL-proline (DHP). This mutant gene encodes a GK which is refractory to allosteric feedback inhibition by proline (DHPR). Comparison of the proB+ and DHPR proB sequences revealed a single base difference, an A-T to C-G transversion localized at nt position 428 within the amino acid (aa) coding region of proB. This mutation predicts an aa change from glutamic acid in the wild-type (wt) enzyme to alanine in the DHPR enzyme.     

Characterization and colocalization of steroid binding and dimerization activities in the mouse estrogen receptor

We have identified a region within the steroid binding domain of the mouse estrogen receptor that is required for both receptor dimerization and high affinity DNA binding. Analysis of sequences in this region revealed that a heptad repeat of hydrophobic residues was conserved in all members of the nuclear receptor superfamily. Single amino acid substitutions of residues in the N-terminal half, but not the C-terminal half, of the repeat prevented receptor dimerization. Steroid binding was abolished by point mutations in the center of the conserved region, implying that the steroid binding and dimerization domains overlap. The role of this region in steroid receptor function is discussed in relation to other models of protein dimerization and DNA binding.     

Proline transport in Salmonella typhimurium: putP permease mutants with altered substrate specificity.

The putP gene encodes a proline permease required for Salmonella typhimurium LT2 to grow on proline as the sole source of nitrogen. The wild-type strain is sensitive to two toxic proline analogs (azetidine-2-carboxylic acid and 3,4-dehydroproline) also transported by the putP permease. Most mutations in putP prevent transport of all three substrates. Such mutants are unable to grow on proline and are resistant to both of the analogs. To define domains of the putP gene that specify the substrate binding site, we used localized mutagenesis to isolate rare mutants with altered substrate specificity. The position of the mutations in the putP gene was determined by deletion mapping. Most of the mutations are located in three small (approximately 100-base-pair) deletion intervals of the putP gene. The sensitivity of the mutants to the proline analogs was quantitated by radial streaking to determine the affinity of the mutant permeases for the substrates. Some of the mutants showed apparent changes in the kinetics of the substrates transported. These results indicate that the substrate specificity mutations are probably due to amino acid substitutions at or near the active site of proline permease.     

NMR investigation of main-chain dynamics of the H80E mutant of bovine neurophysin-I: demonstration of dimerization-induced changes at the hormone-binding site

Neurophysins are hormone-binding proteins composed of two partially homologous domains. Ligand-binding (localized to the amino domain) and dimerization (involves both domains) are cooperatively linked by an as yet undefined allosteric mechanism. To help define this mechanism, we investigated the backbone dynamics of the unliganded monomeric state of the H80E mutant of bovine neurophysin-I by (15)N NMR. Model-free analysis of the NMR relaxation parameters indicated significantly greater flexibility in the carboxyl domain than in the amino domain, particularly at their dimerization interface segments. Amino domain residues critical to hormone binding were highly structured, constraining potential allosteric mechanisms. Model-free analysis additionally demonstrated chemical exchange effects, manifest as R(ex) terms, in 16 residues, 14 of which are located in the amino domain at, or immediately adjacent to, either the dimerization interface or the hormone-binding site. The chemical exchange process was further characterized using relaxation-compensated CPMG measurements, the results allowing assignment of the process to monomer-dimer exchange and calculation of the exchange kinetics, which were slow on the NMR time scale. An apparently different concentration-dependent process, distinguished from normal dimerization by its fast exchange behavior and pH-independence, also principally involved a subset of residues at and immediately adjacent to either the hormone-binding site or the amino domain dimerization interface. The data represent the first direct demonstration of an effect of dimerization in the unliganded state on neurophysin's hormone-binding site, the effect particularly involving residues that interact with hormone residue 2, and specifically identify Ser25 and Ile26 as likely intermediaries between the sites of dimerization and of hormone binding. Consistent with recent views of the role of anchor residues in protein interactions, we propose that dimerization proceeds by a fast pH-independent association of the well-structured amino domain interface that is rapidly communicated to the binding site for hormone residue 2, followed by a rate-determining pH-dependent interaction of the less structured carboxyl domain interface.     

Structure-function analysis of human interleukin-2. Identification of amino acid residues required for biological activity

To locate functional domains of the interleukin-2 (IL-2) protein, a cDNA clone encoding biologically active human IL-2 was mutagenized using synthetic oligonucleotides to incorporate defined amino acid substitutions and deletions in the mature protein. The IL-2 analogs were then produced in Escherichia coli and assayed for the ability to induce proliferation of IL-2-dependent cells and the ability to compete for binding to the IL-2 receptor. Our analysis of over 50 different mutations demonstrated that the integrity of at least three regions of the IL-2 molecule is required for full biological activity: the NH2 terminus (residues 1-20), the COOH terminus (residues 121-133), and 2 of the 3 cysteine residues (58 and 105). Deletion of the NH2-terminal 20 amino acids or the COOH-terminal 10 amino acids resulted in the loss of greater than 99% of bioactivity and binding. Amino acid substitutions at specific positions in these regions also resulted in proteins which retained less than 1% activity. The NH2 terminus and an adjacent internal region were recognized by neutralizing anti-IL-2 antibodies. In combination with the results from epitope competition analysis with neutralizing antibodies, these data are consistent with the IL-2 protein being folded such that the NH2 terminus, the COOH terminus, and the internal 30- to 60-region are juxtaposed to form the binding site recognized by the IL-2 receptor.     

AMP inhibition of pig kidney fructose-1,6-bisphosphatase.

Lys-112 and Tyr-113 in pig kidney fructose-1,6-bisphosphatase (FBPase) make direct interactions with AMP in the allosteric binding site. Both residues interact with the phosphate moiety of AMP while Tyr-113 also interacts with the 3'-hydroxyl of the ribose ring. The role of these two residues in AMP binding and allosteric inhibition was investigated. Site-specific mutagenesis was used to convert Lys-112 to glutamine (K112Q) and Tyr-113 to phenylalanine (Y113F). These amino acid substitutions result in small alterations in k(cat) and increases in K(m). However, both the K112Q and Y113F enzymes show alterations in Mg(2+) affinity and dramatic reductions in AMP affinity. For both mutant enzymes, the AMP concentration required to reduced the enzyme activity by one-half, [AMP](0.5), was increased more than a 1000-fold as compared to the wild-type enzyme. The K112Q enzyme also showed a 10-fold reduction in affinity for Mg(2+). Although the allosteric site is approximately 28 A from the metal binding sites, which comprise part of the active site, these site-specific mutations in the AMP site influence metal binding and suggest a direct connection between the allosteric and the active sites.     

Site-directed mutational analysis of human tumor necrosis factor-alpha receptor binding site and structure-functional relationship.

In order to define the receptor binding site and the structure-functional relationship of tumor necrosis factor (TNF), single amino acid substitutions were made by site-directed mutagenesis at selected residues of human tumor necrosis factor, using a phagemid mutagenesis/expression vector. The recombinant TNF mutants were compared to the wild type TNF in assays using crude bacterial lysates, for protein yield, solubility, subunit trimerization, receptor binding inhibition activity, and in vitro cytotoxic activity. All mutants which did not form cross-linkable trimer also showed little cytotoxic activity or receptor binding inhibition activity, indicating that trimer formation is obligatory for TNF-alpha activity. Most mutations of internal residues yielded no cross-linkable trimer, while most mutations of surface residues yielded cross-linkable trimer. Mutations at surface residues Leu29, Arg31, and Ala35 yielded cross-linkable trimers with good activities, except proline substitutions which may cause conformational changes in the polypeptide chain. This suggested that these residues are near the receptor binding site. Mutations at other strictly conserved internal residues such as Ser60, His78, and Tyr119 form cross-linkable trimer with little activity. These mutations may indirectly affect the receptor binding site by forming trimers with undetectable abnormalities. Mutants of surface residues Tyr87, Ser95, Ser133, and Ser147 affect receptor binding and cytotoxic activity but not trimer formation, suggesting that these residues are involved directly in receptor binding. The fact that residues Arg31, Ala35, Tyr87, Ser95, and Ser147, located on the opposite sides of a monomer, are clustered at the intersubunit grooves of TNF trimer supports the current notion that TNF receptor binding sites are trivalent and are located at the three intersubunit grooves. However, our finding that Ser133, which is outside the groove, can also be involved directly in receptor binding suggested that the receptor binding sites of TNF may not be confined to the intersubunit grooves, but extended to include additional surface residues.     

Isolation and preliminary characterization of amino acid substitution mutations that increase the activity of the osmoregulated ProP protein of Salmonella enterica serovar Typhimurium

In Enterobacteriaceae, the ProP protein, which takes up proline and glycine betaine, is subject to a post-translational control mechanism that increases its activity at high osmolarity. In order to investigate the osmoregulatory mechanism of the Salmonella enterica ProP, we devised a positive selection for mutations that conferred increased activity on this protein at low osmolarity. The selection involved the isolation of mutations in a proline auxotroph that resulted in increased accumulation of proline via the ProP system in the presence of glycine betaine, which is a competitive inhibitor of proline uptake by this permease. This selection was performed by first-year undergraduates in two semesters of a research-based laboratory course. The students generated sixteen mutations resulting in six different single amino acids substitutions. They determined the effects of the mutations on the growth rates of the cells in media of high and low osmolarity in the presence of low concentrations of proline or glycine betaine. Furthermore, they identified the mutations by DNA sequencing and displayed the mutated amino acids on a putative three-dimensional structure of the protein. This analysis suggested that all six amino acid substitutions are residues in trans-membrane helices that have been proposed to contribute to the formation of the transport pore, and, thus, may affect the substrate binding site of the protein.     

Regulation of phenylalanine biosynthesis. Studies on the mechanism of phenylalanine binding and feedback inhibition in the Escherichia coli P-protein.

Isothermal titration calorimetry (ITC) and site-directed mutagenesis were used to study the interaction of Phe with (a) the Escherichia coli P-protein, a bifunctional chorismate mutase/prephenate dehydratase that is feedback inhibited by Phe, (b) PDT32, a 32 kDa P-protein fragment (residues 101-386) containing the prephenate dehydratase and regulatory domains, and (c) R12, a C-terminal 12 kDa P-protein fragment (residues 286-386) containing the regulatory domain. DeltaH(total) values for PDT32, which included the heats of Phe binding, conformational change, and dimerization, established that in developing a mechanism for end product feedback inhibition, the P-protein has evolved a ligand recognition domain that exhibits Phe-binding enthalpies comparable to those reported for other full-fledged amino acid receptor proteins. Sequence alignments of R12 with other Phe-binding enzymes identified two highly conserved regions, GALV (residues 309-312) and ESRP (residues 329-332). Site-directed mutagenesis and ITC established that changes in the GALV and ESRP regions affected Phe binding and feedback inhibition to different extents. Mutagenesis further showed that C374 was essential for feedback inhibition, but not for Phe binding, while W338 was involved in Phe binding, but not in the Phe-induced conformational change required for feedback inhibition.     

A Symmetric Region of the HIV-1 Integrase Dimerization Interface Is Essential for Viral Replication

HIV-1 integrase (IN) is an important target for contemporary antiretroviral drug design research. Historically, efforts at inactivating the enzyme have focused upon blocking its active site. However, it has become apparent that new classes of allosteric inhibitors will be necessary to advance the antiretroviral field in light of the emergence of viral strains resistant to contemporary clinically used IN drugs. In this study we have characterized the importance of a close network of IN residues, distant from the active site, as important for the obligatory multimerization of the enzyme and viral replication as a whole. Specifically, we have determined that the configuration of six residues within a highly symmetrical region at the IN dimerization interface, composed of a four-tiered aromatic interaction flanked by two salt bridges, significantly contributes to proper HIV-1 replication. Additionally, we have utilized a quantitative luminescence assay to examine IN oligomerization and have determined that there is a very low tolerance for amino acid substitutions along this region. Even conservative residue substitutions negatively impacted IN multimerization, resulting in an inactive viral enzyme and a non-replicative virus. We have shown that there is a very low tolerance for amino acid variation at the symmetrical dimeric interface region characterized in this study, and therefore drugs designed to target the amino acid network detailed here could be expected to yield a significantly reduced number of drug-resistant escape mutations compared to contemporary clinically-evaluated antiretrovirals.     

Mutagenic mapping of helical structures in the transmembrane segments of the yeast alpha-factor receptor

The alpha-mating pheromone receptor encoded by the yeast STE2 gene is a G protein coupled receptor that initiates signaling via a MAP kinase pathway that prepares haploid cells for mating. To establish the range of allowed amino acid substitutions within transmembrane segments of this receptor, we conducted extensive random mutagenesis of receptors followed by screening for receptor function. A total of 157 amino acid positions in seven different mutagenic libraries corresponding to the seven predicted transmembrane segments were analyzed, yielding 390 alleles that retain at least 60 % of normal signaling function. These alleles contained a total of 576 unique amino acid substitutions, including 61 % of all the possible amino acid changes that can arise from single base substitutions. The receptor exhibits a surprising tolerance for amino acid substitutions. Every amino acid in the mutagenized regions of the transmembrane regions could be substituted by at least one other residue. Polar amino acids were tolerated in functional receptors at 115 different positions (73 % of the total). Hydrophobic amino acids were tolerated in functional receptors at all mutagenized positions. Substitutions introducing proline residues were recovered at 53 % of all positions where they could be brought about by single base changes. Residues with charged side-chains could also be tolerated at 53 % of all positions where they were accessible through single base changes. The spectrum of allowed amino acid substitutions was characterized in terms of the hydrophobicity, radius of gyration, and charge of the allowed substitutions and mapped onto alpha-helical structures. By comparing the patterns of allowed substitutions with the recently determined structure of rhodopsin, structural features indicative of helix-helix interactions can be discerned in spite of the extreme sequence divergence between these two proteins.     

Characterization of the antigenic structure of herpes simplex virus type 1 glycoprotein C through DNA sequence analysis of monoclonal antibody-resistant mutants.

Earlier studies of a group of monoclonal antibody-resistant (mar) mutants of herpes simplex virus type 1 glycoprotein C (gC) operationally defined two distinct antigenic sites on this molecule, each consisting of numerous overlapping epitopes. In this report, we further define epitopes of gC by sequence analysis of the mar mutant gC genes. In 18 mar mutants studied, the mar phenotype was associated with a single nucleotide substitution and a single predicted amino acid change. The mutations were localized to two regions within the coding sequence of the external domain of gC and correlated with the two previously defined antigenic sites. The predicted amino acid substitutions of site I mutants resided between residues Gln-307 and Pro-373, whereas those of site II mutants occurred between amino acids Arg-129 and Glu-247. Of the 12 site II mutations, 9 induced amino acid substitutions within an arginine-rich segment of 8 amino acids extending from residues 143 to 151. The clustering of the majority of substituted residues suggests that they contribute to the structure of the affected sites. Moreover, the patterns of substitutions which affected recognition by antibodies with similar epitope specificities provided evidence that epitope structures are physically linked and overlap within antigenic sites. Of the nine epitopes defined on the basis of mutations, three were located within site I and six were located within site II. Substituted residues affecting the site I epitopes did not overlap substituted residues of site II, supporting our earlier conclusion that sites I and II reside in spatially distinct antigenic domains. A computer analysis of the distribution of charged residues and the predicted secondary structural features of wild-type gC revealed that the two antigenic sites reside within the most hydrophilic regions of the molecule and that the antigenic residues are likely to be organized as beta sheets which loop out from the surface of the molecule. Together, these data and our previous studies support the conclusion that the mar mutations identified by sequence analysis very likely occur within or near the epitope structures themselves. Thus, two highly antigenic regions of gC have now been physically and genetically mapped to well-defined domains of the protein molecule.     

Domains, amino acid residues, and new isoforms of Caenorhabditis elegans diacylglycerol kinase 1 (DGK-1) important for terminating diacylglycerol signaling in vivo

Diacylglycerol kinases (DGKs) inhibit diacylglycerol (DAG) signaling by phosphorylating DAG. DGK-1, the Caenorhabditis elegans ortholog of human neuronal DGK, inhibits neurotransmission to control behavior. DGK-1, like DGK, has three cysteine-rich domains (CRDs), a pleckstrin homology domain, and a kinase domain. To identify DGK domains and amino acid residues critical for terminating DAG signaling in vivo, we analyzed 20 dgk-1 mutants defective in DGK-1-controlled behaviors. We found by sequencing that the mutations included nine amino acid substitutions and seven premature stop codons that impair the physiological functions of DGK-1. All nine amino acid substitutions are in the second CRD, the third CRD, or the kinase domain. Thus, these domains are important for the termination of DAG signaling by DGK-1 in vivo. Seven of the substituted amino acid residues are present in all human DGKs and likely define key residues required for the function of all DGKs. An ATP-binding site mutation expected to inactivate the kinase domain retained very little physiological function, but we found two stop codon mutants predicted to truncate DGK-1 before its kinase domain that retained significantly more function. We detected novel splice forms of dgk-1 that can reconcile this apparent conflict, as they skip exons containing the stop codons to produce DGK-1 isoforms that contain the kinase domain. Two of these isoforms lack an intact pleckstrin homology domain and yet appear to have significant function. Additional novel isoform(s) account for all of the DGK-1 function necessary for one behavior, dopamine response.     

Cloning and sequence analysis of lily and tobacco guanylate kinases

Guanylate kinase is an essential enzyme in the nucleotide biosynthetic pathway, catalyzing the reversible transfer of the terminal phospharyl group of ATP to GMP or dGMP. This enzyme has been well studied from several organisms and many structural and functional details have been characterized. Animal GMP kinases have also been implicated in signal transduction pathways. However, the corresponding role by plant derived GMP kinases remains to be elucidated. Full-length cDNA clones encoding enzymatically active guanylate kinases were isolated from cDNA libraries of lily and tobacco. Lily cDNA is predicted to encode a 392-amino acid protein with a molecular mass of 43.1 kDa and carries amino- and carboxy- terminal extensions of the guanylate kinase (GK)-like domain. But tobacco cDNA is predicted to encode a smaller protein of 297-amino acids with a molecular mass of 32.7 kDa. The amino acid residues known to participate in the catalytic activity of functionally characterized GMP kinases, are also conserved in GK domains of LGK-1 and NGK-1. The GK domains of NGK-1, LGK-1 and previously characterized AGK-1 from Arabidopsis exhibit 74–84% identity, whereas their N- and C-terminal domains are more divergent with amino acid conservation in the order of 48-55%. Phylogenetic analysis on the deduced amino acid sequences reveals that NGK-1 and LGK-1 form one distinct subgroup along with AGK-1 and AGK-2 homologues from Arabidopsis. Isolation of GMP kinases from diverse plant species like lily and tobacco adds a new dimension in understanding their role in cell signaling pathways that are associated with plant growth and development.     

Role of serine 352 in the allosteric response of Serratia marcescens aspartokinase I-homoserine dehydrogenase I analyzed by using site-directed mutagenesis.

Aspartokinase I and homoserine dehydrogenase I (AKI-HDI) from Serratia marcescens Sr41 are encoded by the thrA gene as a single polypeptide chain. Previously, a single amino acid substitution of Ser-352 with Phe was shown to produce an AKI-HDI enzyme that is not subject to threonine-mediated feedback inhibition. To determine the role of Ser-352 in the allosteric response, the thrA gene was modified by using site-directed mutagenesis so that Ser-352 of the wild-type AKI-HDI was replaced by Ala, Arg, Asn, Gln, Glu, His, Leu, Met, Pro, Thr, Trp, Tyr, or Val. The Thr-352 and Pro-352 replacements rendered AKIs sensitive to threonine. The Tyr-352 and Asn-352 substitutions led to activation, rather than inhibition, of AKI by threonine. The other replacements conferred threonine insensitivity on AKI. The threonine sensitivity of HDI was also changed by the amino acid substitutions at Ser-352. The HDI carried by the Tyr-352 mutant AKI-HDI was activated by threonine. Single amino acid replacements at Ser-352 by Ala, Asn, Gln, His, Phe, Pro, Thr, or Tyr were introduced into truncated AKI-HDIs containing the AKI and the central regions. The AKI activity of the truncated AKI-HDI containing the first 468 amino acid residues was sensitive to threonine, and introduction of the amino acid replacements did not alter the threonine sensitivity of the AKI. Another truncated AKI-HDI containing the first 462 amino acid residues possessed threonine-resistant AKI, whereas the substitutions of Ser-352 with Ala and Pro rendered AKI sensitive to threonine. The replacement of GIn-351 with Phe activated AK1 of the truncated AKI-HDI in the presence of L-threonine. These findings suggest that Ser-352 of the central region of AKI-HDI is possibly a key residue involved with the allosteric regulation of both AKI and HDI activities.     

IIAGlc inhibition of glycerol kinase: a communications network tunes protein motions at the allosteric site

Steady-state and time-resolved fluorescence anisotropy methods applied to an extrinsic fluorophore that is conjugated to non-native cysteine residues demonstrate that amino acids in an allosteric communication network within a protein subunit tune protein backbone motions at a distal site to enable allosteric binding and inhibition. The unphosphorylated form of the phosphocarrier protein IIAGlc is an allosteric inhibitor of Escherichia coli glycerol kinase, binding more than 25 A from the kinase active site. Crystal structures that showed a ligand-dependent conformational change and large temperature factors for the IIAGlc-binding site on E. coli glycerol kinase suggest that motions of the allosteric site have an important role in the inhibition. Three E. coli glycerol kinase amino acids that are located at least 15 A from the active site and the allosteric site were shown previously to be necessary for transplanting IIAGlc inhibition into the nonallosteric glycerol kinase from Haemophilus influenzae. These three amino acids are termed the coupling locus. The apparent allosteric site motions and the requirement for the distant coupling locus to transplant allosteric inhibition suggest that the coupling locus modulates the motions of the IIAGlc-binding site. To evaluate this possibility, variants of E. coli glycerol kinase and the chimeric, allosteric H. influenzae glycerol kinase were constructed with a non-native cysteine residue replacing one of the native residues in the IIAGlc-binding site. The extrinsic fluorophore Oregon Green 488 (2',7'-difluorofluorescein) was conjugated specifically to the non-native cysteine residue. Steady-state and time-resolved fluorescence anisotropy measurements show that the motions of the fluorophore reflect backbone motions of the IIAGlc-binding site and these motions are modulated by the amino acids at the coupling locus.