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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In this selected literature survey, we have seen that the stabilities of duplexes and triplexes are governed by the vertical base stacking, the horizontal specific base-paired H-bonding and the environmental parameters. The entropic contribution in the solvation/desolvation process is important in driving the aggregation of NA strands and duplex formation, but base stacking and specific H-bonding maintain the helical order. Triplex formation shares most of the physical environmental prerequisites with those of duplex NAs. However, some additional environmental conditions are often needed. Only in low pH solution is the polycytidylic strand protonated and, thus, it is possible for the strand to bind to a G.C duplex sequence to give the C+(G.C) triplex. High ionic strength is often necessary for the screening of inter-phosphate repulsion due to the high linear charge density in triplexes. The presence of specific counterions is important for complexation. In the absence of negative supercoiling, existence of an intramolecular triplex is rare except under very acidic conditions for the formation of C+(G.C)-type intramolecular triplex. As expected, the stabilities of both inter- and intramolecular triplexes increase with sequence length. The thermodynamic principles of helix-coil transition of oligo-duplex may be described by the van't Hoff relationship, which assumes a two-state cooperative melting profile. Thus, the enthalpy, entropy and free energy of transition can be evaluated from the experimental melting curves (e.g. OD, DSC). For polynucleotides, because of the non-two-state nature of transition, the simple van't Hoff relationship is no longer valid, and direct calorimetry is needed to obtain reliable thermodynamic parameters. The pH and salt concentration dependence of duplex stability can be formulated and derived from a van't Hoff equation. Base-stacking patterns are simple in duplexes but not so in triplexes due to the diversity in triplet schemes. The sequence dependence of base stacking for duplexes has been characterized and employed to predict the stability of an arbitrary sequence. In conclusion, the stability of duplex is relatively well-characterized by thermodynamic data in terms of both base stacking and specific H-bonding. Thermodynamic studies of triplexes have been far fewer in number. Oligonucleotides have found application in the detection and localization of a mRNA or its gene, the detection of bacterial or viral sequences, and the inhibition of the translation of mRNA and the transcription and replication of DNA (Englisch and Gauss, 1991). In a different approach, oligonucleotides have been targeted directly to a DNA duplex motif of a gene in order to inhibit the expression at the beginning of the transcriptional process.(ABSTRACT TRUNCATED AT 400 WORDS)
Prog Biophys Mol Biol 1992
PMID:Stabilities of double- and triple-strand helical nucleic acids. 138 Jul 19

The phase states and phase transitions of some lipids, lipid mixtures of blood and tissues of some animals and human were examined by the method of scanning calorimetry (DSC-2, "Perkin--Elmer"). It was determined that in monohydrated and in hydrated systems, cholesterol produces a liquefying effect on the total phospholipids of the brain and blood. In complex multicomponent lipid systems changes of the phase states and of temperatures of the phase transitions of some lipids were discovered. The role of these phase transitions and miscibility of lipids for functioning of the cellular membranes and of the key-enzymes in the process of changes of lipoproteins of the blood (LCAT) is being discussed, as well as the connection of the phase transitions of cholesterol esters of the blood with atherosclerosis.
Mol Biol (Mosk)
PMID:[Phase transitions, lipid-lipid interactions and their role in some biostructures]. 732 25

It has been demonstrated that the amino acids Asp537, Asp812, Lys631, His811 and Tyr639 are involved in bacteriophage T7 RNA polymerase catalysis. In the present paper, we report kinetic, spectroscopic and calorimetric characterization of the wild-type and mutant T7 RNA polymerases generated at these five loci (D537N, E; K631M, R; Y639F, S, A, W; H811Q, A; D812N, E). The wild-type enzyme has a substantial amount of secondary structure as determined by CD analysis (alpha-helix, 43%; beta-sheet, 14%; beta-turn, 25%; unordered, 18%). The CD spectra of 12 mutants at five loci are very similar to that of the wild-type, except for the mutant Y639W. Within experimental error, the thermal transition temperatures measured by CD and DSC as well as the lambda max values of the fluorescence spectra were the same for the wild-type and all of the mutants. Therefore, the overall folding and stability of the mutant enzymes are very similar to those of the wild-type enzyme, although small local conformational changes cannot be excluded. For the synthesis of the pentamer pppGGACU, the mutants D537E and D812E showed an approximately two- to threefold decrease in (kcat)app and an approximately two- to threefold increase in (Km)app, relative to the wild-type, in contrast to the mutants D537N and D812N which exhibited no detectable activity. The mutant K631R showed a sevenfold reduction in (kcat)app and a two- to threefold increase in (Km)app, supporting our earlier observation with the mutant K631M that Lys631 may be involved in phosphodiester bond formation. The mutant Y639S can synthesize the trimer GGA with an approximately 50-fold decrease in (kcat)app and a tenfold increase in (Km)app, relative to the wild-type, underlining the importance of the phenyl ring of Tyr639. The mutant H811A, in which the side-chain at position 811 is incapable of forming a hydrogen bond, can synthesize the trimer GGA with an approximately tenfold decrease in (kcat)app and an approximately 35-fold increase in (Km)app. Thus, either the hydrogen-bonding capacity of this residue is non-essential or some other group can functionally substitute for the His811 side-chain. The wild-type enzyme showed significant effects of the base position in the sequence on the apparent binding constants for the NTPs. The kinetics of GpG-primed trimer, tetramer and pentamer synthesis on three 22 bp templates were investigated for the wild-type and mutant enzymes with measurable activity.(ABSTRACT TRUNCATED AT 400 WORDS)
J Mol Biol 1994 Mar 18
PMID:Bacteriophage T7 RNA polymerase and its active-site mutants. Kinetic, spectroscopic and calorimetric characterization. 813 19

Using reverse transcription polymerase chain reactions (RT-PCR), the DNA sequence for the main membrane-spanning region (IS3 through IVS6) of the gene encoding the alpha-subunit of the para sodium channel of the German cockroach, Blattella germanica, has been determined. The overall structure of the open reading frame region of this B. germanica gene is very similar to that of the para gene of Drosophila melanogaster, and that of the partially sequenced para gene of Musca domestica. On the other hand, it is distinctly different from that of the DSC gene (Drosophila sodium channel). As a result of a side-by-side comparison of the para gene sequences of the susceptible CSMA strain and the kdr resistant VT strain of B. germanica, one mutation (TTG to TTC) at the approximate center of the IIS6 membrane-spanning segment was found to result in an amino acid change from L to F. While the functional meaning of this mutation for the operation of the para sodium channel remains to be studied, this region is very highly conserved among all sodium channels identified so far, and is one of the most hydrophobic areas of the entire alpha-subunit. For comparison, we have studied the same region of the para sodium channel of both kdr and susceptible SBO strain of the housefly, Musca domestica. We found the homologous type of mutation, CTT to TTT, resulting in the same amino acid alteration (L to F) at this site. However, in the case of houseflies both kdr and susceptible strains contained both L and F versions of the protein. The ratio of TTT to CTT was significantly higher in the kdr strain of M. domestica than in the three susceptible strains examined.
Mol Gen Genet 1996 Aug 27
PMID:Cloning and sequencing of the para-type sodium channel gene from susceptible and kdr-resistant German cockroaches (Blattella germanica) and house fly (Musca domestica). 880 4

The 8.12 idiotype is expressed in elevated titer in the serum of patients with systemic lupus erythematosus and is a marker for a subpopulation of anti-DNA antibodies that possess a V(lambda)II encoded light chain. This study utilized a eukaryotic expression system to identify the structural basis for expression of this idiotype. Reversion of the 8.12+ DSC light chain to the hslv215.23/DPL11 germline gene reveals that the 8.12 idiotype is encoded in the germline. The 8.12+ DSC and the 8.12 AS17 light chains, both belonging to the V(lambda)II family, were subjected to site directed mutagenesis, to localize amino acids important for expression of the 8.12 idiotype. Point mutations were performed in CDR1, CDR2, FR3 and CDR3, in positions where the 8.12+ DSC differs from the 8.12-AS17. Amino acids in CDR1 and the CDR2 proximal region of FR3, but not the J proximal region of CDR3, play a crucial role in 8.12 reactivity. The 3-D structure of Mcg, a human IgG1, with which DSC shares a sequence homology of 92.3% has been examined to visualize the effect of each of the mutations and to identify the surface on DSC that comprises the idiotype.
Mol Immunol 1996 Nov
PMID:Molecular mapping of the 8.12 SLE-associated idiotype specificity at the single amino acid level. 912 62

The effect of saturated solutions of polyglutamate on the thermal stabilities of the Met-80-heme iron bond and of the ferricytochrome c as a whole were studied by absorption spectroscopy and differential scanning calorimetry at pH 7.0. According to spectral data the midtransition temperature of the cleavage of the sulfur-iron bond was 57.4 +/- 0.5 degrees C and 66.8 +/- 0.5 degrees C for cytochrome c and cytochrome c-polyglutamate complex, respectively. Addition of polyglutamate to cytochrome c at pH 7.0 alters the denaturation properties of the protein. As follows from DSC scans, the denaturation temperature for cytochrome c is decreased from 85.4 +/- 0.2 degrees C to 68.7 +/- 0.2 degrees C in the presence of the saturated amount of polyglutamate. The protein stability in terms of Gibbs energy change at protein unfolding amount to delta G(25 degrees C) = 22.7 +/- 2.7 and 32.0 +/- 2.2 kJ/mol, for cytochrome c and cytochrome c-polyglutamate complex, respectively, at pH 7.0. It is evident that polyglutamate increases the thermal stability of the sulfur-iron bond and decreases the denaturation temperature of the cytochrome c molecule as a whole. The complex thermal stability in terms of Gibbs energy is not lower than that of cytochrome c in the range of physiological temperatures.
Biochem Mol Biol Int 1997 Nov
PMID:Effect of polyglutamate on the thermal stability of ferricytochrome c. 938 49

Previous DSC and X-ray studies on RM6, a loop deletion mutant of wtROP protein, have shown that removal of five amino acids from the loop causes a dramatic reorganization of the wild-type structure. The new tetrameric molecule exhibits a significantly higher stability (Lassalle, M.W. et al., J. Mol. Biol., 1998, 279, 987-1000) and unfolds in a second order reaction (Lassalle, M.W. and Hinz, H.-J., Biochemistry, 1998, 37, 8465-8472). In the present investigation we report extensive refolding studies of RM6 at different temperatures and GdnHCl concentrations monitored by CD and fluorescence to probe for changes in secondary and tertiary structure, respectively. The measurements permitted us to determine activation parameters as a function of denaturant concentration. The results demonstrate convincingly that the variation with GdnHCl concentration of the activation parameters deltaH#, deltaS# and deltaG# is very similar for unfolding and refolding. For both processes the activation properties approach a maximum in the vicinity of the denaturant concentration, c(K=1), where the equilibrium constant equals 1, i.e. deltaG0 equals zero. CD and fluorescence refolding kinetics are described by identical constants suggesting that the formation of secondary and tertiary structure occurs simultaneously. Refolding is, however, characterized by a more complex mechanism than unfolding. Although the general pattern is dominated by the sequence monomers to dimers to tetramers, parallel side reactions involving dimers and monomers have to be envisaged in the initial folding phase, supporting the view that the native state of RM6 can be reached by several rather than a single pathway.
 ...
PMID:Refolding studies on the tetrameric loop deletion mutant RM6 of ROP protein. 1035 32

Stefin A folds as a monomer under strongly native conditions. We have observed that under partially denaturing conditions in the temperature range from 74 to 93 degrees C it folds into a dimer, while it is monomeric above the melting temperature of 95 degrees C. Below 74 degrees C the dimer is trapped and it does not dissociate. The dimer is a folded and structured protein as judged by CD and NMR, nevertheless it is no more functional as an inhibitor of cysteine proteases. The monomer-dimer transition proceeds at a slow rate and the activation energy of dimerization at 99 kcal/mol is comparable to the unfolding enthalpy. A large and negative dimerization enthalpy of -111(+/- 8) kcal/mol was calculated from the temperature dependence of the dissociation constant. An irreversible pretransition at 10-15 deg. below the global unfolding temperature has been observed previously by DSC and can now be assigned to the monomer-dimer transition. Backbone resonances of all the dimer residues were assigned using 15N isotopically enriched protein. The dimer is symmetric and the chemical shift differences between the monomer and dimer are localized around the tripartite hydrophobic wedge, which otherwise interacts with cysteine proteases. Hydrogen exchange protection factors of the residues affected by dimer formation are higher in the dimer than in the monomer. The monomer to dimer transition is accompanied by a rapid exchange of all of the amide protons which are protected in the dimer, indicating that the transition state is unfolded to a large extent. Our results demonstrate that the native monomeric state of stefin A is actually metastable but is favored by the kinetics of folding. The substantial energy barrier which separates the monomer from the more stable dimer traps each state under native conditions.
J Mol Biol 1999 Sep 03
PMID:Accessing the global minimum conformation of stefin A dimer by annealing under partially denaturing conditions. 1051 44

Retroviruses employ -1 translational frameshifting to regulate the relative concentrations of structural and non-structural proteins critical to the viral life cycle. The 1.6 A crystal structure of the -1 frameshifting pseudoknot from beet western yellows virus reveals, in addition to Watson-Crick base-pairing, many loop-stem RNA tertiary structural interactions and a bound Na(+). Investigation of the thermodynamics of unfolding of the beet western yellows virus pseudoknot reveals strongly pH-dependent loop-stem tertiary structural interactions which stabilize the molecule, contributing a net of DeltaH approximately -30 kcal mol(-1) and DeltaG degrees (37) of -3.3 kcal mol(-1) to a total DeltaH and DeltaG degrees (37) of -121 and -16 kcal mol(-1), respectively, at pH 6.0, 0.5 M K(+) by DSC. Characterization of mutant RNAs supports the presence of a C8(+).G12-C26 loop 1-stem 2 base-triple (pK(a)=6.8), protonation of which contributes nearly -3.5 kcal mol(-1) in net stability in the presence of a wild-type loop 2. Substitution of the nucleotides in loop 2 with uridine bases, which would eliminate the minor groove triplex, destroys pseudoknot formation. An examination of the dependence of the monovalent ion and type on melting profiles suggests that tertiary structure unfolding occurs in a manner quantitatively consistent with previous studies on the stabilizing effects of K(+), NH(4)(+) and Na(+) on other simple duplex and pseudoknotted RNAs.
J Mol Biol 2000 Feb 18
PMID:Energetics of a strongly pH dependent RNA tertiary structure in a frameshifting pseudoknot. 1066 15

Hyperactivation of Cdc2 in fission yeast causes cells to undergo a lethal premature mitosis called mitotic catastrophe. This phenotype is observed in cdc2-3w wee1-50 cells at high temperature. Eleven of 17 mutants that suppress this phenotype define a single complementation group, mcs1. The mcs1-77 mutant also suppresses lethal inactivation of the Wee1 and Mik1 tyrosine kinases and thus delays mitosis independently of Cdc2 tyrosine phosphorylation. We have cloned mcs1 by isolating suppressors of the cell cycle arrest phenotype of mcs1-77 cdc25-22 cells and found that it encodes Res2, a component of the START gene-specific transcription factor complex MBF (also known as DSC-1). The mcs1-77 mutant bears a single point mutation in the DNA-binding domain of Res2 that causes glycine 68 to be replaced by a serine residue. Importantly, two substrates of the anaphase-promoting complex (APC), the major B-type cyclin, Cdc13, and the anaphase inhibitor, Cut2, are unstable in G2-phase mcs1-77 cells. Consistent with this, we observe abnormal sister chromatid separation in mcs1-77 cdc25-22 cells at the restrictive temperature. Mutation of either Cdc10 or Res1 also deregulates MBF-dependent transcription and causes a G2 delay. We find that this cell cycle delay is abolished in the absence of the APC regulator Ste9/Srw1 and that the periodic expression of Ste9/Srw1 is controlled by the MBF complex. These data suggest that in fission yeast the MBF complex plays a key role in the inactivation of cyclin B and Cut2 destruction by controlling the periodic production of APC regulators.
Mol Biol Cell 2000 Oct
PMID:A role for the START gene-specific transcription factor complex in the inactivation of cyclin B and Cut2 destruction. 1102 45


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