Ser Mutation Research Articles

Free energy simulation to investigate the effect of amino acid sequence environment on the severity of osteogenesis imperfecta by glycine mutations in collagen

Molecular dynamics simulations were carried out to calculate the free energy change difference of two collagen-like peptide models for Gly --> Ser mutations causing two different osteogenesis imperfecta phenotypes. These simulations were performed to investigate the impact of local amino acid sequence environment adjacent to a mutation site on the stability of the collagen. The average free energy differences for a Gly --> Ser mutant relative to a wild type are 3.4 kcal/mol and 8.2 kcal/mol for a nonlethal site and a lethal site, respectively. The free energy change differences of mutant containing two Ser residues relative to the wild type at the nonlethal and lethal mutation sites are 4.6 and 9.8 kcal/mol, respectively. Although electrostatic interactions stabilize mutants containing one or two Ser residues at both mutation sites, van der Waals interactions are of sufficient magnitude to cause a net destabilization. The presence of Gln and Arg near the mutation site, which contain large and polar side chains, provide more destabilization than amino acids containing small and nonpolar side chains.

Ser120 of Ubc2/Rad6 Regulates Ubiquitin-dependent N-end Rule Targeting by E3α/Ubr1

In CHO cells, CDK1/2-dependent phosphorylation of Ubc2/Rad6 at Ser(120) stimulates its ubiquitin conjugating activity and can be replicated by a S120D point mutant (Sarcevic, B., Mawson, A., Baker, R. T., and Sutherland, R. L. (2002) EMBO J. 21, 2009-2018). In contrast, we find that ectopic expression of wild type Ubc2b but not Ubc2bS120D or Ubc2bS120A in T47D human breast cancer cells specifically stimulates N-end rule-dependent degradation but not the Ubc2-independent unfolded protein response pathway, indicating that the former is E2 limiting in vivo and likely down-regulated by Ser(120) phosphorylation, as modeled by the S120D point mutation. In vitro kinetic analysis shows the in vivo phenotype of Ubc2bS120D and Ubc2bS120A is not due to differences in activating enzyme-catalyzed E2 transthiolation. However, the Ser(120) mutants possess marked differences in their abilities to support in vitro conjugation by the N-end rule-specific E3α/Ubr1 ligase that presumably accounts for their in vivo effects. Initial rate kinetics of human E3α-catalyzed conjugation of the human α-lactalbumin N-end rule substrate shows Ubc2bS120D is 20-fold less active than wild type E2, resulting from an 8-fold increase in K(m) and a 2.5-fold decrease in V(max), the latter reflecting a decreased ability to support the initial step in target protein conjugation; Ubc2bS120A is 8-fold less active than wild type E2 due almost exclusively to a decrease in V(max), reflecting a defect in polyubiquitin chain elongation. These studies suggest a mechanism for the integrated regulation of diverse ubiquitin-dependent signaling pathways through E2 phosphorylation that yields differential effects on its cognate ligases.

Structural basis of triclosan resistance

Triclosan (5-chloro-2-(2,4-dichloro-phenoxy)-phenol, TCL) is a well known inhibitor against enoyl-acyl carrier protein reductase (ENR), an enzyme critical for cell-wall synthesis of bacteria. The inhibitory concentration at 50% inhibition (IC(50)) of TCL against the Escherichia coli ENR is 150nM for wild type (WT), 380, 470 and 68,500nM for Ala, Ser and Val mutants, respectively. To understand this high TCL resistance in the G93V mutant, we obtained the crystal structures of mutated ENRs complexed with TCL and NAD(+). The X-ray structural analysis along with the ab initio calculations and molecular dynamics simulations explains the serious consequence in the G93V mutant complex. The major interactions around TCL due to the aromatic(cation)-aromatic and hydrogen bonding interactions are found to be conserved both in WT and mutant complexes. Thus, the overall structural change of protein is minimal except that a flexible α-helical turn around TCL is slightly pushed away due to the presence of the bulky valine group. However, TCL shows substantial edge-to-face aromatic (π)-interactions with both the flexible R192-F203 region and the residues in the close vicinity of G93. The weakening of some edge-to-face aromatic interactions around TCL in the G93V mutant results in serious resistance to TCL. This understanding is beneficial to design new generation of antibiotics which will effectively act on the mutant ENRs.

A Novel p53 Phosphorylation Site within the MDM2 Ubiquitination Signal: II. A MODEL IN WHICH PHOSPHORYLATION AT SER269 INDUCES A MUTANT CONFORMATION TO p53

The p53 DNA-binding domain harbors a conformationally flexible multiprotein binding site that regulates p53 ubiquitination. A novel phosphorylation site exists within this region at Ser(269), whose phosphomimetic mutation inactivates p53. The phosphomimetic p53 (S269D) exhibits characteristics of mutant p53: stable binding to Hsp70 in vivo, elevated ubiquitination in vivo, inactivity in DNA binding and transcription, increased thermoinstability using thermal shift assays, and λ(max) of intrinsic tryptophan fluorescence at 403 nm rather than 346 nm, characteristic of wild type p53. These data indicate that p53 conformational stability is regulated by a phosphoacceptor site within an exposed flexible surface loop and that this can be destabilized by phosphorylation. To test whether other motifs within p53 have similarly evolved, we analyzed the effect of Ser(215) mutation on p53 function because Ser(215) is another inactivating phosphorylation site in the conformationally flexible PAb240 epitope. The p53(S215D) protein is inactive like p53(S269D), whereas p53(S215A) is as active as p53(S269A). However, the double mutant p53(S215A/S269A) was transcriptionally inactive and more thermally unstable than either individual Ser-Ala loop mutant. Molecular dynamics simulations suggest that (i) solvation of phospho-Ser(215) and phospho-Ser(269) by positive charged residues or solvent water leads to local unfolding, which is accompanied by local destabilization of the N-terminal loop and global destabilization of p53, and (ii) the double alanine 215/269 mutation disrupts hydrogen bonding normally stabilized by both Ser(215) and Ser(269). These data indicate that p53 has evolved two serine phosphoacceptor residues within conformationally flexible epitopes that normally stabilize the p53 DNA-binding domain but whose phosphorylation induces a mutant conformation to wild type p53.

DHHC protein-dependent palmitoylation protects regulator of G-protein signaling 4 from proteasome degradation

Regulator of G-protein signaling 4 (RGS4), an intracellular modulator of G-protein coupled receptor (GPCR)-mediated signaling, is regulated by multiple processes including palmitoylation and proteasome degradation. We found that co-expression of DHHC acyltransferases (DHHC3 or DHHC7), but not their acyltransferase-inactive mutants, increased expression levels of RGS4 but not its Cys2 to Ser mutant (RGS4C2S). DHHC3 interacts with and palmitoylates RGS4 but not RGS4C2S in vivo. Palmitoylation prolongs the half-life of RGS4 by over 8-fold and palmitoylated RGS4 blocked α 1A-adrenergic receptor-stimulated intracellular Ca 2+ mobilization. Together, our findings revealed that DHHC proteins could regulate GPCR-mediated signaling by increasing RGS4 stability. Structured summary MINT- 8049215: Rgs4 (uniprotkb: P49799) physically interacts (MI: 0915) with DHHC3 (uniprotkb: Q8R173) by anti-tag coimmunoprecipitation (MI: 0007)

The severity of osteogenesis imperfecta: A comparison to the relative free energy differences of collagen model peptides

Molecular dynamics simulations were carried out to calculate free energy differences between the folded and unfolded states of wild type and mutant collagen model peptides. The calculated stability of the collagen models was compared with the severity of osteogenesis imperfecta. Free energy differences of Gly → Xaa (Xaa: Ser, Cys, Glu, and Asp) mutations between the wild type and the mutants at position 15 of the model peptide were 3.8, 4.2, 5.6, and 8.8 kcal/mol, respectively. The corresponding free energy differences of a second Gly mutation at the same position in different chains were, on average, 1.3, 1.5, 2.9, and 5.4 kcal/mol, respectively. Free energy simulations were also performed to estimate the relative stability between an oxidized form and a reduced form of the mutants containing two Cys residues, which indicated that the mutant of the collagen-like peptide containing an intramolecular disulfide bond was more stable than the mutant containing one Cys residue but less stable than the wild type. The calculated free energy differences between an oxidized and a reduced form of the mutants containing two Cys residues are 0.8 and 2.6 kcal/mol for the disulfide bonds between Chains A and B and between Chains A and C, respectively.

Kinetic studies of Gly28:Ser mutant form of Bacillus pumilus lipase: Changes in kcat and thermal dependence

Lipases are useful catalysts for a wide variety of industrial purposes. Herein we report the stability and thermal dependence of the activity of wild-type Bacillus pumilus lipase (BplA) and four site-directed mutants designed to improve its thermal stability. The Gly28:Ser mutation produces a dramatic four-fold increase in its k cat and a remarkable increase in its stability. While the increase in k cat is temperature-independent, the increase in stability shows that the resultant interactions of this mutation have a strong enthalpic component. Thermal dependence of stability, k cat, K M and k cat/ K M were analysed to gain insight on the structural effects of mutations on BplA. Our results are consistent with a gain in enzyme mobility for those mutants displaying enhanced catalytic properties; the analysis of thermal dependence of kinetic parameters indicates that the mutations did not change either the catalytic mechanism or the rate-limiting step of catalysis.

Solvation Effects on S K-Edge XAS Spectra of Fe−S Proteins: Normal and Inverse Effects on WT and Mutant Rubredoxin

S K-edge X-ray absorption spectroscopy (XAS) was performed on wild type Cp rubredoxin and its Cys --> Ser mutants in both solution and lyophilized forms. For wild type rubredoxin and for the mutants where an interior cysteine residue (C6 or C39) is substituted by serine, a normal solvent effect is observed, that is, the S covalency increases upon lyophilization. For the mutants where a solvent accessible surface cysteine residue is substituted by serine, the S covalency decreases upon lyophilization which is an inverse solvent effect. Density functional theory (DFT) calculations reproduce these experimental results and show that the normal solvent effect reflects the covalency decrease due to solvent H-bonding to the surface thiolates and that the inverse solvent effect results from the covalency compensation from the interior thiolates. With respect to the Cys --> Ser substitution, the S covalency decreases. Calculations indicate that the stronger bonding interaction of the alkoxide with the Fe relative to that of thiolate increases the energy of the Fe d orbitals and reduces their bonding interaction with the remaining cysteines. The solvent effects support a surface solvent tuning contribution to electron transfer, and the Cys --> Ser result provides an explanation for the change in properties of related iron-sulfur sites with this mutation.

Gene methylation of SFRP2, P16, DAPK1, HIC1, and MGMT and KRAS mutations in sporadic colorectal cancer

The aim of this study was to investigate the methylation of the SFRP2, P16, DAPK1, HIC1, and MGMT genes, as well as the mutation of amino acid codons 12 and 13 of the KRAS gene in normal and tumor tissue DNA of patients diagnosed with sporadic colorectal cancer (SCRC). The methylation of gene regions and the KRAS mutations of normal (N) and tumor tissue (T) DNA obtained from 17 patients diagnosed with SCRC and 20 healthy controls were investigated using the polymerase chain reaction and reverse-hybridization methods. There was an Asp mutation in four patients, an Asp and Ser mutations in one patient in codon 12 of the KRAS gene, and an Asp mutation in codon 13 in eight patients. Overall promoter methylation (OPM) in the SFRP2 gene was observed in one N and four T, whereas partial promoter methylation (PPM) was observed in two N and five T. OPM in the P16 gene was present in one T. In the DAPK1 gene, OPM existed in seven T and five N, while PPM was present in two N. In the HIC1 gene, OPM was demonstrated in three T, while PPM was noted in two N; however, no methylation existed in N. In the MGMT gene, OPM occurred in five T and two N, and PPM was present in one T. KRAS mutations in Turkish patients with SCRC are similar to those of other population groups. Methylations in the genes, which underwent methylation analysis, were higher in T in comparison with N, and it has been suggested that significant results would be obtained by making a study with a larger population.

Redox Regulation of Plasmodium falciparum Ornithine δ-Aminotransferase

Ornithine δ-aminotransferase (OAT) of the malaria parasite Plasmodium falciparum catalyzes the reversible conversion of ornithine into glutamate-5-semialdehyde and glutamate and is—in contrast to its human counterpart—activated by thioredoxin (Trx) by a factor of 10. Trx, glutaredoxin, and plasmoredoxin are redox-active proteins that play a crucial role in the maintenance and control of redox reactions, and were shown to interact with P. falciparum OAT. OAT, which is involved in ornithine homeostasis and proline biosynthesis, is essential for mitotic cell division in rapidly growing cells, thus representing a potential target for chemotherapeutic intervention. Here we report the three-dimensional crystal structure of P. falciparum OAT at 2.3 Å resolution. The overall structure is very similar to that of the human OAT. However, in plasmodial OAT, the loop involved in substrate binding contains two cysteine residues, which are lacking in human OAT. Site-directed mutagenesis of these cysteines and functional analysis demonstrated that Cys154 and Cys163 mediate the interaction with Trx. Interestingly, the Cys154 → Ser mutant has a strongly reduced specific activity, most likely due to impaired binding of ornithine. Cys154 and Cys163 are highly conserved in Plasmodium but do not exist in other organisms, suggesting that redox regulation of OAT by Trx is specific for malaria parasites. Plasmodium might require a tight Trx-mediated control of OAT activity for coordinating ornithine homeostasis, polyamine synthesis, proline synthesis, and mitotic cell division.

Resolving Disulfide Structural Isoforms of IgG2 Monoclonal Antibodies by Ion Mobility Mass Spectrometry

Recombinant monoclonal antibodies are an important class of therapeutic agents that have found widespread use for the treatment of many human diseases. Here, we have examined the utility of ion mobility mass spectrometry (IMMS) for the rapid characterization of disulfide variants in intact IgG2 monoclonal antibodies. It is shown that IMMS reveals 2 to 3 gas-phase conformer populations for IgG2s. In contrast, a single gas-phase conformer is revealed using IMMS for both an IgG1 antibody and a Cys-232 --> Ser mutant IgG2, both of which are homogeneous with respect to disulfide bonding. This provides strong evidence that the observed IgG2 gas-phase conformers are related to disulfide bond heterogeneity. Additionally, IMMS analysis of redox enriched disulfide isoforms allows assignment of the mobility peaks to established disulfide bonding patterns. These data clearly illustrate how IMMS can be used to quickly provide information on the higher order structure of antibody therapeutics.

Role of External Loops of Human Ceruloplasmin in Copper Loading by ATP7B and Ccc2p

Ceruloplasmin is a multicopper oxidase required for correct iron homeostasis.Previously, we have identified a ceruloplasmin mutant associated with the iron overload disease aceruloplasminemia, which was unable to acquire copper from the mammalian pump ATP7B but could be produced in an enzymatically active form in yeast. Here, we report the expression of recombinant ceruloplasmin in the yeast Pichia pastoris and the study of the role of five surface-exposed loops in copper incorporation by comparing the efficiencies of mammalian ATP7B and yeast Ccc2p. The possibility to "mix and match" mammalian and yeast multicopper oxidases and copper ATPases can provide clues on the molecular features underlying the process of copper loading in multicopper oxidases.

Cooperation of tumor‐derived HBx mutants and p53‐249ser mutant in regulating cell proliferation, anchorage‐independent growth and aneuploidy in a telomerase‐immortalized normal human hepatocyte‐derived cell line

Hepatocellular carcinoma (HCC) is a common cancer, and hepatitis B virus (HBV) is a major etiological agent. Convincing epidemiological and experimental evidence also links HCC to aflatoxin, a naturally occurring mycotoxin that produces a signature p53-249(ser) mutation. Recently, we have reported that tumor-derived HBx variants encoded by HBV exhibited attenuated transactivation and proapoptotic functions but retained their ability to block p53-mediated apoptosis. These results indicate that mutations in HBx may contribute to the development of HCC. In this study, we determined whether tumor-derived HBx mutants along, or in cooperation with p53-249(ser), could alter cell proliferation and chromosome stability of normal human hepatocytes. To test this hypothesis, we established a telomerase immortalized normal human hepatocycte line HHT4 that exhibited a near diploid karyotype and expressed many hepatocyte-specific genes. We found that overexpression one of the tumor-derived HBx mutants, CT, significantly increased colony forming efficiency (CFE) while its corresponding wild-type allele CNT significantly decreased CFE in HHT4 cells. p53-249(ser) rescued CNT-mediated inhibition of colony formation. Although HHT4 cells lacked an anchorage independent growth capability as they did not form any colonies in soft agar, the CT-expressing HHT4 cells could form colonies, which could be significantly enhanced by p53-249(ser). Induction of aneuploidy could be observed in HHT4 cells expressing CT, but additionally recurring chromosome abnormalities could only be detected in cells coexpressing CT and p53-249(ser). Our results are consistent with the hypothesis that certain mutations in HBx and p53 at codon 249 may cooperate in contributing to liver carcinogenesis.

Stabilization of Luciola mingrelica firefly luciferase by genetic engineering methods

The results of Luciola mingrelica firefly luciferase stabilization by genetic engineering methods are reviewed. The Cys62, Cys146, and Cys164 to Ser mutant enzymes with an enhanced thermostability and lower sensitivity to dithiothreitol were obtained by site-directed mutagenesis. The double mutant G216N, A217L was obtained, which displayed a higher thermostability and resistance to DMSO in comparison with WT luciferase. Random mutagenesis of the gene region encoding residues 1–225 and subsequent screening of the mutants resulted in the production of the mutant MT8 with a higher thermostability, as well as mutants MT3 and MT4 with higher resistance to dimethyl sulfoxide. The mutant 4TS was obtained by the method of directed evolution of the gene site encoding residues 130–390, which was shown to contain eight replacements after four cycles of mutagenesis and had two-fold higher specific activity, eight-fold lower K m value for ATP, and stability at 42°C, which was 65-fold higher that of WT luciferase. The stabilization mechanism of this mutant is discussed.

Palmitoylation stabilizes unliganded rod opsin

S-palmitoylation is a conserved feature in many G protein-coupled receptors (GPCRs) involved in a broad array of signaling processes. The prototypical GPCR, rhodopsin, is S-palmitoylated on two adjacent C-terminal Cys residues at its cytoplasmic surface. Surprisingly, absence of palmitoylation has only a modest effect on in vitro or in vivo signaling. Here, we report that palmitoylation-deficient (Palm(-/-)) mice carrying two Cys to Thr and Ser mutations in the opsin gene displayed profound light-induced retinal degeneration that first involved rod and then cone cells. After brief bright light exposure, their retinas exhibited two types of deposits containing nucleic acid and invasive phagocytic macrophages. When Palm(-/-) mice were crossed with Lrat(-/-) mice lacking lecithin:retinol acyl transferase to eliminate retinoid binding to opsin and thereby rendering the eye insensitive to light, rapid retinal degeneration occurred even in 3- to 4-week-old animals. This rapid degeneration suggests that nonpalmitoylated rod opsin is unstable. Treatment of 2-week-old Palm(-/-)Lrat(-/-) mice with an artificial chromophore precursor prevented this retinopathy. In contrast, elimination of signaling to G protein in Palm(-/-)Gnat1(-/-) mice had no effect, indicating that instability of unpalmitoylated opsin lacking chromophore rather than aberrant signal transduction resulted in retinal pathology. Together, these observations provide evidence for a structural role of rhodopsin S-palmitoylation that may apply to other GPCRs as well.

Globin-like proteins in Caenorhabditis elegans: in vivo localization, ligand binding and structural properties

BackgroundThe genome of the nematode Caenorhabditis elegans contains more than 30 putative globin genes that all are transcribed. Although their translated amino acid sequences fit the globin fold, a variety of amino-acid substitutions and extensions generate a wide structural diversity among the putative globins. No information is available on the physicochemical properties and the in vivo expression.ResultsWe expressed the globins in a bacterial system, characterized the purified proteins by optical and resonance Raman spectroscopy, measured the kinetics and equilibria of O2 binding and determined the crystal structure of GLB-1* (CysGH2 → Ser mutant). Furthermore, we studied the expression patterns of glb-1 (ZK637.13) and glb-26 (T22C1.2) in the worms using green fluorescent protein technology and measured alterations of their transcript abundances under hypoxic conditions.GLB-1* displays the classical three-over-three α-helical sandwich of vertebrate globins, assembled in a homodimer associated through facing E- and F-helices. Within the heme pocket the dioxygen molecule is stabilized by a hydrogen bonded network including TyrB10 and GlnE7.GLB-1 exhibits high ligand affinity, which is, however, lower than in other globins with the same distal TyrB10-GlnE7 amino-acid pair. In the absence of external ligands, the heme ferrous iron of GLB-26 is strongly hexacoordinated with HisE7, which could explain its extremely low affinity for CO. This globin oxidizes instantly to the ferric form in the presence of oxygen and is therefore incapable of reversible oxygen binding.ConclusionThe presented data indicate that GLB-1 and GLB-26 belong to two functionally-different globin classes.

Analysis of the assembly mechanism for multimeric lamprey VLRB antibodies (43.19)

Abstract Instead of the immunoglobulin-type antigen receptors in jawed vertebrates, the surviving jawless vertebrates use variable lymphocyte receptors (VLRs) that are constructed with variable leucine-rich repeat (LRR) sequences to recognize antigens. Two VLR genes in lamprey and hagfish are somatically assembled during lymphopoiesis using a gene conversion-like mechanism in which highly diverse LRR flanking sequences are copied in serial fashion to construct mature VLRs. Lamprey have been shown to secrete polymeric VLRB antibodies that consist of 8-12 identical disulfide-linked subunits. The overall structure of VLRB antibodies resembles that of IgM antibodies in that both are composed of 4-6 dimers. Previous studies have shown that eight cysteines in the C-terminal hydrophobic region are involved in assembly of the multivalent VLRB antibodies through formation of disulfide bonds. We have characterized a panel of VLRB Cys to Ser mutants using site-directed mutagenesis to determine which cysteines are used for disulfide bond formation and VLRB polymerization. Two or more disulfide bonds were found to be essential for VLRB dimerization and polymerization. Our data further suggest that the carboxy-terminal cysteines 5, 6, 7, and 8 form the cornerstone for VLRB dimerization and polymerization, whereas cysteines 1, 2, 3, and 4 contribute to the stability of the polymer structure.

Spectroscopic and computational investigation of three Cys-to-Ser mutants of nickel superoxide dismutase: insight into the roles played by the Cys2 and Cys6 active-site residues.

Nickel-dependent superoxide dismutase (NiSOD) is a member of a class of metalloenzymes that protect aerobic organisms from the damaging superoxide radical (O(2) (.-)). A distinctive and fascinating feature of NiSOD is the presence of active-site nickel-thiolate interactions involving the Cys2 and Cys6 residues. Mutation of one or both Cys residues to Ser prevents catalysis of O(2) (.-), demonstrating that both residues are necessary to support proper enzymatic activity (Ryan et al., J Biol Inorg Chem, 2010). In this study, we have employed a combined spectroscopic and computational approach to characterize three Cys-to-Ser (Cys --> Ser) mutants (C2S, C6S, and C2S/C6S NiSOD). Similar electronic absorption and magnetic circular dichroism spectra are observed for these mutants, indicating that they possess nearly identical active-site geometric and electronic structures. These spectroscopic data also reveal that the Ni(2+) ion in each mutant adopts a high-spin (S = 1) configuration, characteristic of a five- or six-coordinate ligand environment, as opposed to the low-spin (S = 0) configuration observed for the four-coordinate Ni(2+) center in the native enzyme. An analysis of the electronic absorption and magnetic circular dichroism data within the framework of density functional theory computations performed on a series of five- and six-coordinate C2S/C6S NiSOD models reveals that the active site of each Cys --> Ser mutant possesses an essentially six-coordinate Ni(2+) center with a rather weak axial bonding interaction. Factors contributing to the lack of catalytic activity displayed by the Cys --> Ser NiSOD mutants are explored.

Importance of Alanine at Position 178 in Proteorhodopsin for Absorption of Prevalent Ambient Light in the Marine Environment

It is usually assumed that only amino acids located near the retinal chromophore are responsible for color tuning of rhodopsins. However, we recently found that replacement of Ala178 with Arg in the E-F loop of proteorhodopsin (PR), an archaeal-type rhodopsin in marine bacteria, shifts the lambda(max) from 525 to 545 nm at neutral pH [Yoshitsugu, M., Shibata, M., Ikeda, D., Furutani, Y., and Kandori, H. (2008) Angew. Chem., Int. Ed. 47, 3923-3926]. Since the location of Ala178 is distant from the retinal chromophore (approximately 25 A), the molecular mechanism of the unusual mutation effect on color tuning is intriguing. A recent mutation study revealed that the observed color change was highly specific to position 178 [Yoshitsugu, M., Yamada, J., and Kandori, H. (2009) Biochemistry 48, 4324-4330]. Thus, in the study presented here, we replaced Ala178 with 19 different amino acids and measured the absorption spectra and the pK(a) of the Schiff base counterion, Asp97. Most of the mutants exhibited a spectral red shift and increased pK(a) of Asp97. None of charged amino acids at position 178 influences color tuning of PR specifically, being similar to the Arg replacement studied earlier. Only Cys and Thr replacements exhibited color and a pK(a) similar to that of the wild type. Ser, Val, and Gly mutants behave like the wild type only with respect to the lambda(max) of the species with deprotonated Asp97. We conclude that the E-F loop region contains a unique structure in PR, disruption of which causes large-scale rearrangement of alpha-helices. Ala178 in PR contributes to the blue-shifted absorption (525 nm at neutral pH) and lowering of the counterion pK(a), which is important for proton-pump function in the marine environment, even though its position is far removed from the chromophore binding domain.

The protein function of lipoprotein lipase with Asn291Ser and Lys312insC compound mutation

To study the function of lipoprotein lipase (LPL)with Asn291 Ser and Lys312insC compound mutation in LPL gene knockout heterozygous (LPL~(+/-)) mice. The results showed that triglycerides, free fatty acids, blood glucose and weight of LPL~(+/-) mice were higher than those of c57 mice(P<0. 05). The expressions of LPL Mrna and LPL protein of LPL~(+/-) group were lower than those of c57 group(P<0.05). The injection of Asn291Ser +Lys312insC protein caused little change of the lipid mass and LPL activity,but the injection of normal LPL protein induced obvious decrease of lipid mass and increase of the LPL activity. Key words: Lipoprotein lipase; Gene knockout; Compound mutation