Dephosphorylation of Ser-226 facilitates plasma membrane retention of Ntcp

Ntcp is a phosphoprotein, and its translocation by cAMP to the plasma membrane is associated with dephosphorylation. However, the phosphorylation site(s) of Ntcp is not known. Thus, the aim of the present study was to determine the potential Ntcp phosphorylation sites and whether any of these phosphorylation sites is involved in Ntcp translocation. To determine the potential phosphorylation sites, metabolically labeled [32P]Ntcp isolated from hepatocytes was digested with clostripain and then subjected to SDS-PAGE followed by autoradiography. Clostripain digestion resulted in two phosphorylated peptides, and cAMP decreased phosphorylation of one of the peptides (7.8 K(d)), which contains the putative third cytoplasmic loop with three serine (Ser-213, Ser-226, and Ser-227) and two threonine (Thr-219 and Thr-225) residues. To determine whether any one of these serine/threonine residues is phosphorylated and/or is involved in Ntcp translocation, each of these serine/threonine residues were mutated to alanine. HuH-7 cells were transiently transfected with the wild-type and the mutated Ntcps followed by determination of taurocholate uptake and Ntcp expression, translocation and phosphorylation. Mutation of only Ser-226 resulted in 30% decrease in Ntcp phosphorylation and in 2.5 and 3.2-fold increases in taurocholate uptake and Ntcp retention in the plasma membrane, respectively. Cyclic AMP failed to further decrease phosphorylation and increase translocation of S226A-Ntcp. Taken together, these results suggest that the Ser-226 in the third cytoplasmic loop of Ntcp is phosphorylated and cAMP may increase Ntcp translocation to the plasma membrane by dephosphorylating Ntcp at this site.     

Identification of inhibitory autophosphorylation sites in casein kinase I epsilon.

Casein kinase I epsilon (CKIepsilon) is a widely expressed protein kinase implicated in the regulation of diverse cellular processes including DNA replication and repair, nuclear trafficking, and circadian rhythm. CKIepsilon and the closely related CKIdelta are regulated in part through autophosphorylation of their carboxyl-terminal extensions, resulting in down-regulation of enzyme activity. Treatment of CKIepsilon with any of several serine/threonine phosphatases causes a marked increase in kinase activity that is self-limited. To identify the sites of inhibitory autophosphorylation, a series of carboxyl-terminal deletion mutants was constructed by site-directed mutagenesis. Truncations that eliminated specific phosphopeptides present in the wild-type kinase were used to guide construction of specific serine/threonine to alanine mutants. Amino acids Ser-323, Thr-325, Thr-334, Thr-337, Ser-368, Ser-405, Thr-407, and Ser-408 in the carboxyl-terminal tail of CKIepsilon were identified as probable in vivo autophosphorylation sites. A recombinant CKIepsilon protein with serine and threonine to alanine mutations eliminating these autophosphorylation sites was 8-fold more active than wild-type CKIepsilon using IkappaBalpha as a substrate. The identified autophosphorylation sites do not conform to CKI substrate motifs identified in peptide substrates.     

Vibrational spectroscopy of bacteriorhodopsin mutants. Evidence that Thr-46 and Thr-89 form part of a transient network of hydrogen bonds.

The role of Thr-46 and Thr-89 in the bacteriorhodopsin photocycle has been investigated by Fourier transform infrared difference spectroscopy and time-resolved visible absorption spectroscopy of site-directed mutants. Substitutions of Thr-46 and Thr-89 reveal alterations in the chromophore and protein structure during the photocycle, relative to wild-type bacteriorhodopsin. The mutants T89D and to a lesser extent T89A display red shifts in the visible lambda max of the light-adapted states compared with wild type. During the photocycle, T89A exhibits an increased decay rate of the K intermediate, while a K intermediate is not detected in the photocycle of T89D at room temperature. In the carboxyl stretch region of the Fourier transform infrared difference spectra of T89D, a new band appears as early as K formation which is attributed to the deprotonation of Asp-89. Along with this band, an intensity increase occurs in the band assigned to the protonation of Asp-212. In the mutant T46V, a perturbation in the environment of Asp-96 is detected in the L and M intermediates which corresponds to a drop in its pK alpha. These data indicate that Thr-89 is located close to the chromophore, exerts steric constraints on it during all-trans to 13-cis isomerization, and is likely to participate in a hydrogen-bonding network that extends to Asp-212. In addition, a transient interaction between Thr-46 and Asp-96 occurs early in the photocycle. In order to explain these results, a previously proposed model of proton transport is extended to include the existence of a transient network of hydrogen-bonded residues. This model can account for the protonation changes of key amino acid residues during the photocycle of bacteriorhodopsin.     

Structure-function studies on bacteriorhodopsin. X. Individual substitutions of arginine residues by glutamine affect chromophore formation, photocycle, and proton translocation

We have individually replaced all 7 of the arginine residues in bacteriorhodopsin by glutamine. The mutants with substitutions at positions 7, 164, 175, and 225 showed essentially the wild-type phenotype in regard to chromophore regeneration, chromophore lambda max, and proton pumping, although the mutant Arg-175----Gln showed decreased rate of chromophore regeneration. Glutamine substitutions of Arg-82, -134, and -227 affected proton pumping ability, and caused specific alterations in the bacteriorhodopsin photocycle. Finally, electrostatic interactions are proposed between Arg-82 and -227, and specific carboxylic acid residues in helices C and G, which regulate the purple to blue transition and proton transfers during the photocycle.     

Substitution of Pro206 and Ser86 residues in the retinal binding pocket of Anabaena sensory rhodopsin is not sufficient for proton pumping function

Anabaena sensory rhodopsin is a seven transmembrane protein that uses all-trans/13-cis retinal as a chromophore. About 22 residues in the retinal-binding pocket of microbial rhodopsins are conserved and important to control the quality of absorbing light and the function of ion transport or sensory transduction. The absorption maximum is 550 nm in the presence of all-trans retinal at dark. Here, we mutated Pro206 to Glu or Asp, of which the residue is conserved as Asp among all other microbial rhodopsins, and the absorption maximum and pKa of the proton acceptor group were measured by absorption spectroscopy at various pHs. Anabaena rhodopsin was expressed best in Escherichia coli in the absence of extra leader sequence when exogenous all-trans retinal was added. The wild-type Anabaena rhodopsin showed small absorption maximum changes between pH 4 and 11. In addition, Pro206Asp showed 46 nm blue-shift at pH 7.0. Pro206Glu or Asp may change the contribution to the electron distribution of the retinal that is involved in the major role of color tuning for this pigment. The critical residue Ser86 (Asp 96 position in bacteriorhodopsin: proton donor) for the pumping activity was replaced with Asp, but it did not change the proton pumping activity of Anabaena rhodopsin.     

Screening and characterization of proteorhodopsin color-tuning mutations in Escherichia coli with endogenous retinal synthesis

Proteorhodopsin is photoactive 7-transmembrane protein, which uses all-trans retinal as a chromophore. Proteorhodopsin subfamilies are spectrally tuned in accordance with the depth of habitat of the host organisms, numerous species of marine picoplankton. We try to find residues critical for the spectral tuning through the use of random PCR mutagenesis and endogenous retinal biosynthesis. We obtained 16 isolates with changed color by screening in Escherichia coli with internal retinal biosynthesis system containing genes for beta-carotene biosynthesis and retinal synthase. Some isolates contained multiple substitutions, which could be separated to give 20 single mutations influencing the spectral properties. The color-changing residues are distributed through the protein except for the helix A, and about a half of the mutations is localized on the helices C and D, implying their importance for color tuning. In the pumping form of the pigment, absorption maxima in 8 mutants are red-shifted and in 12 mutants are blue-shifted compared to the wild-type. The results of flash-photolysis showed that most of the low pumping activity mutants possess slower rates of M decay and O decay. These results suggest that the color-tuning residues are not restricted to the retinal binding pocket, in accord with a recent evolutionary analysis.     

Active-site mutants of beta-lactamase: use of an inactive double mutant to study requirements for catalysis

We have studied the catalytic activity and some other properties of mutants of Escherichia coli plasmid-encoded RTEM beta-lactamase (EC 3.5.2.6) with all combinations of serine and threonine residues at the active-site positions 70 and 71. (All natural beta-lactamases have conserved serine-70 and threonine-71.) From the inactive double mutant Ser-70----Thr, Thr-71----Ser [Dalbadie-McFarland, G., Cohen, L. W., Riggs, A. D., Morin, C., Itakura, K., & Richards, J. H. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 6409-6413], an active revertant, Thr-71----Ser (i.e., residue 70 in the double mutant had changed from threonine to the serine conserved at position 70 in the wild-type enzyme), was isolated by an approach that allows identification of active revertants in the absence of a background of wild-type enzyme. This mutant (Thr-71----Ser) has about 15% of the catalytic activity of wild-type beta-lactamase. The other possible mutant involving serine and threonine residues at positions 70 and 71 (Ser-70----Thr) shows no catalytic activity. The primary nucleophiles of a serine or a cysteine residue [Sigal, I. S., Harwood, B. G., & Arentzen, R. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 7157-7160] at position 70 thus seem essential for enzymatic activity. Compared to wild-type enzyme, all three mutants show significantly reduced resistance to proteolysis; for the active revertant (Thr-71----Ser), we have also observed reduced thermal stability and reduced resistance to denaturation by urea.     

Importance of Thr-353 of the conserved phosphorylation loop of the sarcoplasmic reticulum Ca2+-ATPase in MgATP binding and catalytic activity.

Mutants in which Thr-353 of the Ca(2+)-ATPase of sarcoplasmic reticulum had been replaced with alanine, serine, glutamine, cysteine, valine, aspartate, or tyrosine were analyzed functionally. All the mutations severely affected MgATP binding, whereas ATP binding was close to normal in the alanine, serine, glutamine, and valine mutants. In the serine and valine mutants, the maximum rate of phosphorylation from MgATP was 8- and 600-fold lower, respectively, compared with wild type. Replacement of Mg(2+) with Mn(2+) led to a 1.5-fold enhancement of the maximum phosphorylation rate in the valine mutant and a 5-fold reduction in the wild type. The turnover of the phosphoenzyme formed from MgATP was slowed 1-2 orders of magnitude relative to wild type in the alanine, serine, and valine mutants, but was close to normal in the aspartate and cysteine mutants. Only the serine mutant formed a phosphoenzyme in the backward reaction with P(i), and the hydrolysis of this intermediate was greatly enhanced. Analysis of the functional changes in the mutants in the light of the recent high resolution structure of the Ca(2+)-ATPase crystallized without the MgATP substrate suggests that, in the native activated state of the enzyme, the side chain hydroxyl of Thr-353 participates in important interactions with nucleotide and phosphate, possibly in catalysis, whereas the main chain carbonyl of Thr-353, but not the side chain, may coordinate the catalytic Mg(2+).     

An analysis of the side chain requirement at position 177 within the lactose permease which confers the ability to recognize maltose.

In previous work (Brooker, R. J., and Wilson, T. H. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 3959-3963), lactose permease mutants were isolated which possessed an enhanced recognition for maltose. In some of these mutants, the wild-type alanine residue at position 177 was changed to valine or threonine. To gain further insight into the side chain requirement at position 177 that confers maltose recognition, further substitutions of isoleucine, leucine, phenylalanine, proline, and serine have been made via site-directed mutagenesis. Permeases containing alanine or serine exhibited poor maltose recognition whereas those containing isoleucine, leucine, phenylalanine, proline, or valine showed moderate or good recognition. As far as galactosides are concerned, the Val-177, Pro-177, and Ser-177 mutants were able to transport lactose as well as, or slightly better than, the wild-type strain. The other mutants displayed moderately reduced levels of lactose transport. For example, the Phe-177 mutant, which was the most defective, showed a level of downhill transport which was approximately 20% that of the wild-type strain. In uphill transport assays, all of the position 177 mutants were markedly defective in their ability to accumulate beta-D-thiomethylgalactopyranoside against a concentration gradient. Finally, the position 177 mutants were analyzed for their ability to catalyze an H+ leak. Interestingly, even though the wild-type permease does not leak H+ across the bacterial membrane, all of the position 177 mutants were shown to transport H+ in the absence of sugars. For most of the mutants, this H+ leak was blocked by the addition of beta-D-thiodigalactoside. Overall, these results are discussed with regard to the effects of position 177 substitutions on the sugar recognition site and H+ transport.     

The role of a conserved serine residue within hydrogen bonding distance of FAD in redox properties and the modulation of catalysis by Ca2+/calmodulin of constitutive nitric-oxide synthases

The crystal structure of the neuronal nitric-oxide synthase (nNOS) NADPH/FAD binding domain indicated that Ser-1176 is within hydrogen bonding distance of Asp-1393 and the O4 atom of FAD and is also near the N5 atom of FAD (3.7 A). This serine residue is conserved in most of the ferredoxin-NADP+ reductase family of proteins and is important in electron transfer. In the present study, the homologous serines of both nNOS (Ser-1176) and endothelial nitric-oxide synthase (eNOS) (Ser-942) were mutated to threonine and alanine. Both substitutions yielded proteins that exhibited decreased rates of electron transfer through the flavin domains, in the presence and absence of Ca2+/CaM, as measured by reduction of potassium ferricyanide and cytochrome c. Rapid kinetics measurements of flavin reduction of all the mutants also showed a decrease in the rate of flavin reduction, in the absence and presence of Ca2+/CaM, as compared with the wild type proteins. The serine to alanine substitution caused both nNOS and eNOS to synthesize NO more slowly; however, the threonine mutants gave equal or slightly higher rates of NO production compared with the wild type enzymes. The midpoint redox potential measurements of all the redox centers revealed that wild type and threonine mutants of both nNOS and eNOS are very similar. However, the redox potentials of the FMN/FMNH* couple for alanine substitutions of both nNOS and eNOS are >100 mV higher than those of wild type proteins and are positive. These data presented here suggest that hydrogen bonding of the hydroxyl group of serine or threonine with the isoalloxazine ring of FAD and with the amino acids in its immediate milieu, particularly nNOS Asp-1393, affects the redox potentials of various flavin states, influencing the rate of electron transfer.     

Vibrational spectroscopy of bacteriorhodopsin mutants. Evidence for the interaction of aspartic acid 212 with tyrosine 185 and possible role in the proton pump mechanism

The role of Asp-212 in the proton pumping mechanism of bacteriorhodopsin (bR) has been studied by a combination of site-directed mutagenesis and Fourier transform infrared difference spectroscopy. Difference spectra were recorded at low temperature for the bR----K and bR----M photoreactions of the mutants Asp-212----Glu, Asp-212----Asn, and Asp-212----Ala. Despite an increased proportion of the 13-cis form of bR (normally associated with dark adaptation), all of the mutants exhibited a light-adapted form containing as a principal component the normal all-trans retinal chromophore. The absence of a shift in the retinal C = C stretching frequency in these mutants indicates that Asp-212 is not a major determinant of the visible absorption wavelength maximum in light-adapted bR. It is unlikely that Asp-212 is the acceptor group for the Schiff base proton since both the Asp-212----Glu and Asp-212----Ala mutants formed an M intermediate. All of the Asp-212 mutants were missing a Fourier transform infrared difference band that had been assigned previously to protonation changes of Tyr-185. These results are discussed in terms of a model in which Tyr-185 and Asp-212 form a polarizable hydrogen bond and are positioned near the C13-Schiff base portion of the chromophore. These 2 residues may be involved in stabilizing the relative orientation of the F and G helices and isomerizing the retinal in a regioselective manner about the C13 = C14 double bond.     

Critical amino acid residues in transmembrane domain 1 of the human organic anion transporter hOAT1

Human organic anion transporter 1 (hOAT1) belongs to a superfamily of organic anion transporters, which play critical roles in the body disposition of clinically important drugs, including anti-human immunodeficiency virus therapeutics, anti-tumor drugs, antibiotics, anti-hypertensives, and anti-inflammatories. Previously we suggested that the predicted transmembrane domain 1 (TM1) of hOAT1 might be important for its function. In the present study, we examined the role of each residue within TM1 of hOAT1 in substrate recognition and transport. Alanine scanning was used to construct mutants of hOAT1, and the uptake of model substrate para-aminohippurate was studied in COS-7 cells expressing the mutant transporters. This approach led to the discovery of two critical amino acid residues, Leu-30 and Thr-36. A substitution of Leu-30 or Thr-36 with alanine resulted in a complete loss of transport activities. We then further characterized Leu-30 and Thr-36 by mutagenizing these residues to amino acids with different physicochemical properties. Leu-30 was replaced with amino acids with varying sizes of side chains, including glycine, valine, and isoleucine. We showed that progressively smaller side chains at position 30 increasingly impaired hOAT1 function mainly because of the impaired surface expression of the transporter. Thr-36, another critical amino acid in TM1, was replaced by serine and cysteine. Similar to the substitution of Thr-36 by alanine, substitution by serine and cysteine at this position abolished transport activity without affecting the surface expression of the transporter. The fact that Thr-36 cannot be substituted with serine and that the side chains of alanine, serine, and cysteine are smaller than that of threonine by a methyl group indicate that both the methyl group and the hydroxyl group of Thr-36 could be critical for hOAT1 activity. Together we conclude that Leu-30 and Thr-36 play distinct roles in hOAT1 function. Leu-30 is important in targeting the transporter to the plasma membrane. In contrast, Thr-36 is critical for substrate recognition. The present study provided the first molecular evidence that transmembrane domain 1 is a critical determinant of hOAT1 function and may provide important insights into the structure-function relationships of the organic anion transporter family.     

Investigation of invariant serine/threonine residues in mevalonate kinase. Tests of the functional significance of a proposed substrate binding motif and a site implicated in human inherited disease

Mevalonate kinase serine/threonine residues have been implicated in substrate binding and inherited metabolic disease. Alignment of >20 mevalonate kinase sequences indicates that Ser-145, Ser-146, Ser-201, and Thr-243 are the only invariant residues with alcohol side chains. These residues have been individually mutated to alanine. Structural integrity of the mutants has been demonstrated by binding studies using fluorescent and spin-labeled ATP analogs. Kinetic characterization of the mutants indicates only modest changes in K(m)((ATP)). K(m) for mevalonate increases by approximately 20-fold for S146A, approximately 40-fold for T243A, and 100-fold for S201A. V(max) changes for S145A, S201A, and T243A are < or =3-fold. Thus, the 65-fold activity decrease associated with the inherited human T243I mutation seems attributable to the nonconservative substitution rather than any critical catalytic function. V(max) for S146A is diminished by 4000-fold. In terms of V/K(MVA), this substitution produces a 10(5)-fold effect, suggesting an active site location and catalytic role for Ser-146. The large k(cat) effect suggests that Ser-146 productively orients ATP during catalysis. K(D(Mg-ATP)) increases by almost 40-fold for S146A, indicating a specific role for Ser-146 in liganding Mg(2+)-ATP. Instead of mapping within a proposed C-terminal ATP binding motif, Ser-146 is situated in a centrally located motif, which characterizes the galactokinase/homoserine kinase/ mevalonate kinase/phosphomevalonate kinase protein family. These observations represent the first functional demonstration that this region is part of the active site in these related phosphotransferases.     

Vibrational spectroscopy of bacteriorhodopsin mutants: evidence for the interaction of proline-186 with the retinylidene chromophore

Fourier-transform infrared difference spectroscopy has been used to study the role of the three membrane-embedded proline residues, Pro-50, Pro-91, and Pro-186, in the structure and function of bacteriorhodopsin. All three prolines were replaced by alanine and glycine; in addition, Pro-186 was changed to valine. Difference spectra were recorded for the bR----K and bR----M photoreactions of each of these mutants and compared to those of wild-type bacteriorhodopsin. Only substitutions of Pro-186 caused significant perturbations in the frequency of the C = C and C - C stretching modes of the retinylidene chromophore. In addition, these substitutions reduced bands in the amide I and II region associated with secondary structural changes and altered signals assigned to the adjacent Tyr-185. Pro-186----Val caused the largest alterations, producing a second species similar to bR548 and nearly blocking chromophore isomerization at 78 K but not at 250 K. These results are consistent with a model of the retinal binding site in which Pro-186 and Tyr-185 are located in direct proximity to the chromophore and may be involved in linking chromophore isomerization to protein structural changes. Evidence is also found that Pro-50 may be structurally active during the bR----K transition and that substitution of this residue by glycine preserves the normal protein structural changes during the photocycle.     

Role of Specific Phosphorylation Sites of <Emphasis Type="Italic">Arabidopsis</Emphasis> Brassinosteroid-Insensitive 1 Receptor Kinase in Plant Growth and Development

Brassinosteroid-insensitive 1 (BRI1), the receptor of brassinosteroids (BRs), is a dual-function serine/threonine/tyrosine protein kinase which initiates BR signaling and regulates plant growth via its protein kinase activity. Previous research has identified phosphorylation sites of Arabidopsis BRI1 in vivo and in vitro, but the significance of which to BR signaling and plant development has not been discussed comprehensively. To investigate this, we systematically characterized Arabidopsis BRI1 site-directed mutants in the weak bri1-5 background. For vegetative organ development regulation, we demonstrated that Thr-1039, Ser-1042, and Ser-1044 were critical for vegetative development because mutants with eliminated phosphorylation at these residues exhibited aberrant leaf growth, whereas Ser-1172 and Ser-1187 slightly inhibited leaf growth. For reproductive organ development regulation, first, the notion that Thr-1039, Ser-1042, and Ser-1044 were essential for normal plant height is supported by the evidence that mutations preventing phosphorylation at Thr-1039, Ser-1042, and Ser-1044 decreased plant height. Second, comparison of seed yield-related traits showed that unphosphorylated Ser-1168-Ala, Ser-1172-Ala, and Ser-1179-Ala+Thr-1180-Ala mutants reduced seed yield dramatically, whereas eliminating phosphorylation at Ser-1042 caused increased seed production. In addition, we found that Ser-1042 and Ser-1044 were essential for BR signaling. The unphosphorylated Ser-1042-Ala and Ser-1044-Ala mutants displayed hyposensitive phenotypes accompanied with decreased accumulation of dephosphorylated BRI1-EMS suppressor 1 (BES1) protein and increased Constitutive Photomorphogenesis Dwarf expression levels as well as limited inhibition of hypocotyl and root elongation under exogenous brassinolide. Taken together, our data suggest that BRI1 phosphorylation at specific sites differentially affects growth and development which may provide novel approaches to precisely regulate economic yield through modifying specific BRI1 phosphorylation sites in crop species.     

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.     

Conversion of trypsin to a functional threonine protease

The hydroxyl group of a serine residue at position 195 acts as a nucleophile in the catalytic mechanism of the serine proteases. However, the chemically similar residue, threonine, is rarely used in similar functional context. Our structural modeling suggests that the Ser 195 --> Thr trypsin variant is inactive due to negative steric interaction between the methyl group on the beta-carbon of Thr 195 and the disulfide bridge formed by cysteines 42 and 58. By simultaneously truncating residues 42 and 58 and substituting Ser 195 with threonine, we have successfully converted the classic serine protease trypsin to a functional threonine protease. Substitution of residue 42 with alanine and residue 58 with alanine or valine in the presence of threonine 195 results in trypsin variants that are 10(2) -10(4) -fold less active than wild type in kcat/KM but >10(6)-fold more active than the Ser 195 --> Thr single variant. The substitutions do not alter the substrate specificity of the enzyme in the P1'- P4' positions. Removal of the disulfide bridge decreases the overall thermostability of the enzyme, but it is partially rescued by the presence of threonine at position 195.     

Structural studies on transmembrane proteins. 1. Model study using bacteriorhodopsin mutants containing single cysteine residues

In developing new approaches to structural studies of polytopic transmembrane proteins, we have prepared bacteriorhodopsin mutants containing single cysteine residues at selected sites in different topological domains. Four such mutants were prepared: Gly-72----Cys and Ser-169----Cys in the presumed looped-out regions on the opposite sides of the membrane bilayer and Thr-90----Cys and Leu-92----Cys in the membrane-embedded helix C. The four mutants folded and regenerated the characteristic chromophore in detergent/phospholipid micelles and pumped protons like the wild-type bacteriorhodopsin. After reconstitution in asolectin vesicles, the sulfhydryl groups in the mutants Gly-72----Cys and Ser-169----Cys reacted with iodo[2-3H]acetic acid, while the sulfhydryl groups in the membrane-embedded mutants, Thr-90----Cys and Leu-92----Cys, did not. The sulfhydryl groups in all four mutants could be derivatized in the denatured state by reaction with iodoacetic acid or 6-acryloyl-2-(dimethylamino)naphthalene. Of these derivatives, the two from the mutants Gly-72----Cys and Ser-169----Cys folded like the wild-type bacterioopsin, whereas of the two from the helix C mutants, Thr-90----Cys and Leu-92----Cys, only the latter folded normally. However, the folding of Leu-92----Cys was also impaired when treated with the bulky 5-(iodoacetamido)fluorescein. The reactivity and the folding behavior of the cysteine mutants can thus report on the topographic domain as well as on the orientation of the helices within the membrane.     

Identification of in vivo phosphorylation sites and their functional significance in the sodium iodide symporter

The Na+/I- symporter (NIS)-mediated iodide uptake activity is the basis for targeted radioiodide ablation of thyroid cancers. Although it has been shown that NIS protein is phosphorylated, neither the in vivo phosphorylation sites nor their functional significance has been reported. In this study, Ser-43, Thr-49, Ser-227, Thr-577, and Ser-581 were identified as in vivo NIS phosphorylation sites by mass spectrometry. Kinetic analysis of NIS mutants of the corresponding phosphorylated amino acid residue indicated that the velocity of iodide transport of NIS is modulated by the phosphorylation status of Ser-43 and Ser-581. We also found that the phosphorylation status of Thr-577 may be important for NIS protein stability and that the phosphorylation status of Ser-227 is functionally silent. Thr-49 appears to be critical for proper local structure/conformation of NIS because mutation of Thr-49 to alanine, aspartic acid, or serine results in reduced NIS activity without alterations in total or cell surface NIS protein levels. Taken together, we showed that NIS protein levels and functional activity could be modulated by phosphorylation through distinct mechanisms.     

Structure-function studies on bacteriorhodopsin. V. Effects of amino acid substitutions in the putative helix F

To test structural and mechanistic proposals about bacteriorhodopsin, a series of analogues with single amino acid substitutions has been studied. Mutants in the proposed helix F of bacteriorhodopsin were chosen for investigation because of the probable interaction of this part of the protein with the retinal chromophore. Seven mutants of the bacteriorhodopsin gene were constructed by site-directed mutagenesis, and the gene products were expressed in Escherichia coli. The resulting mutant proteins were purified and assayed for their ability to interact with retinal in phospholipid/detergent micelles to form a bacteriorhodopsin-like chromophore. Four mutants, Ser-183----Ala, Tyr-185----Phe, Ser-193----Ala, and Glu-194----Gln, bound retinal to give pigments with absorption maxima approximately the same as the wild type. Three mutant opsins bound retinal to give chromophores that were blue-shifted relative to the wild type. Two Trp----Phe substitutions at positions 182 and 189 gave absorption maxima of 480 and 524 nm, respectively, and the mutant Pro-186----Leu gave a pigment with an absorption maximum of 470 nm. However, none of the amino acid substitutions eliminated the ability of the mutant bacteriorhodopsin to pump protons in response to illumination.