Mutation of Actin Tyr-53 Alters the Conformations of the DNase I-binding Loop and the Nucleotide-binding Cleft

All but 11 of the 323 known actin sequences have Tyr at position 53, and the 11 exceptions have the conservative substitution Phe, which raises the following questions. What is the critical role(s) of Tyr-53, and, if it can be replaced by Phe, why has this happened so infrequently? We compared the properties of purified endogenous Dictyostelium actin and mutant constructs with Tyr-53 replaced by Phe, Ala, Glu, Trp, and Leu. The Y53F mutant did not differ significantly from endogenous actin in any of the properties assayed, but the Y53A and Y53E mutants differed substantially; affinity for DNase I was reduced, the rate of nucleotide exchange was increased, the critical concentration for polymerization was increased, filament elongation was inhibited, and polymerized actin was in the form of small oligomers and imperfect filaments. Growth and/or development of cells expressing these actin mutants were also inhibited. The Trp and Leu mutations had lesser but still significant effects on cell phenotype and the biochemical properties of the purified actins. We conclude that either Tyr or Phe is required to maintain the functional conformations of the DNase I-binding loop (D-loop) in both G- and F-actin, and that the conformation of the D-loop affects not only the properties that directly involve the D-loop (binding to DNase I and polymerization) but also allosterically modifies the conformation of the nucleotide-binding cleft, thus increasing the rate of nucleotide exchange. The apparent evolutionary “preference” for Tyr at position 53 may be the result of Tyr allowing dynamic modification of the D-loop conformation by phosphorylation (Baek, K., Liu, X., Ferron, F., Shu, S., Korn, E. D., and Dominguez, R. (2008) Proc. Natl. Acad. Sci. U.S.A. 105, 11748–11753) with effects similar, but not identical, to those of the Ala and Glu mutations.     

Mutant profilin suppresses mutant actin-dependent mitochondrial phenotype in Saccharomyces cerevisiae

In the Saccharomyces cerevisiae actin-profilin interface, Ala(167) of the actin barbed end W-loop and His(372) near the C terminus form a clamp around a profilin segment containing residue Arg(81) and Tyr(79). Modeling suggests that altering steric packing in this interface regulates actin activity. An actin A167E mutation could increase interface crowding and alter actin regulation, and A167E does cause growth defects and mitochondrial dysfunction. We assessed whether a profilin Y79S mutation with its decreased mass could compensate for actin A167E crowding and rescue the mutant phenotype. Y79S profilin alone caused no growth defect in WT actin cells under standard conditions in rich medium and rescued the mitochondrial phenotype resulting from both the A167E and H372R actin mutations in vivo consistent with our model. Rescue did not result from effects of profilin on actin nucleotide exchange or direct effects of profilin on actin polymerization. Polymerization of A167E actin was less stimulated by formin Bni1 FH1-FH2 fragment than was WT actin. Addition of WT profilin to mixtures of A167E actin and formin fragment significantly altered polymerization kinetics from hyperbolic to a decidedly more sigmoidal behavior. Substitution of Y79S profilin in this system produced A167E behavior nearly identical to that of WT actin. A167E actin caused more dynamic actin cable behavior in vivo than observed with WT actin. Introduction of Y79S restored cable movement to a more normal phenotype. Our studies implicate the importance of the actin-profilin interface for formin-dependent actin and point to the involvement of formin and profilin in the maintenance of mitochondrial integrity and function.     

Site-specific protein dynamics in communication pathway from sensor to signaling domain of oxygen sensor protein, HemAT-Bs: Time-resolved Ultraviolet Resonance Raman Study

HemAT-Bs is a heme-based signal transducer protein responsible for aerotaxis. Time-resolved ultraviolet resonance Raman (UVRR) studies of wild-type and Y70F mutant of the full-length HemAT-Bs and the truncated sensor domain were performed to determine the site-specific protein dynamics following carbon monoxide (CO) photodissociation. The UVRR spectra indicated two phases of intensity changes for Trp, Tyr, and Phe bands of both full-length and sensor domain proteins. The W16 and W3 Raman bands of Trp, the F8a band of Phe, and the Y8a band of Tyr increased in intensity at hundreds of nanoseconds after CO photodissociation, and this was followed by recovery in ～50 μs. These changes were assigned to Trp-132 (G-helix), Tyr-70 (B-helix), and Phe-69 (B-helix) and/or Phe-137 (G-helix), suggesting that the change in the heme structure drives the displacement of B- and G-helices. The UVRR difference spectra of the sensor domain displayed a positive peak for amide I in hundreds of nanoseconds after photolysis, which was followed by recovery in ～50 μs. This difference band was absent in the spectra of the full-length protein, suggesting that the isolated sensor domain undergoes conformational changes of the protein backbone upon CO photolysis and that the changes are restrained by the signaling domain. The time-resolved difference spectrum at 200 μs exhibited a pattern similar to that of the static (reduced - CO) difference spectrum, although the peak intensities were much weaker. Thus, the rearrangements of the protein moiety toward the equilibrium ligand-free structure occur in a time range of hundreds of microseconds.     

Hydrogen exchange kinetics of bovine pancreatic trypsin inhibitor beta-sheet protons in trypsin-bovine pancreatic trypsin inhibitor, trypsinogen-bovine pancreatic trypsin inhibitor, and trypsinogen-isoleucylvaline-bovine pancreatic trypsin inhibitor

Hydrogen exchange rates of six beta-sheet peptide amide protons in bovine pancreatic trypsin inhibitor (BPTI) have been measured in free BPTI and in the complexes trypsinogen-BPTI, trypsinogen-Ile-Val-BPTI, bovine trypsin-BPTI, and porcine trypsin-BPTI. Exchange rates in the complexes are slower for Ile-18, Arg-20, Gln-31, Phe-33, Tyr-35, and Phe-45 NH, but the magnitude of the effect is highly variable. The ratio of the exchange rate constant in free BPTI to the exchange rate constant in the complex, k/kcpIx, ranges from 3 to much greater than 10(3). Gln-31, Phe-45, and Phe-33 NH exchange rate constants are the same in each of the complexes. For Ile-18 and Tyr-35, k/kcpIx is much greater than 10(3) for the trypsin complexes but is in the range 14-43 for the trypsinogen complexes. Only the Arg-20 NH exchange rate shows significant differences between trypsinogen-BPTI and trypsinogen-Ile-Val-BPTI and between porcine and bovine trypsin-BPTI.     

Phospholipase A2 engineering. The structural and functional roles of aromaticity and hydrophobicity in the conserved phenylalanine-22 and phenylalanine-106 aromatic sandwich.

The highly conserved phenylalanine-22 and phenylalanine-106, arranged as an aromatic sandwich, form part of an invariant hydrophobic wall that shields the active site of bovine pancreatic phospholipase A2 (PLA2) from bulk solvent [Dijkstra, B. W., Drenth, J., & Kalk, K. H. (1981) Nature 289, 604-606]. The residues have also been suggested to interact with the sn-2 acyl chain of bound phospholipid substrate [White, S. P., Scott, D. L., Otwinowski, Z., Gelb, M. H., & Sigler, P. B. (1990) Science 250, 1560-1563]. We now report the importance of these two residues in the structure and function of PLA2 in terms of aromaticity (changing to Ile) and hydrophobic (changing to Ala) and hydrophilic (changing to Tyr) character of these residues. The structural properties of the mutants were analyzed by proton NMR and by guanidine hydrochloride-induced denaturation. The functional properties were determined by measuring kinetic parameters toward various substrates in the forms of monomers, micelles, and vesicles, and by measuring equilibrium dissociation constants at the interface. The results show that (i) The conformational stability of each mutant was as good as that of wild-type PLA2; none of the mutants was significantly perturbed structurally as judged from detailed 1H NMR analysis. These results suggest that neither the Phe-22/Phe-106 face-to-face pair nor the Phe-22/Tyr-111 edge-to-face pair plays a significant structural role. (ii) Mutations to Ile at either position 22 or position 106 resulted in only minor perturbations in activity. This suggests that the aromaticity is not important to the function of these two residues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stabilization of Pseudomonas aeruginosa cytochrome c(551) by systematic amino acid substitutions based on the structure of thermophilic Hydrogenobacter thermophilus cytochrome c(552)

A heterologous overexpression system for mesophilic Pseudomonas aeruginosa holocytochrome c(551) (PA c(551)) was established using Escherichia coli as a host organism. Amino acid residues were systematically substituted in three regions of PA c(551) with the corresponding residues from thermophilic Hydrogenobacter thermophilus cytochrome c(552) (HT c(552)), which has similar main chain folding to PA c(551), but is more stable to heat. Thermodynamic properties of PA c(551) with one of three single mutations (Phe-7 to Ala, Phe-34 to Tyr, or Val-78 to Ile) showed that these mutants had increased thermostability compared with that of the wild-type. Ala-7 and Ile-78 may contribute to the thermostability by tighter hydrophobic packing, which is indicated by the three dimensional structure comparison of PA c(551) with HT c(552). In the Phe-34 to Tyr mutant, the hydroxyl group of the Tyr residue and the guanidyl base of Arg-47 formed a hydrogen bond, which did not exist between the corresponding residues in HT c(552). We also found that stability of mutant proteins to denaturation by guanidine hydrochloride correlated with that against the thermal denaturation. These results and others described here suggest that significant stabilization of PA c(551) can be achieved through a few amino acid substitutions determined by molecular modeling with reference to the structure of HT c(552). The higher stability of HT c(552) may in part be attributed to some of these substitutions.     

Tryptophan fluorescence of yeast actin resolved via conserved mutations

Actin contains four tryptophan residues, W79, W86, W340, and W356, all located in subdomain 1 of the protein. Replacement of each of these residues with either tyrosine (W79Y and W356Y) or phenylalanine (W86F and W340F) generated viable proteins in the yeast Saccharomyces cerevisiae, which, when purified, allowed the analysis of the contribution of these residues to the overall tryptophan fluorescence of actin. The sum of the relative contributions of these tryptophans was found to account for the intrinsic fluorescence of wild-type actin, indicating that energy transfer between the tryptophans is not the main determinant of their quantum yield, and that these mutations induce little conformational change to the protein. This was borne out by virtually identical polymerization rates and similar myosin interactions of each of the mutants and the wild-type actin. In addition, these mutants allowed the dissection of the microenvironment of each tryptophan as actin undergoes conformational changes upon metal cation exchange and polymerization. Based on the relative tryptophan contributions determined from single mutants, a triple mutant of yeast actin (W79) was generated that showed small intrinsic fluorescence and should be useful for studies of actin interactions with actin-binding proteins.     

Actin-inhibition and folding of vertebrate deoxyribonuclease I are affected by mutations at residues 67 and 114

Amino acid (aa) residues (Val-67 and Ala-114) have been suggested as being mainly responsible for actin-binding in human and bovine deoxyribonucleases I (DNase I). This study presents evidence of these two aa mutational mechanisms, not only for actin-binding but also for folding of DNase I in mammals, reptiles and amphibians. Human and viper snake (Agkistrodon blomhoffii) enzymes are inhibited by actin, whereas porcine, rat snake (Elaphe quadrivirgata), and African clawed frog (Xenopus laevis) enzymes are not. To investigate the role of aa at 67, mutants of rat snake (Ile67Val) and viper snake (Val67Ile) enzymes were constructed. After substitution, the rat snake was inhibited by actin, while the viper snake was not. For the role of aa at 114, mutants of viper snake (Phe114Ala), rat snake (Phe114Ala), African clawed frog (Phe114Ala), and porcine (Ser114Ala/Ser114Phe) enzymes were constructed. Strikingly, the substitute mutants for viper snake, rat snake and African clawed frog expressed no protein. The porcine (Ser114Ala) enzyme was inhibited by actin, but not the porcine (Ser114Phe) enzyme. These results suggest that Val-67 may be essential for actin-binding, that Phe-114 may be related to the folding of DNase I in reptiles and amphibians, and that Ala-114 may be indispensable for actin-binding in mammals.     

NMR docking of a substrate into the X-ray structure of staphylococcal nuclease.

The conformation of the staphylococcal nuclease-bound metal-dTdA complex, previously determined by NMR methods [Weber, D.J., Mullen, G.P., Mildvan, A.S. (1991) Biochemistry 30:7425-7437] was docked into the X-ray structure of the enzyme-Ca(2+)-3',5'-pdTp complex [Loll, P.J., Lattman, E.E. (1989) Proteins: Struct., Funct., Genet. 5:183-201] by superimposing the metal ions, taking into account intermolecular nuclear Overhauser effects from assigned aromatic proton resonances of Tyr-85, Tyr-113, and Tyr-115 to proton resonances of the leaving dA moiety of dTdA, and energy minimization to relieve small overlaps. The proton resonances of the Phe, Tyr, and Trp residues of the enzyme in the ternary enzyme-La(3+)-dTdA complex were sequence specifically assigned by 2D phase-sensitive NOESY, with and without deuteration of the aromatic protons of the Tyr residues, and by 2D heteronuclear multiple quantum correlation (HMQC) spectroscopy and 3D NOESY-HMQC spectroscopy with 15N labeling. While resonances of most Phe, Tyr and Trp residues were unshifted by the substrate dTdA from those found in the enzyme-La(3+)-3',5'-pdTp complex and the enzyme-Ca(2+)-3',5'-pdTp complex, proton resonances of Tyr-85, Tyr-113, Tyr-115, and Phe-34 were shifted by 0.08 to 0.33 ppm and the 15N resonance of Tyr-113 was shifted by 2.1 ppm by the presence of substrate. The optimized position of enzyme-bound dTdA shows the 5'-dA leaving group to partially overlap the inhibitor, 3',5'-pdTp (in the X-ray structure). The 3'-TMP moiety of dTdA points toward the solvent in a channel defined by Ile-18, Asp-19, Thr-22, Lys-45, and His-46. The phosphate of dTdA is coordinated by the metal, and an adjacent inner sphere water ligand is positioned to donate a hydrogen bond to the general base Glu-43 and to attack the phosphorus with inversion. Arg-35 and Arg-87 donate monodentate hydrogen bonds to different phosphate oxygens of dTdA, with Arg-87 positioned to protonate the leaving 5'-oxygen of dA, thus clarifying the mechanism of hydrolysis. Model building of an additional 5'-dGMP onto the 3'-oxygen of dA placed this third nucleotide onto a surface cleft near residues Glu-80, Asp-83, Lys-84, and Tyr-115 with its 3'-OH group accessible to the solvent, thus defining the size of the substrate binding site as accommodating a trinucleotide.     

M-CSF regulates the cytoskeleton via recruitment of a multimeric signaling complex to c-Fms Tyr-559/697/721

M-CSF is known to induce cytoskeletal reorganization in macrophages and osteoclasts by activation of phosphatidylinositol 3-kinase (PI3K) and c-Src, but the detailed mechanisms remain unclear. We find, unexpectedly, that tyrosine (Tyr) to phenylalanine (Phe) mutation of Tyr-721, the PI3K binding site in the M-CSF receptor c-Fms, fails to suppress cytoskeletal remodeling or actin ring formation. In contrast, mutation of c-Fms Tyr-559 to Phe blocks M-CSF-induced cytoskeletal reorganization by inhibiting formation of a Src Family Kinase SFK.c-Cbl.PI3K complex and the downstream activation of Vav3 and Rac, two key mediators of actin remodeling. Using an add-back approach in which specific Tyr residues are reinserted into c-Fms inactivated by the absence of all seven functionally important Tyr residues, we find that Tyr-559 is necessary but not sufficient to transduce M-CSF-dependent cytoskeletal reorganization. Furthermore, this same add-back approach identifies important roles for Tyr-697 and Tyr-721 in collaborating with Tyr-559 to recruit a multimeric signaling complex that can transduce signals from c-Fms to the actin cytoskeleton.     

Aromatic residues located close to the active center are essential for the catalytic reaction of flap endonuclease-1 from hyperthermophilic archaeon Pyrococcus horikoshii

Flap endonuclease-1 (FEN-1) possessing 5'-flap endonuclease and 5'-->3' exonuclease activity plays important roles in DNA replication and repair. In this study, the kinetic parameters of mutants at highly conserved aromatic residues, Tyr33, Phe35, Phe79, and Phe278-Phe279, in the vicinity of the catalytic centers of FEN-1 were examined. The substitution of these aromatic residues with alanine led to a large reduction in kcat values, although these mutants retained Km values similar to that of the wild-type enzyme. Notably, the kcat of Y33A and F79A decreased 333-fold and 71-fold, respectively, compared with that of the wild-type enzyme. The aromatic residues Tyr33 and Phe79, and the aromatic cluster Phe278-Phe279 mainly contributed to the recognition of the substrates without the 3' projection of the upstream strand (the nick, 5'-recess-end, single-flap, and pseudo-Y substrates) for the both exo- and endo-activities, but played minor roles in recognizing the substrates with the 3' projection (the double flap substrate and the nick substrate with the 3' projection). The replacement of Tyr33, Phe79, and Phe278-Phe279, with non-charged aromatic residues, but not with aliphatic hydrophobic residues, recovered the kcat values almost fully for the substrates without the 3' projection of the upstream strand, suggesting that the aromatic groups of Tyr33, Phe79, and Phe278-Phe279 might be involved in the catalytic reaction, probably via multiple stacking interactions with nucleotide bases. The stacking interactions of Tyr33 and Phe79 might play important roles in fixing the template strand and the downstream strand, respectively, in close proximity to the active center to achieve the productive transient state leading to the hydrolysis.     

GTP-yeast actin

Because of the apparently greater conformational flexibility of yeast versus muscle actin and the ability of other members in the actin protein superfamily to efficiently use both ATP and GTP, we assessed the ability of yeast actin to function with GTP. Etheno-ATP exchange studies showed that the binding of GTP to yeast actin is about 1/9 as tight as that of ATP in contrast to the 1/1,240 ratio for muscle actin. Proteolysis of GTP-bound G-yeast actin suggests that the conformation of subdomain 2 is very much like that of ATP-bound actin, but CD studies show that GTP-bound actin is less thermostable than ATP-bound actin. GTP-actin polymerizes with an apparent critical concentration of 1.5 microm, higher than that of ATP-actin (0.3 microm) although filament structures observed by electron microscopy were similar. Yeast actin hydrolyzes GTP in a polymerization-dependent manner, and GTP-bound F-actin decorates with the myosin S1. Conversion of Phe(306) in the nucleotide binding site to the Tyr found in muscle actin raised the nucleotide discrimination ratio from the 1/9 of wild-type actin to 1/125. This result agrees with modeling that predicts that removal of the Tyr hydroxyl will create a space for the C2 amino group of the GTP guanine.     

Substitution of Trp1242 of TM17 alters substrate specificity of human multidrug resistance protein 3

Multidrug resistance protein 3 (MRP3) is an ATP-dependent transporter of 17beta-estradiol 17beta(d-glucuronide) (E(2)17betaG), leukotriene C(4) (LTC(4)), methotrexate, and the bile salts taurocholate and glycocholate. In the present study, the role of a highly conserved Trp residue at position 1242 on MRP3 transport function was examined by expressing wild-type MRP3 and Ala-, Cys-, Phe-, Tyr-, and Pro-substituted mutants in human embryonic kidney 293T cells. Four MRP3-Trp(1242) mutants showed significantly increased E(2)17betaG uptake, whereas transport by the Pro mutant was undetectable. Similarly, the Pro mutant did not transport LTC(4). By comparison, LTC(4) transport by the Ala, Cys, Phe, and Tyr mutants was reduced by approximately 35%. The Ala, Cys, Phe, and Tyr mutants all showed greatly reduced methotrexate and leucovorin transport, except the Tyr mutant, which transported leucovorin at levels comparable with wild-type MRP3. In contrast, the MRP3-Trp(1242) substitutions did not significantly affect taurocholate transport or taurocholate and glycocholate inhibition of E(2)17betaG uptake. Thus Trp(1242) substitutions markedly alter the substrate specificity of MRP3 but leave bile salt binding and transport intact.     

Isolation of four novel tachykinins from frog (Rana catesbeiana) brain and intestine

In a survey for unknown bioactive peptides in frog (Rana catesbeiana) brain and intestine, we isolated four novel peptides that exhibit potent stimulant effects on smooth muscle preparation of guinea pig ileum. By microsequencing and synthesis, these peptides were identified as Lys- Pro- Ser- Pro- Asp- Arg- Phe- Tyr- Gly- Leu- Met- NH2 (ranatachykinin A), Tyr- Lys- Ser- Asp- Ser- Phe- Tyr- Gly- Leu- Met- NH2 (ranatachykinin B), His- Asn- Pro- Ala- Ser- Phe- Ile- Gly- Leu- Met- NH2 (ranatachykinin C) and Lys- Pro- Ans- Pro- Glu- Arg- Phe- Tyr- Ala- Pro- Met- NH2 (ranatachykinin D). Ranatachykinin (RTK) A, B and C conserve the C- terminal sequence, Phe- X- Gly- Leu- Met- NH2, which is common to known members of the tachykinin family. On the other hand, RTK-D has a striking feature in its C-terminal sequence, Phe- Tyr- Ala- Pro- Met- NH2, which has never been found in other known tachykinins, and may constitute a new subclass in the tachykinin family.     

Processing mutations located throughout the human multidrug resistance P-glycoprotein disrupt interactions between the nucleotide binding domains

The most common cause of cystic fibrosis is misfolding of the cystic fibrosis transmembrane conductance regulator (CFTR) protein because of deletion of residue Phe-508 (DeltaF508). P-glycoprotein (P-gp) is an ideal model protein for studying how mutations disrupt folding of ATP-binding cassette proteins such as CFTR because specific chemical chaperones can be used to correct folding defects. Interactions between the nucleotide binding domains (NBDs) are critical because ATP binds at the interface between the NBDs. Here, we used disulfide cross-linking between cysteines in the Walker A sites and the LSGGQ signature sequences to test whether processing mutations located throughout P-gp disrupted interactions between the NBDs. We found that mutations present in the cytoplasmic loops, transmembrane segments, and linker regions or deletion of Tyr-490 (equivalent to Phe-508 in CFTR) inhibited cross-linking between the NBDs. Deletion of Phe-508 in the P-gp/CFTR chimera also inhibited cross-linking between the NBDs. Cross-linking was restored, however, when the mutants were expressed in the presence of the chemical chaperone cyclosporin A. The "rescued" mutants exhibited drug-stimulated ATPase activity, and cross-linking between the NBDs was inhibited by vanadate trapping of nucleotide. These results together with our previous findings (Loo, T. W., Bartlett, M. C., and Clarke, D. M. (2002) J. Biol. Chem. 277, 27585-27588) indicate that processing mutations disrupt interactions among all four domains. It appears that cross-talk between the cytoplasmic and the transmembrane domains is required for establishment of proper domain-domain interactions that occur during folding of ATP-binding cassette protein transporters.     

Conformational stability of CopC and roles of residues Tyr79 and Trp83

CopC is a periplasmic copper Chaperone protein that has a β‐barrel fold and two metal‐binding sites distinct for Cu(II) and Cu(I). In the article, four mutants (Y79F, Y79W, Y79WW83L, Y79WW83F) were obtained by site‐directed mutagenesis. The far‐UV CD spectra of the proteins were similar, suggesting that mutations did not bring any significant changes in secondary structures. Meanwhile the effects of mutations on the protein's function were manifested by Cu(II) binding. Fluorescence lifetime measurement and quenching of tryptophan fluorescence by acrylamide and KI showed that the microenvironment around Trp83 was more hydrophobic than that around Tyr79 in apoCopC. Unfolding experiments induced by guanidinium chloride (GdnHCl), urea provided the conformational stability of each protein. The Δ<ΔG⁰_element> obtained using the model of structural elements was used to show the role of Tyr79 and Trp83. On the one hand, the <ΔG⁰_element> induced by urea for Y79F, Y79W have a loss of 6.51, 2.03 kJ/mol, respectively, compared with apoCopC, proving that replacement of Tyr79 by Phe or Trp all decreased the protein stability, meaning that the hydrogen bonds interactions between Tyr79 and Thr75 played an important role in stabilizing apoCopC. On the other hand, the <ΔG⁰_element> induced by urea for Y79WW83L have a loss of 11.44 kJ/mol, but for Y79WW83F did a raise of 1.82 kJ/mol compared with Y79W. The replacement of Trp83 by Phe and Leu yields opposite effects on protein stability, which suggested that the aromatic ring of Trp83 was important in maintaining the hydrophobic core of apoCopC.     

H-NMR study of rabbit skeletal muscle troponin C: Ca(2+)-dependent interaction with mastoparan.

1H-NMR spectroscopy is employed to study the interaction between rabbit skeletal muscle troponin (C (TnC) and wasp venom tetradecapeptide mastoparan. We monitored the spectral change of the following species of TnC as a function of mastoparan concentration: apoTnC, Ca(2+)-saturated TnC (Ca4TnC) and Ca(2+)-half loaded TnC (Ca2TnC). When apo-TnC is titrated with mastoparan, line-broadening is observed for the ring-current shifted resonance of Phe-23, Ile-34, Val-62 and Phe-72 and the downfield-shifted CH alpha-resonances of Asp-33, Thr-69 and Asp-71; these residues are located in the N-domain. When Ca4TnC is titrated with mastoparan, chemical shift change is observed for the ring-current shifted resonances of Phe-99, Ile-110 and Phe-148 and the downfield-shifted CH alpha-resonances of Asn-105, Ala-106, Ile-110 and Ile-146 and aromatic resonance of Tyr-109 and His-125; these residues are located in the C-domain. The resonance of Phe-23, Asp-33, Asp-71, Phe-72, Phe-99, Tyr-109, Ile-146, His-125 and Phe-148 in both N- and C-domains changes when Ca2TnC is titrated with mastoparan. These results suggest that mastoparan binds to the N-domain of apo-TnC, the C-domain of Ca4TnC and the N- and C-domains of Ca2TnC; the hydrophobic cluster in each domain is involved in binding. As mastoparan binds to TnC, the above resonances shift to their normal chemical shift positions. The stability of the cluster and the beta-sheet is reduced by mastoparan-binding. These results suggest that the conformation of the hydrophobic cluster and the neighboring beta-sheet change to a loose form. The stability of the N-domain of Ca2TnC and Ca4TnC increases when these species bind 1 mol of mastoparan at the C-domain. These results suggest a mastoparan-induced interaction between the N- and C-domains of TnC.     

慢性肾炎体液氨基酸与正常比较

慢性肾炎体液氨基酸与正常比较蒋滢,黄美英,何达纯ＣｈａｎｇｅｏｆＦｌｕｉｄＦｒｅｅＡｍｉｎｏＡｃｉｄｉｎＰａｔｉｅｎｔｓＷｉｔｈＣｈｒｏｎｉｃＮｅｐｈｒｉｔｉｓ￥ＪｉａｎｇＹｉｎｇ;ＨｅＤａｃｈｕｎａｎｄＨｕａｎｇＭｅｉｙｉｎｇ（Ｄｅｐａｒｔｍｅｎｔ...     

Sequential side-chain residue motions transform the binary into the ternary state of DNA polymerase lambda

The nature of conformational transitions in DNA polymerase lambda (pol lambda), a low-fidelity DNA repair enzyme in the X-family that fills short nucleotide gaps, is investigated. Specifically, to determine whether pol lambda has an induced-fit mechanism and open-to-closed transition before chemistry, we analyze a series of molecular dynamics simulations from both the binary and ternary states before chemistry, with and without the incoming nucleotide, with and without the catalytic Mg(2+) ion in the active site, and with alterations in active site residues Ile(492) and Arg(517). Though flips occurred for several side-chain residues (Ile(492), Tyr(505), Phe(506)) in the active site toward the binary (inactive) conformation and partial DNA motion toward the binary position occurred without the incoming nucleotide, large-scale subdomain motions were not observed in any trajectory from the ternary complex regardless of the presence of the catalytic ion. Simulations from the binary state with incoming nucleotide exhibit more thumb subdomain motion, particularly in the loop containing beta-strand 8 in the thumb, but closing occurred only in the Ile(492)Ala mutant trajectory started from the binary state with incoming nucleotide and both ions. Further connections between active site residues and the DNA position are also revealed through our Ile(492)Ala and Arg(517)Ala mutant studies. Our combined studies suggest that while pol lambda does not demonstrate large-scale subdomain movements as DNA polymerase beta (pol beta), significant DNA motion exists, and there are sequential subtle side chain and other motions-associated with Arg(514), Arg(517), Ile(492), Phe(506), Tyr(505), the DNA, and again Arg(514) and Arg(517)-all coupled to active site divalent ions and the DNA motion. Collectively, these motions transform pol lambda to the chemistry-competent state. Significantly, analogs of these residues in pol beta (Lys(280), Arg(283), Arg(258), Phe(272), and Tyr(271), respectively) have demonstrated roles in determining enzyme efficiency and fidelity. As proposed for pol beta, motions of these residues may serve as gate-keepers by controlling the evolution of the reaction pathway before the chemical reaction.