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PubMed2005

Kinetics of acid-induced spectral changes in the GFPmut2 chromophore.

We have used a nanosecond pH-jump technique, coupled with simultaneous transient absorption and fluorescence emission detection, to characterize the dynamics of the acid-induced spectral changes in the GFPmut2 chromophore. Disappearance of the absorbance at 488 nm and the green fluorescence emission occurs with a thermally activated, double exponential relaxation. To understand the source of the two transients we have introduced mutations in amino acid residues that interact with the chromophore (H148G, T203V, and E222Q). Results indicate that the faster transient is associated with proton binding from the solution, while the second process, smaller in amplitude, is attributed to structural rearrangement of the amino acids surrounding the chromophore. The protonation rate shows a 3-fold increase for the H148G mutant, demonstrating that His148 plays a key role in protecting the chromophore from the solvent. The deprotonation rate for T203V is an order of magnitude smaller, showing that the hydrogen bond with the hydroxyl of Thr203 is important in stabilizing the deprotonated form of the chromophore. A kinetic model suggests that, in addition to protecting the chromophore from the solvent, His148 may act as the primary acceptor for the protons on the way to the chromophore.

Green Fluorescent Proteins↗
PubMed2000

The SOS response regulates adaptive mutation.

Upon starvation some Escherichia coli cells undergo a transient, genome-wide hypermutation (called adaptive mutation) that is recombination-dependent and appears to be a response to a stressful environment. Adaptive mutation may reflect an inducible mechanism that generates genetic variability in times of stress. Previously, however, the regulatory components and signal transduction pathways controlling adaptive mutation were unknown. Here we show that adaptive mutation is regulated by the SOS response, a complex, graded response to DNA damage that includes induction of gene products blocking cell division and promoting mutation, recombination, and DNA repair. We find that SOS-induced levels of proteins other than RecA are needed for adaptive mutation. We report a requirement of RecF for efficient adaptive mutation and provide evidence that the role of RecF in mutation is to allow SOS induction. We also report the discovery of an SOS-controlled inhibitor of adaptive mutation, PsiB. These results indicate that adaptive mutation is a tightly regulated response, controlled both positively and negatively by the SOS system.

Adaptation, Physiological↗
PubMed2004

Expression studies of mutations underlying Taiwanese Hunter syndrome (mucopolysaccharidosis type II).

Nearly 300 different mutations underlying mucopolysaccharidosis type II (MPS II) have been identified worldwide. To investigate the molecular lesions underlying Taiwanese MPS II, probands and families were identified and screened for iduronate-2-sulfatase (IDS) mutation by single-strand conformation polymorphism and DNA sequencing. Five novel and five previously reported mutations were found. Together with those previously reported, a total of 17 identified missense, small deletion, and nonsense mutations were further characterized by transient expression studies. Transfection of COS-7 cells by the mutated cDNA did not yield active enzyme, demonstrating the deleterious nature of the mutations. A 57% decrease in IDS mRNA level was seen with the 231del6 mutation. Among the 11 missense mutations examined, K347E substitution showed apparent normal maturation and targeting on immunoblot and confocal fluorescence microscopy examination. The other 10 missense mutations showed apparent normal precursor with little or reduced mature forms, indicating normal maturation but incorrect targeting of the mutant enzymes. Among the six deletion and nonsense mutations examined, 1055del12 and E521X showed abnormal maturation. The staining pattern of the truncated W267X and 1184delG proteins suggested retention within early vacuolar compartments. The mutated 231del6 and 1421delAG proteins were unstable and largely degraded. Molecular analysis of the IDS gene will clearly identify the cause of the disease within patients and allow antenatal and family studies. The further characterization of gene mutations may delineate their functional consequences on IDS activity and processing and may enable future studies of genotype-phenotype correlation to estimate a prognosis and to lead to possible therapeutic interventions.

Animals↗
PubMed1997

Fourteen novel mucopolysaccharidosis IVA producing mutations in GALNS gene.

Mucopolysaccharidosis IVA (MPS IVA) is an autosomal recessive disorder caused by a deficiency of the lysosomal N-acetylgalactosamine-6-sulfate sulfatase. Here, we report our analysis of data on 21 patients of diverse ethnic and geographic origins studied by SSCP and sequencing analysis. Sixteen mutations were detected, including 14 new mutations (11 missense, one premature termination, one splice site alteration, and one cryptic site alteration). The donor splice site mutation (IVS4 + 1G-->A) predicts that normal splicing will be abolished and that translation would lead to an immediate premature termination (W141X). Another novel nucleotide change outside the coding sequence is an intronic alteration (IVS9-42C-->T:ggtcggtgcggttggtgc) creating a potential cryptic donor site. The nucleotide sequence surrounding this alteration is highly suggestive of a consensus donor splice site. All 12 missense and nonsense mutations were shown by transient expression to abolish or greatly reduce GALNS activity, thereby providing an explanation as to why they produce MPS IVA. All mutations were readily confirmed by restriction enzyme or by allelic specific oligonucleotide analysis (ASO). These findings, coupled with previously reported mutations, bring the total of different mutations to 41 among independent families with MPS IVA, illustrating the extensive allelic heterogeneity among mutations producing MPS IVA.

Chondroitinsulfatases↗
PubMed1992

Separation of the transcriptional activation and replication functions of the bovine papillomavirus-1 E2 protein.

Replication of bovine papillomavirus-1 (BPV-1) DNA requires two viral gene products, the E1 protein and the full-length E2 protein. The 48 kDa E2 protein is a site-specific DNA-binding protein that binds to several sites which lie adjacent to the BPV-1 origin of replication. The 85 amino acid C-terminal domain contains the specific DNA binding and dimerization properties of the protein. The approximately 200 amino acid N-terminal domain is crucial for transcriptional activation. Both of these domains are highly conserved among different papillomaviruses. An internal hinge region separates the two functional domains. The region varies in amino acid sequence and length among the E2 proteins of different papillomaviruses. A series of mutations were constructed within the E2 open reading frame which delete various regions of the conserved DNA binding and transactivation domains as well as the internal hinge region. Two mutated E2 proteins that lack portions of the conserved DNA-binding domain but which support DNA replication were identified using transient replication assays. These mutated E2 proteins were unable to function as transcriptional activators. Conversely, two E2 proteins containing large deletions of the hinge region were able to activate transcription, but were defective for replication. Thus, the replication and transactivation functions of the E2 protein are separable.

Animals↗
PubMed2002

Transcriptional regulation of the BCL-6 gene: mechanistic dissection using mutant cell lines.

BACKGROUND: Mice lacking the BCL-6 gene product exhibit overexpression of T helper cell type 2 cytokines, and multi-organ inflammatory responses characterized by eosinophilia and infiltration by IgE+ B lymphocytes. Elevated IgE levels appear to result from loss of BCL-6's role in repression of the Stat6-dependent processes of I-epsilon transcription and IgE class-switching. Understanding the regulation of the BCL-6 gene expression is therefore relevant to the allergic response, due to the IgE-dependent activation of mast cells and basophils. MATERIALS AND METHODS: We analyzed a pair of isogenic cell lines that exhibit differential expression of the BCL-6 gene and mapped the control point to transcription using nuclear run-on assays. Direct analyses of the BCL-6 gene and transfection experiments were carried out to map the molecular basis for the differential expression. RESULTS: In this study, we report that pair of isogenic cell lines exhibits differential expression of BCL-6. BCL-6 mRNA is barely detectable in Jijoye, a Burkitt lymphoma B cell line, but is abundant in Clone-13, a mutant cell line derived from Jijoye. Corresponding to the mRNA level, BCL-6 protein is detected only in Clone-13, but not in Jijoye. Nuclear run-on assays indicate that BCL-6 expression is regulated mainly at the transcriptional level in these two cell lines. Gene structural alterations are not detected in a region including a 3-kb promoter and the first intron, where most of the chromosomal translocations are observed in diffuse large cell lymphoma (DLCL). Interestingly, multiple, heterozygous point mutations are identified in the first intron, the hypermutation region of BCL-6, in both of the two cell lines. However, in transient transfection experiments these mutations have no effects on gene expression. CONCLUSIONS: Our data suggest that the distinct profile of BCL-6 expression in Jijoye/Clone-13 is due to either a missing negative element in a different portion of the gene or that there is an issue of chromatin accessibility operating in BCL-6 gene regulation. This pair of isogenic cell lines (Jijoye/Clone-13) thus provides a new system to dissect the regulation of BCL-6 gene expression.

Cell Line↗
PubMed1996

A D255H substitution in the arylsulphatase A gene of two unrelated Belgian patients with late-infantile metachromatic leukodystrophy.

Metachromatic leukodystrophy (MLD) is an autosomal recessive disease of myelin metabolism caused by a deficiency in the lysosomal enzyme arylsulphatase A (ARSA). We have identified a new mutation in exon 4 of the ARSA gene of two unrelated Belgian patients with late-infantile MLD. The mutation predicts an aspartic acid-to-histidine substitution at position 255 in arylsulphatase A (D255H), in a highly conserved region among sulphatases. Transient expression of the mutation in COS cells did not show an increase in ARSA activity. Both patients were compound heterozygotes carrying the frequent splice site mutation in intron 2 (459 + IG -->A) on the other allele.

Animals↗
PubMed2004

Overlapping binding sites in protein phosphatase 2A for association with regulatory A and alpha-4 (mTap42) subunits.

Diverse functions of protein Ser/Thr phosphatases depend on the distribution of the catalytic subunits among multiple regulatory subunits. In cells protein phosphatase 2A catalytic subunit (PP2Ac) mostly binds to a scaffold subunit (A subunit or PR65); however, PP2Ac alternatively binds to alpha-4, a subunit related to yeast Tap42 protein, which also associates with phosphatases PP4 or PP6. We mapped alpha-4 binding to PP2Ac to the helical domain, residues 19-165. We mutated selected residues and transiently expressed epitope-tagged PP2Ac to assay for association with A and alpha-4 subunits by co-precipitation. The disabling H118N mutation at the active site or the presence of the active site inhibitor microcystin-LR did not interfere with binding of PP2Ac to either the A subunit or alpha-4, showing that these are allosteric regulators. Positively charged side chains Lys(41), Arg(49), and Lys(74) on the back surface of PP2Ac are unique to PP2Ac, compared with phosphatases PP4, PP6, and PP1. Substitution of one, two, or three of these residues with Ala produced a progressive loss of binding to the A subunit, with a corresponding increase in binding to alpha-4. Conversely, mutation of Glu(42) in PP2Ac essentially eliminated PP2Ac binding to alpha-4, with an increase in binding to the A subunit. Reciprocal changes in binding because of mutations indicate competitive distribution of PP2Ac between these regulatory subunits and demonstrate that the mutated catalytic subunits retained a native conformation. Furthermore, neither the Lys(41)-Arg(49)-Lys(74) nor Glu(42) mutations affected the phosphatase-specific activity or binding to microcystin-agarose. Binding of PP2Ac to microcystin and to alpha-4 increased with temperature, consistent with an activation energy barrier for these interactions. Our results reveal that the A subunit and alpha-4 (mTap42) require charged residues in separate but overlapping surface regions to associate with the back side of PP2Ac and modulate phosphatase activity.

Alanine↗
PubMed1995

Induction of cell death by myristylated death domain of p55 TNF receptor is not abolished by Iprcg-like point mutation in death domain.

We transiently expressed the intracellular domains of p55 TNF receptor (TNFR1) as either a cytosolic- or a membrane-associated form and examined their effects on the endogenous receptor-mediated gene expression as well as on cell viability. We found that gene expression as measured by luciferase activity under NF-kappa B-controlling elements was blocked by all forms of the intracellular domain of TNFR1. The blockade of reporter gene expression was due to the cell death induced by the intracellular domain of TNFR1 per se. The killing mechanism of the intracellular domain peptides appeared to be apoptotic. Interestingly, myristylated form of the intracellular domain, consisting of mainly death domain showed the most potent cell-killing activity. Moreover, this myristylated death domain could still induce cell death even if lprcg-like point mutation (Leu351 to Ala), which has been reported to abrogate TNF-induced cytotoxicity, was introduced. This result suggests that the myristylated death domain activates an additional death signaling pathway which is not involved in TNF-induced cell death.

Apoptosis↗
PubMed1998

The amino-terminal Src homology 2 domain of phospholipase C gamma 1 is essential for TCR-induced tyrosine phosphorylation of phospholipase C gamma 1.

TCR engagement activates phospholipase C gamma 1 (PLC gamma 1) via a tyrosine phosphorylation-dependent mechanism. PLC gamma 1 contains a pair of Src homology 2 (SH2) domains whose function is that of promoting protein interactions by binding phosphorylated tyrosine and adjacent amino acids. The role of the PLC gamma 1 SH2 domains in PLC gamma 1 phosphorylation was explored by mutational analysis of an epitope-tagged protein transiently expressed in Jurkat T cells. Mutation of the amino-terminal SH2 domain (SH2(N) domain) resulted in defective tyrosine phosphorylation of PLC gamma 1 in response to TCR/CD3 perturbation. In addition, the PLC gamma 1 SH2(N) domain mutant failed to associate with Grb2 and a 36- to 38-kDa phosphoprotein (p36-38), which has previously been recognized to interact with PLC gamma 1, Grb2, and other molecules involved in TCR signal transduction. Conversely, mutation of the carboxyl-terminal SH2 domain (SH2(C) domain) did not affect TCR-induced tyrosine phosphorylation of PLC gamma 1. Furthermore, binding of p36-38 to PLC gamma 1 was not abrogated by mutations of the SH2(C) domain. In contrast to TCR/CD3 ligation, treatment of cells with pervanadate induced tyrosine phosphorylation of either PLC gamma 1 SH2(N) or SH2(C) domain mutants to a level comparable with that of the wild-type protein, indicating that pervanadate treatment induces an alternate mechanism of PLC gamma 1 phosphorylation. These data indicate that the SH2(N) domain is required for TCR-induced PLC gamma 1 phosphorylation, presumably by participating in the formation of a complex that promotes the association of PLC gamma 1 with a tyrosine kinase.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Effects of cystic fibrosis and congenital bilateral absence of the vas deferens-associated mutations on cystic fibrosis transmembrane conductance regulator-mediated regulation of separate channels.

The protein defective in cystic fibrosis (CF), the CF transmembrane-conductance regulator (CFTR), functions as an epithelial chloride channel and as a regulator of separate ion channels. Although the consequences that disease-causing mutations have on the chloride-channel function have been studied extensively, little is known about the effects that mutations have on the regulatory function. To address this issue, we transiently expressed CFTR-bearing mutations associated with CF or its milder phenotype, congenital bilateral absence of the vas deferens, and determined whether mutant CFTR could regulate outwardly rectifying chloride channels (ORCCs). CFTR bearing a CF-associated mutation in the first nucleotide-binding domain (NBD1), DeltaF508, functioned as a chloride channel but did not regulate ORCCs. However, CFTR bearing disease-associated mutations in other domains retained both functions, regardless of the associated phenotype. Thus, a relationship between loss of CFTR regulatory function and disease severity is evident for NBD1, a region of CFTR that appears important for regulation of separate channels.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1989

Developmental and tissue-specific expression of nuclear proteins that bind the regulatory element of the major histocompatibility complex class I gene.

Expression of MHC class I genes varies according to developmental stage and type of tissues. To study the basis of class I gene regulation in tissues in vivo, we examined binding of nuclear proteins to the conserved cis sequence of the murine H-2 gene, class I regulatory element (CRE), which contains two independent factor-binding sites, region I and region II. In gel mobility shift analyses we found that extracts from adult tissues that express class I genes, such as spleen and liver, had binding activity to region I. In contrast, extracts from brain, which does not express class I genes, did not show region I binding activity. In addition, fetal tissues that express class I gene at very low levels, also did not reveal region I binding activity. Binding activity to region I became detectable during the neonatal period when class I gene expression sharply increases. Most of these tissues showed binding activity to region II, irrespective of class I gene expression. Although region II contained a sequence similar to the AP-1 recognition site, AP-1 was not responsible for the region II binding activity detected in this work. These results illustrate a correlation between region I binding activity and developmental and tissue-specific expression of MHC class I genes. The CRE exerts an enhancer-like activity in cultured fibroblasts. We evaluated the significance of each factor binding to CRE. Single 2-bp mutations were introduced into the CRE by site-directed mutagenesis and the ability of each mutant to elicit the enhancer activity was tested in transient CAT assays. A mutation that eliminated region I protein binding greatly impaired enhancer activity. A mutation that eliminated region II binding also caused a lesser but measurable effect. We conclude that region I and region II are both capable of enhancing transcription of the class I gene. These results indicate that in vivo regulation of MHC class I gene expression is mediated by binding of trans-acting factors to the CRE.

Animals↗
PubMed2002

Contributions of the non-alpha subunit residues (loop D) to agonist binding and channel gating in the muscle nicotinic acetylcholine receptor.

The agonist binding site of the nicotinic acetylcholine receptor has a loop-based structure, and is formed by residues located remotely to each other in terms of primary structure. Amino acid residues in sites delta57 and delta59, and the equivalent residues in the epsilon; subunit, have been identified as part of the agonist binding site and designated as loop D. The effects of point mutations in sites delta57, delta59, epsilon;55 and epsilon;57 on agonist binding and channel gating were studied. The mutated receptors were expressed transiently in HEK 293 cells and the currents were recorded using the cell-attached single-channel patch clamp technique. The results demonstrate that the mutations mainly affect channel gating with the major portion of the effect due to a reduction in the channel opening rate constant. For both the delta57/epsilon;55 and the delta59/epsilon;57 site, a mutation in the epsilon; subunit had a greater effect on channel gating than a mutation in the delta subunit. In all instances, agonist binding was affected to a lesser degree than channel gating. Previous data have placed the delta57 and delta59 residues in or near the agonist binding pocket. The data presented here suggest that these two residues (and the homologous sites in the epsilon; subunit) are not involved in specific interactions with the nicotinic agonist and that they affect the activation of the nicotinic receptor by shaping the overall structure of the agonist binding site.

Acetylcholine↗
PubMed2005

Mutations in the pleckstrin homology domain of dynamin 2 cause dominant intermediate Charcot-Marie-Tooth disease.

Charcot-Marie-Tooth (CMT) disease is a clinically and genetically heterogeneous group of peripheral neuropathies. Different chromosomal loci have been linked with three autosomal dominant, 'intermediate' types of CMT: DI-CMTA, DI-CMTB and DI-CMTC. We refined the locus associated with DI-CMTB on chromosome 19p12-13.2 to 4.2 Mb in three unrelated families with CMT originating from Australia, Belgium and North America. After screening candidate genes, we identified unique mutations in dynamin 2 (DNM2) in all families. DNM2 belongs to the family of large GTPases and is part of the cellular fusion-fission apparatus. In transiently transfected cell lines, mutations of DNM2 substantially diminish binding of DNM2 to membranes by altering the conformation of the beta3/beta4 loop of the pleckstrin homology domain. Additionally, in the Australian and Belgian pedigrees, which carry two different mutations affecting the same amino acid, Lys558, CMT cosegregated with neutropenia, which has not previously been associated with CMT neuropathies.

Animals↗
PubMed1999

Altered gene expression pattern in the fatty liver dystrophy mouse reveals impaired insulin-mediated cytoskeleton dynamics.

The mouse fatty liver dystrophy (fld) mutation is characterized by transient hypertriglyceridemia and fatty liver during the neonatal period, followed by development of a peripheral neuropathy. To uncover the metabolic pathway that is disrupted by the fld mutation, we analyzed the altered pattern of gene expression in the fatty liver of fld neonates by representational difference analysis of cDNA. Differentially expressed genes detected include a novel member of the Ras superfamily of small GTP-binding proteins, a novel Ser/Thr kinase, and several actin cytoskeleton-associated proteins including actin, profilin, alpha-actinin, and myosin light chain. Because these proteins have a potential functional link in the propagation of hormone signals, we investigated cytoskeleton dynamics in fld cells in response to hormone treatment. These studies revealed that preadipocytes from fld mice exhibit impaired formation of actin membrane ruffles in response to insulin treatment. These findings suggest that the altered mRNA expression levels detected in fld tissue represent a compensatory response for the nonfunctional fld gene and that the fld gene product may be required for development of normal insulin response.

Amino Acid Sequence↗
PubMed1998

R to Q amino acid substitution in the GFFKR sequence of the cytoplasmic domain of the integrin IIb subunit in a patient with a Glanzmann's thrombasthenia-like syndrome.

The integrin IIbbeta3 mediates platelet aggregation through its fibrinogen and adhesive protein-binding properties. Particular interest concerns the role of the cytoplasmic domains of IIb and beta3. We now report the molecular analysis of IIbbeta3 from a patient with a Glanzmann's thrombasthenia-like syndrome for whom the principal characteristics are an approximate 50% total platelet content of IIbbeta3 but with a much lower proportion in the surface pool (Hardisty et al, Blood 80:696, 1992). Polymerase chain reaction (PCR) single-strand conformational polymorphism and DNA sequencing showed a heterozygous mutation giving rise to amino acid substitution R995 to Q in the GFFKR sequence of the cytoplasmic domain of IIb. Reverse transcriptase-PCR and polymorphism analysis only detected mRNA for the mutated allele of the IIb gene and a single allele of the beta3 gene in his platelets, suggesting other unidentified defects. Site-directed mutagenesis followed by transient expression of the mutated IIb together with wild-type beta3 in Cos-7 cells resulted in a markedly decreased expression of the complex at the cell surface when compared with cells transfected with wild-type IIb and beta3. Flow cytometry with PAC-1 and a stable Chinese hamster ovary-transfected cell line showed that the mutated receptor was not locked into a high activation state, although it became so in the presence of the activating antibody, anti-LIBS6. This is the first reported natural mutation in the highly conserved GFFKR sequence of the IIb cytoplasmic domain.

Amino Acid Substitution↗
PubMed2002

Human papillomavirus type 31 replication modes during the early phases of the viral life cycle depend on transcriptional and posttranscriptional regulation of E1 and E2 expression.

The E1 and E2 proteins are both required for papillomavirus DNA replication, and replication efficiency is controlled by the abundance of these factors. In human papillomaviruses (HPVs), the regulation of E1 and E2 expression and its effect on viral replication are not well understood. In particular, it is not known if E1 and E2 modulate their own expression and how posttranscriptional mechanisms may affect the levels of the replication proteins. Previous studies have implicated splicing within the E6 open reading frame (ORF) as being important for modulating replication of HPV type 31 (HPV31) through altered expression of E1 and E2. To analyze the function of the E6 intron in viral replication more specifically, we examined the effects of E6 splicing mutations in the context of entire viral genomes in transient assays. HPV31 genomes which had mutations in the splice donor site (E6SD) or the splice acceptor site (E6SA), a deletion of the intron (E6ID), or substituted heterologous intron sequences (E6IS) were constructed. Compared to wild-type (wt) HPV31, pHPV31-E6SD, -E6SA, and -E6IS replicated inefficiently while pHPV31-E6ID replicated at an intermediate level. Cotransfection of the E6 mutant genomes with an E1 expression vector strongly activated their replication levels, indicating that efficient expression of E1 requires E6 internal splicing. In contrast, replication was activated only moderately with an E2 expression vector. Replacing the wt E6 intron in HPV31 with a heterologous intron from simian virus 40 (E6SR2) resulted in replication levels similar to that of the wt in the absence of expression vectors, suggesting that mRNA splicing upstream of the E1 ORF is important for high-level replication. To examine the effects of E6 intron splicing on E1 and E2 expression directly, we constructed reporter DNAs in which the luciferase coding sequences were fused in frame to the E1 (E1Luc) or E2 (E2Luc) gene. Reporter activities were then analyzed in transient assays with cotransfected E1 or E2 expression vectors. Both reporters were moderately activated by E1 in a dose-dependent manner. In addition, E1Luc was activated by low doses of E2 but was repressed at high doses. In contrast, E2 had little effect on E2Luc activity. These data indicate that E1 expression and that of E2 are interdependent and regulated differentially. When the E6 splicing mutations were analyzed in both reporter backgrounds, only E1Luc activities correlated with splicing competence in the E6 ORF. These findings support the hypothesis that the E6 intron primarily regulates expression of E1. Finally, in long-term replication assays, none of the E6 mutant genomes could be stably maintained. However, cotransfection of the E6 splicing mutant genomes with pHPV31-E7NS, which contains a nonsense mutation in the E7 coding sequence, restored stable replication of some mutants. Our observations indicate that E1 expression and that of E2 are differentially regulated at multiple levels and that efficient expression of E1 is required for transient and stable viral replication. These regulatory mechanisms likely act to control HPV copy number during the various phases of the viral life cycle.

Carcinoma, Squamous Cell↗
PubMed2002

Plastocyanin-cytochrome f interactions: the influence of hydrophobic patch mutations studied by NMR spectroscopy.

Transient complex formation between plastocyanin from Prochlorothrix hollandica and cytochrome f from Phormidium laminosum was investigated using nuclear magnetic resonance (NMR) spectroscopy. Binding curves derived from NMR titrations at 10 mM ionic strength reveal a 1:1 stoichiometry and a binding constant of 6 (+/-2) x 10(3) M(-1) for complex formation, 1 order of magnitude larger than that for the physiological plastocyanin-cytochrome f complex from Ph. laminosum. Chemical-shift perturbation mapping indicates that the hydrophobic patch of plastocyanin is involved in the complex interface. When the unusual hydrophobic patch residues of P. hollandica plastocyanin were reverted to the conserved residues found in most other plastocyanins (Y12G/P14L), the binding constant for the interaction with cytochrome f was unaffected. However, the chemical shift perturbation map was considerably different, and the size of the average perturbation decreased by 40%. The complexes of both the wild-type and double mutant plastocyanin with cytochrome f were sensitive to ionic strength, contrary to the physiological complex. The possible implications of these findings for the mechanism of transient complex formation are discussed.

Binding Sites↗