Modulation of the remote heme site geometry of recombinant mouse neuronal nitric-oxide synthase by the N-terminal hook region

The role of two essential residues at the N-terminal hook region of neuronal nitric-oxide synthase (nNOS) in nitric-oxide synthase activity was investigated. Full-length mouse nNOS proteins containing single-point mutations of Thr-315 and Asp-314 to alanine were produced in the Escherichia coli and baculovirus-insect cell expression systems. The molecular properties of the mutant proteins were analyzed in detail by biochemical, optical, and electron paramagnetic resonance spectroscopic techniques and compared with those of the wild-type enzyme. Replacement of Asp-314 by Ala altered the geometry around the heme site and the substrate-binding pocket of the heme domain and abrogated the ability of nNOS to form catalytically active dimers. Replacement of Thr-315 by Ala reduced the protein stability and altered the geometry around the heme site, especially in the absence of bound (6R)-5,6,7, 8-tetrahydro-L-biopterin cofactor. These results suggest that Asp-314 and Thr-315 both play critical structural roles in stabilizing the heme domain and subunit interactions in mouse nNOS.     

Arabidopsis PDK1: identification of sites important for activity and downstream phosphorylation of S6 kinase

The Arabidopsis thaliana protein kinase AtPDK1 was identified as a homologue of the mammalian 3-phosphoinositide-dependent protein kinase-1 (PDK1), which is involved in a number of physiological processes including cell growth and proliferation. We now show that AtPDK1, expressed in E. coli as a recombinant protein, undergoes autophosphorylation at several sites. Using mass spectrometry, three phosphorylated amino acid residues, Ser-177, Ser-276 and Ser-382, were identified, followed by mutational analyses to reveal their roles. These residues are not conserved in mammalian PDK1s. Mutation of Ser-276 in AtPDK1 to alanine resulted in an enzyme with no detectable autophosphorylation. Autophosphorylation was significantly reduced in the Ser177Ala mutant but was only slightly reduced in the Ser382Ala mutant. Other identified sites of importance for autophosphorylation and/or activity of AtPDK1 were Asp-167, Thr-176, and Thr-211. Sites in the mammalian PDK1 corresponding to Asp-167 and Thr-211 are essential for PDK1 autophosphorylation and activity. Autophosphorylation was absent in the Asp167Ala mutant while the Thr176Ala and The211Ala mutants exhibited very low but detectable autophosphorylation, pointing to both similarity and difference between mammalian and plant enzymes. We also demonstrate that AtS6k2, an A. thaliana homologue to the mammalian S6 kinases, is an in vitro target of AtPDK1. Our data clearly show that Asp-167, Thr-176, Ser-177, Thr-211, and Ser-276 in AtPDK1 are important for the downstream phosphorylation of AtS6k2. The results confirm that AtPDK1, like mammalian PDK1, needs phosphorylation at several sites for full downstream phosphorylation activity. Finally, we investigated A. thaliana 14-3-3 proteins as potential AtPDK1 regulatory proteins and the effect of phospholipids on the AtPDK1 activity. Nine of the 12 14-3-3 isoforms tested enhanced AtPDK1 activity whereas one isoform suppressed the activity. No significant effects on AtPDK1 activity by the various phospholipids (including phosphoinositides) were evident.     

Involvement of glycine and aspartate residues in the binding capacity of FAD in the NADH dehydrogenase from an alkaliphilic Bacillus

There is a region exhibiting a similarity of amino acid sequence near the carboxyl-terminal segment of each FAD-containing oxidoreductase. In this region, four amino acid residues-Thr, Ala, Gly, and Asp-are highly conserved. To determine the involvement of the four amino acid residues (Thr-469, Ala-476, Gly-478, and Asp-479) in the activity of NADH dehydrogenase of an alkaliphilic Bacillus, mutations of these amino acid residues were conducted. In spite of high conservation, mutations of Thr-469 and Ala-476 to Ala and Ser, respectively, did not lead to a critical loss of enzyme activity. However, mutations of Gly-478 and Asp-479 to Ala caused a complete loss of the activity, which appears to result from the loss of binding capacity of FAD.     

Involvement of Glycine and Aspartate Residues in the Binding Capacity of FAD in the NADH Dehydrogenase from an Alkaliphilic <Emphasis Type="Italic">Bacillus</Emphasis>

There is a region exhibiting a similarity of amino acid sequence near the carboxyl-terminal segment of each FAD-containing oxidoreductase. In this region, four amino acid residues—Thr, Ala, Gly, and Asp—are highly conserved. To determine the involvement of the four amino acid residues (Thr-469, Ala-476, Gly-478, and Asp-479) in the activity of NADH dehydrogenase of an alkaliphilic Bacillus, mutations of these amino acid residues were conducted. In spite of high conservation, mutations of Thr-469 and Ala-476 to Ala and Ser, respectively, did not lead to a critical loss of enzyme activity. However, mutations of Gly-478 and Asp-479 to Ala caused a complete loss of the activity, which appears to result from the loss of binding capacity of FAD. Received: 3 July 2002 / Accepted: 29 July 2002     

Structure-function analysis of human IL-6: identification of two distinct regions that are important for receptor binding.

Interleukin-6 (IL-6) is a multifunctional cytokine that plays an important role in host defense. It has been predicted that IL-6 may fold as a 4 alpha-helix bundle structure with up-up-down-down topology. Despite a high degree of sequence similarity (42%) the human and mouse IL-6 polypeptides display distinct species-specific activities. Although human IL-6 (hIL-6) is active in both human and mouse cell assays, mouse IL-6 (mIL-6) is not active on human cells. Previously, we demonstrated that the 5 C-terminal residues of mIL-6 are important for activity, conformation, and stability (Ward LD et al., 1993, Protein Sci 2:1472-1481). To further probe the structure-function relationship of this cytokine, we have constructed several human/mouse IL-6 hybrid molecules. Restriction endonuclease sites were introduced and used to ligate the human and mouse sequences at junction points situated at Leu-62 (Lys-65 in mIL-6) in the putative connecting loop AB between helices A and B, at Arg-113 (Val-117 in mIL-6) at the N-terminal end of helix C, at Lys-150 (Asp-152 in mIL-6) in the connecting loop CD between helices C and D, and at Leu-178 (Thr-180 in mIL-6) in helix D. Hybrid molecules consisting of various combinations of these fragments were constructed, expressed, and purified to homogeneity. The conformational integrity of the IL-6 hybrids was assessed by far-UV CD. Analysis of their biological activity in a human bioassay (using the HepG2 cell line), a mouse bioassay (using the 7TD1 cell line), and receptor binding properties indicates that at least 2 regions of hIL-6, residues 178-184 in helix D and residues 63-113 in the region incorporating part of the putative connecting loop AB through to the beginning of helix C, are critical for efficient binding to the human IL-6 receptor. For human IL-6, it would appear that interactions between residues Ala-180, Leu-181, and Met-184 and residues in the N-terminal region may be critical for maintaining the structure of the molecule; replacement of these residues with the corresponding 3 residues in mouse IL-6 correlated with a significant loss of alpha-helical content and a 200-fold reduction in activity in the mouse bioassay. A homology model of mIL-6 based on the X-ray structure of human granulocyte colony-stimulating factor is presented.     

Site-directed mutagenesis suggests close functional relationship between a human rhinovirus 3C cysteine protease and cellular trypsin-like serine proteases

Human rhinoviruses, like other picornaviruses, encode a cysteine protease (designated 3C) which cleaves mainly at viral Gln-Gly pairs. There are significant areas of homology between picornavirus 3C cysteine proteases and cellular serine proteases (e.g. trypsin), suggesting a functional relationship between their catalytic regions. To test this functional relationship, we made single substitutions in human rhinovirus type 14 protease 3C at seven amino acid positions which are highly conserved in the 3C proteases of animal picornaviruses. Substitutions at either His-40, Asp-85, or Cys-146, equivalent to the trypsin catalytic triad His-57, Asp-102, and Ser-195, respectively, completely abolished 3C proteolytic activity. Single substitutions were also made at either Thr-141, Gly-158, His-160, or Gly-162, which are equivalent to the trypsin specificity pocket region. Only the mutant with a conservative Thr-141 to Ser substitution exhibited proteolytic activity, which was much reduced compared with the parent. These results, together with immunoprecipitation data which indicate that Asp-85, Thr-141, and Cys-146 lie in accessible surface regions, suggest that the catalytic mechanism of picornavirus 3C cysteine proteases is closely related to that of cellular trypsin-like serine proteases.     

Identification of critical, conserved vicinal aspartate residues in mammalian and bacterial ADP-ribosylarginine hydrolases.

NAD:arginine ADP-ribosyltransferases and ADP-ribosylarginine hydrolases catalyze opposing arms of a putative ADP-ribosylation cycle. ADP-ribosylarginine hydrolases from mammalian tissues and Rhodospirillum rubrum exhibit three regions of similarity in deduced amino acid sequence. We postulated that amino acids in these consensus regions could be critical for hydrolase function. To test this hypothesis, hydrolase, cloned from rat brain, was expressed as a glutathione S-transferase fusion protein in Escherichia coli and purified by glutathione-Sepharose affinity chromatography. Conserved amino acids in each of these regions were altered by site-directed mutagenesis. Replacement of Asp-60 or Asp-61 with Ala, Gln, or Asn, but not Glu, significantly reduced enzyme activity. The double Asp-60 --> Glu/Asp-61 --> Glu mutant was inactive, as were Asp-60 --> Gln/Asp-61 --> Gln or Asp-60 --> Asn/Asp-61 --> Asn. The catalytically inactive single and double mutants appeared to retain conformation, since they bound ADP-ribose, a substrate analogue and an inhibitor of enzyme activity, with affinity similar to that of the wild-type hydrolase and with the expected stoichiometry of one. Replacing His-65, Arg-139, Asp-285, which are also located in the conserved regions, with alanine did not change specific activity. These data clearly show that the conserved vicinal aspartates 60 and 61 in rat ADP-ribosylarginine hydrolase are critical for catalytic activity, but not for high affinity binding of the substrate analogue, ADP-ribose.     

A receptor binding domain of mouse interleukin-4 defined by a solid-phase binding assay and in vitro mutagenesis.

Interleukin 4 (IL-4) is a potent, pleiotropic lymphokine that affects a variety of cells, especially those of hematopoietic origin. Although murine and human IL-4 are homologous proteins, they display a species specificity in which murine IL-4 acts only upon mouse cells, and human IL-4 only upon human cells. We have used a mutagenesis strategy to define both the structural determinants of this specificity and a receptor binding domain of murine IL-4. To do this, we developed convenient solid-phase binding assays for mouse and for human IL-4, each utilizing receptor-immunoglobulin fusion proteins and alkaline phosphatase-tagged ligands. These were employed to assess the receptor binding activities of wild type and mutant forms of IL-4. In a separate biological assay, we measured the ability of each version of IL-4 to induce proliferation of a cultured mouse T-cell line. By replacing regions of mouse IL-4 with homologous segments of human IL-4, we found that the amino-terminal 16 residues and the carboxyl-terminal 20 residues of murine IL-4 are required for species-specific receptor binding as well as for T-cell proliferation. A major portion of the amino acid sequence between these regions can be substituted between mouse and human without loss of receptor binding or biological activity. Further, alanine-scanning mutagenesis revealed specific residues in the amino- and carboxyl-terminal regions (Glu-12, Ile-14, Leu-104, Asp-106, Phe-107, and Leu-111) that bear side chains critical for function. An analysis of the carboxyl-terminal region of murine IL-4 and its comparison with carboxyl-terminal regions of other related cytokines suggest an evolutionary conservation of structural and functional features.     

Genetic linkage studies of the human glycosphingolipid β-galactosidases

The genetic linkage relationships of the human glycosphingolipid beta-galactosidases were determined using human--mouse somatic cell hybrids. A new method was devised for the estimation of human galactosylceramide, lactosylceramide, and GMI-ganglioside beta-galactosidase activities in the presence of their mouse counterparts, which takes advantage of the reproducible specific activity of lysosomal hydrolases under a given set of culture conditions and is based on differences in both pH optima and sensitivity to chloride ion. Human and mouse chromosomes were identified by their characteristic banding patterns obtained after quinacrine staining, and the optimum glycolipid beta-galactosidase activity was determined for three different substrates. A ratio was defined for each activity which was the specific activity at the human pH optimum divided by the specific activity at the mouse pH optimum. Linear regression analysis was used to test for concordant segregation between pH ratios for each enzyme and the frequency of occurrence of different human chromosomes in the man--mouse somatic hybrid clones. The results obtained from two independent series of hybrid clones indicated that human beta-galactosidase activities consistently segregated with human chromosome 12 in these somatic cell hybrids.     

Protein kinase C phosphorylation of the metabotropic glutamate receptor mGluR5 on Serine 839 regulates Ca2+ oscillations

The activation of Group 1 metabotropic glutamate receptors, mGluR5 and mGluR1alpha, triggers intracellular calcium release; however, mGluR5 activation is unique in that it elicits Ca2+ oscillations. A short region of the mGluR5 C terminus is the critical determinant and differs from the analogous region of mGluR1alpha by a single amino acid residue, Thr-840, which is an aspartic acid (Asp-854) in mGluR1alpha. Previous studies show that mGluR5-elicited Ca2+ oscillations require protein kinase C (PKC)-dependent phosphorylation and identify Thr-840 as the phosphorylation site. However, direct phosphorylation of mGluR5 has not been studied in detail. We have used biochemical analyses to directly investigate the phosphorylation of the mGluR5 C terminus. We showed that Ser-839 on mGluR5 is directly phosphorylated by PKC, whereas Thr-840 plays a permissive role. Although Ser-839 is conserved in mGluR1alpha (Ser-853), it is not phosphorylated, as the adjacent residue (Asp-854) is not permissive; however, mutagenesis of Asp-854 to a permissive alanine residue allows phosphorylation of Ser-853 on mGluR1alpha. We investigated the physiological consequences of mGluR5 Ser-839 phosphorylation using Ca2+ imaging. Mutations that eliminate Ser-839 phosphorylation prevent the characteristic mGluR5-dependent Ca2+ oscillations. However, mutation of Thr-840 to alanine, which prevents potential Thr-840 phosphorylation but is still permissive for Ser-839 phosphorylation, has no effect on Ca2+ oscillations. Thus, we showed that it is phosphorylation of Ser-839, not Thr-840, that is absolutely required for the unique Ca2+ oscillations produced by mGluR5 activation. The Thr-840 residue is important only in that it is permissive for the PKC-dependent phosphorylation of Ser-839.     

An activation switch in the rhodopsin family of G protein-coupled receptors: the thyrotropin receptor

We aimed at understanding molecular events involved in the activation of a member of the G protein-coupled receptor family, the thyrotropin receptor. We have focused on the transmembrane region and in particular on a network of polar interactions between highly conserved residues. Using molecular dynamics simulations and site-directed mutagenesis techniques we have identified residue Asn-7.49, of the NPxxY motif of TM 7, as a molecular switch in the mechanism of thyrotropin receptor (TSHr) activation. Asn-7.49 appears to adopt two different conformations in the inactive and active states. These two states are characterized by specific interactions between this Asn and polar residues in the transmembrane domain. The inactive gauche+ conformation is maintained by interactions with residues Thr-6.43 and Asp-6.44. Mutation of these residues into Ala increases the constitutive activity of the receptor by factors of approximately 14 and approximately 10 relative to wild type TSHr, respectively. Upon receptor activation Asn-7.49 adopts the trans conformation to interact with Asp-2.50 and a putatively charged residue that remains to be identified. In addition, the conserved Leu-2.46 of the (N/S)LxxxD motif also plays a significant role in restraining the receptor in the inactive state because the L2.46A mutation increases constitutive activity by a factor of approximately 13 relative to wild type TSHr. As residues Leu-2.46, Asp-2.50, and Asn-7.49 are strongly conserved, this molecular mechanism of TSHr activation can be extended to other members of the rhodopsin-like family of G protein-coupled receptors.     

Human and mouse homologues of the Drosophila melanogaster tweety (tty) gene: a novel gene family encoding predicted transmembrane proteins

We have cloned cDNA for TTYH1, a human homologue of the Drosophila melanogaster tweety (tty) gene. The 450-residue predicted protein shows 27% amino acid sequence identity (51% similarity) to the Drosophila protein, which contains an additional C-terminal repetitive region. A second Drosophila homologue exhibits 42% identity (65% similarity) to the tty protein. Mouse (Ttyh1), macaque, and Caenorhabditis elegans homologues were also identified, and the complete coding sequence for the mouse gene was determined. The mouse protein is 91% identical to the human protein. Hydrophobicity analysis of the tty-related proteins indicates that they represent a new family of membrane proteins with five potential membrane-spanning regions. The yeast FTR1 and FTH1 iron transporter proteins and the mammalian neurotensin receptors 1 and 2 have a similar hydrophobicity profile, although there is no detectable sequence homology to the tty-related proteins. This suggests that the tweety-related proteins could be involved in transport of iron or other divalent cations or alternatively that they may be membrane-bound receptors. TTYH1 was mapped to chromosome 19q13.4 by FISH and by radiation hybrid mapping using the Stanford G3 panel.     

Tax responsiveness of the GM-CSF promoter is mediated by mitogen-inducible sequences other than kappa B.

The mechanism by which the human T-cell leukemia viruses type I and II (HTLV-I and -II) transform T cells is unknown, but the nonstructural Tax protein that these viruses produce is known to be essential for viral replication and to have the capacity to trans-activate cellular gene expression. The HTLV-I and -II Tax proteins have been shown to activate the promoter of both the human and mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) genes in mature T-cell lines. T-cell-specific Tax-responsive sequences were previously localized to the 90-bp region extending from base pairs -53 to +37 in the human GM-CSF promoter. In this study, a series of site-directed and deletion mutations were created in the human GM-CSF promoter, which was linked to the chloramphenicol acetyltransferase (CAT) gene, and the constructs were assayed for their response to Tax by using a Tax-expressing plasmid in transient cotransfection assays. The results demonstrated that both copies of the repeated sequence CATTA (A/T), located between base pairs -48 and -36, are required for Tax responsiveness in T cells and that these sequences bind nuclear factors present in T cells. The Tax-responsiveness of other sequences located 5' of base pair -53 was also examined, including an NF-kappa B consensus sequence and the CK1, CK2, and GC-rich regions identified in both the mouse and human GM-CSF promoters. These sequences did not have Tax-responsive regulatory activity when they were examined in the context of the intact human GM-CSF promoter in T cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Exclusion of catalytic and regulatory subunits of cAMP-dependent protein kinase as candidate genes for the defect causing cystic fibrosis.

Cystic fibrosis (CF) is a common autosomal recessive disease with significant morbidity and mortality. Defects in cAMP control mechanisms are implicated in the pathophysiology of the disease. The mutation causing CF has been localized to chromosome 7q22-7q31.1. We have used (1) somatic-cell hybrids containing this region of the human genome in a mouse background and (2) segregation analysis in families to exclude both the genes coding for a catalytic subunit and three distinct regulatory subunits of cAMP-dependent protein kinase as candidates for the gene defect in CF. Two of these genes--those for the human homologue of the mouse type I regulatory subunit and the human homologue of the rat type II regulatory subunit--map to human chromosome 7.     

Characterization of upFc, a fragment of human immunoglobulin G1 produced by pepsin in urea.

The digestion of human IgG1/K myeloma proteins with pepsin in the presence of 8 M-urea produces fragments that differ from those produced by aqueous peptic digestion, and from other characteristic immunoglobulin fragments. Fb'2, the larger urea/pepsin fragment, was previously shown to consist of the constant regions of the light chains, and the CH1 domains and hinge regions of the heavy chains. The smaller fragment, upFc, has now been characterized. After reduction, three peptides were released from fragment upFc. Amino acid sequencing, N- and C-terminal determinations and amino acid compositions have enabled these peptides to be identified as residues Ile-253 to Leu-306, residues Thr-307 to Asp-376 and residues Thr-411 to Gly-446 of the heavy chain. Fragment upFc therefore contains the entire Fc region, beginning at residue Ile-253, except for a 34-residue section from within the CH3-domain disulphide loop. Peptic digestion of IgG1/K proteins in 8M-urea therefore provides a method for isolating from gamma1 heavy chains five homogeneous peptides in good yield, which account for almost the entire constant region. Characterization of fragments Fb'2 and upFc has shown that the action of pepsin in urea is entirely different from that of aqueous pepsin. Two gamma1 heavy chains have been shown to differ in sequence at three positions from the sequence reported for protein Eu.     

Cell cycle-dependent interaction of Mad2 with conserved Box1/2 region of human granulocyte-macrophage colony-stimulating factor receptor common betac

Box1 and 2 (box1/2) are conserved cytoplasmic motifs located in the membrane proximal region of cytokine receptors, including the human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor common betac. Deletion of box1/2 abrogated all the examined activities of GM-CSF, and this phenomenon is explained by the loss of binding by Jak2. To test if a molecule other than Jak2 interacting with the box1/2 region plays a role in GM-CSF receptor signal transduction, we screened for molecules interacting with the box1/2 region by a pull-down assay using recombinant purified protein of GST fused with the betac box1/2 region and a Ba/F3 cell lysate. The mouse homologue of Mad2 protein, which plays an important role in the M phase of the cell cycle, was revealed to associate with the box1/2 region specifically. Peptides corresponding to the box1 sequence also bound to Mad2, and mutation of the box1 decreased the Mad2 interaction. Deletion analysis indicated that interaction with box1/2 occurred through the C-terminal portion of Mad2. Mad2 is known to change affinity for binding partners cell cycle dependently. Binding affinity of Mad2 to box1/2 increased in the late M phase, suggesting the possibility that GM-CSF participates in regulation of the M phase check point through interaction with Mad2.     

Genomic organization of adrenergic and serotonin receptors in the mouse: linkage mapping of sequence-related genes provides a method for examining mammalian chromosome evolution.

Five sequence-related genes encoding four adrenergic receptors and a serotonin receptor were localized to specific regions of four mouse chromosomes with respect to 11 other genetic markers. Linkage was established by the analysis of the haplotypes of 114 interspecific backcross mice. Adra2r (alpha 2-C10) and Adrb1r (beta 1) receptors mapped to the distal region of mouse chromosome 19. These genes were separated by 2.6 +/- 1.5 cM in a segment of mouse chromosome 19 that has a similar organization of these genes on the long arm of human chromosome 10. The Adra1r (alpha 1B), Adrb2r (beta 2), and Htra1 (5HT1A) genes mapped to proximal mouse chromosome 11, proximal mouse chromosome 18, and distal mouse chromosome 13, respectively. The organization of genes linked to these loci on regions of the three mouse chromosomes is consistent with the organization of homologous human genes on human chromosome 5. These findings further define the relationship of linkage groups conserved during the evolution of the mouse and human genomes. We have identified a region that may have been translocated during evolution and suggest that the human genomic organization of adrenergic receptors more closely resembles that of a putative primordial ancestor.     

Catalytic residues of group VIB calcium-independent phospholipase A2 (iPLA2gamma)

Although human group VIB calcium-independent phospholipase A(2) (iPLA(2)gamma) contains the lipase-consensus sequence Gly-Xaa-Ser-Xaa-Gly in the C-terminal half, its overall sequence exhibits a week similarity to those of other PLA(2)s, and thus no information on the catalytic site has been available. Here we show that the C-terminal region of human iPLA(2)gamma is responsible for the enzymatic activity. Comparison of this catalytic domain with those of the mouse homologue, human cytosolic PLA(2) (cPLA(2)), and the plant PLA(2) patatin reveals that an amino acid sequence of a short segment around Asp-627 of iPLA(2)gamma is conserved among these PLA(2)s, in addition to the Ser-483-containing lipase motif; the corresponding serine and aspartate in cPLA(2) and patatin are known to form a catalytic dyad. Since substitution of alanine for either Ser-483 or Asp-627 results in a loss of the PLA(2) activity, we propose that Ser-483 and Asp-627 of human iPLA(2)gamma constitute an active site similar to the Ser-Asp dyad in cPLA(2) and patatin.     

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.