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1.
SAMEM (System for Analysis of Molecular Evolution Modes), a web-based pipeline system for inferring modes of molecular evolution in genes and proteins (http://pixie.bionet.nsc.ru/samem/), is presented. Pipeline 1 performs analyses of protein-coding gene evolution; pipeline 2 performs analyses of protein evolution; pipeline 3 prepares datasets of genes and/or proteins, performs their primary analysis, and builds BLOSUM matrices; pipeline 4 checks if these genes really are protein-coding. Pipeline 1 has an all-new feature, which allows the user to obtain K(R)/K(C) estimates using several different methods. An important feature of pipeline 2 is an original method for analyzing the rates of amino acid substitutions at the branches of a phylogenetic tree. The method is based on Markov modeling and a non-parametric permutation test, which compares expected and observed frequencies of amino acid substitutions, and infers the modes of molecular evolution at deep inner branches. 相似文献
2.
Ronny Martinez Felix Jakob Ran Tu Petra Siegert Karl‐Heinz Maurer Ulrich Schwaneberg 《Biotechnology and bioengineering》2013,110(3):711-720
Bacillus gibsonii Alkaline Protease (BgAP) is a recently reported subtilisin protease exhibiting activity and stability properties suitable for applications in laundry and dish washing detergents. However, BgAP suffers from a significant decrease of activity at low temperatures. In order to increase BgAP activity at 15°C, a directed evolution campaign based on the SeSaM random mutagenesis method was performed. An optimized microtiter plate expression system in B. subtilis was established and classical proteolytic detection methods were adapted for high throughput screening. In parallel, the libraries were screened for increased residual proteolytic activity after incubation at 58°C. Three iterative rounds of directed BgAP evolution yielded a set of BgAP variants with increased specific activity (Kcat) at 15°C and increased thermal resistance. Recombination of both sets of amino acid substitutions resulted finally in variant MF1 with a 1.5‐fold increased specific activity (15°C) and over 100 times prolonged half‐life at 60°C (224 min compared to 2 min of the WT BgAP). None of the introduced amino acid substitutions were close to the active site of BgAP. Activity‐altering amino acid substitutions were from non‐charged to non‐charged or from sterically demanding to less demanding. Thermal stability improvements were achieved by substitutions to negatively charged amino acids in loop areas of the BgAP surface which probably fostered ionic and hydrogen bonds interactions. Biotechnol. Bioeng. 2013; 110: 711–720. © 2012 Wiley Periodicals, Inc. 相似文献
3.
Robert J. DiMario Ashley N. Kophs Varsha S. Pathare James C. Schnable Asaph B. Cousins 《The Plant journal : for cell and molecular biology》2021,105(6):1677-1688
The high rates of photosynthesis and the carbon-concentrating mechanism (CCM) in C4 plants are initiated by the enzyme phosphoenolpyruvate (PEP) carboxylase (PEPC). The flow of inorganic carbon into the CCM of C4 plants is driven by PEPC’s affinity for bicarbonate (KHCO3), which can be rate limiting when atmospheric CO2 availability is restricted due to low stomatal conductance. We hypothesize that natural variation in KHCO3 across C4 plants is driven by specific amino acid substitutions to impact rates of C4 photosynthesis under environments such as drought that restrict stomatal conductance. To test this hypothesis, we measured KHCO3 from 20 C4 grasses to compare kinetic properties with specific amino acid substitutions. There was nearly a twofold range in KHCO3 across these C4 grasses (24.3 ± 1.5 to 46.3 ± 2.4 μm ), which significantly impacts modeled rates of C4 photosynthesis. Additionally, molecular engineering of a low-HCO3− affinity PEPC identified key domains that confer variation in KHCO3. This study advances our understanding of PEPC kinetics and builds the foundation for engineering increased-HCO3− affinity and C4 photosynthetic efficiency in important C4 crops. 相似文献
4.
Summary Rare mutations that alter the substrate specificity of proline permease cluster in discrete regions of theputP gene, suggesting that they may replace amino acids at the active site of the enzyme. IfputP substrate specificity mutations directly alter the active site of proline permease, the mutants should show specific defects in the kinetics of proline transport. In order to test this prediction, we examined the kinetics of threeputP substrate specificity mutants. One class of mutation increases theK
m
over 120-fold but only decreases theV
max fourfold. SuchK
m
mutants may be specifically defective in substrate recognition, thus identifying an amino acid critical for substrate binding. Another class of mutation decreases theV
max 80-fold without changing theK
m
.V
max mutants appear to alter the rate of substrate translocation without affecting the substrate binding site. The last class of mutation alters both theK
m
andV
max of proline transport. These results indicate that substrate specificity mutations alter amino acids critical for Na+/proline symport. 相似文献
5.
Protein recovery by continuous flotation 总被引:2,自引:0,他引:2
Summary Bovine serum albumin (BSA) was recovered from aqueous solutions by foam flotation. The protein concentrations in foam liquid C
S, in feed C
Pand in residue C
Rwere determined. The protein enrichment C
S/CPand the separation C
S/CRas well as the protein fraction in the foam liquid % BSA and foam liquid volume flow were determined as functions of the medium properties, operational conditions, and equipment parameters as well as concentrations of solid particles. At low protein concentrations in feed (e.g., C
P=40 mg · l-1), and at 40° C, high performance was attained (C
X=2,000 mg · l-1, C
R=4.4 mg · l-1, C
S/CP=50, C
S/CR=450, 90% BSA. Continuous foam flotation is an efficient procedure for the recovery of low concentrations of proteins from liquid cultures.Abbreviations BSA
bovine serum albumine
-
C
P
protein concentration in feed (mg · l-1)
-
C
R
protein concentration in residue (mg · l-1)
-
C
S
protein concentration in foam liquid (mg · l-1)
-
C
S/CR
protein separation (-)
-
C
S/CP
protein enrichment (-)
-
V
P
feed rate (ml · min-1)
-
V
R
residue flow rate (ml · min-1)
-
V
S
foam liquid volume flow (ml · min-1)
-
N
number of theoretical stages in an ideal cascade (-)
-
temperature (° C)
-
mean residence time (min) 相似文献
6.
Norman Wang William F. Smith Brian R. Miller Dikran Aivazian Alexey A. Lugovskoy Mitchell E. Reff Scott M. Glaser Lisa J. Croner Stephen J. Demarest 《Proteins》2009,76(1):99-114
Engineered antibodies are a large and growing class of protein therapeutics comprising both marketed products and many molecules in clinical trials in various disease indications. We investigated naturally conserved networks of amino acids that support antibody VH and VL function, with the goal of generating information to assist in the engineering of robust antibody or antibody‐like therapeutics. We generated a large and diverse sequence alignment of V‐class Ig‐folds, of which VH and VL domains are family members. To identify conserved amino acid networks, covariations between residues at all possible position pairs were quantified as correlation coefficients (?‐values). We provide rosters of the key conserved amino acid pairs in antibody VH and VL domains, for reference and use by the antibody research community. The majority of the most strongly conserved amino acid pairs in VH and VL are at or adjacent to the VH–VL interface suggesting that the ability to heterodimerize is a constraining feature of antibody evolution. For the VH domain, but not the VL domain, residue pairs at the variable‐constant domain interface (VH–CH1 interface) are also strongly conserved. The same network of conserved VH positions involved in interactions with both the VL and CH1 domains is found in camelid VHH domains, which have evolved to lack interactions with VL and CH1 domains in their mature structures; however, the amino acids at these positions are different, reflecting their different function. Overall, the data describe naturally occurring amino acid networks in antibody Fv regions that can be referenced when designing antibodies or antibody‐like fragments with the goal of improving their biophysical properties. Proteins 2009. © 2008 Wiley‐Liss, Inc. 相似文献
7.
Directed evolution has been used to enhance the catalytic activity and alkaline pH stability of Thermobifida fusca xylanase A, which is one of the most thermostable xylanases. Under triple screened traits of activity, alkaline pH stability
and thermostability, through two rounds of random mutagenesis using DNA shuffling, a mutant 2TfxA98 with approximately 12-fold
increased k
cat/K
m and 4.5-fold decreased K
m compared with its parent was obtained. Moreover, the alkaline pH stability of 2TfxA98 is increased significantly, with a
thermostability slightly lower than that of its parent. Five amino acid substitutions (T21A, G25P, V87P, I91T, and G217L),
three of them are near the catalytic active site, were identified by sequencing the genes encoding this evolved enzyme. The
activity and stabilizing effects of each amino acid mutation in the evolved enzyme were evaluated by site-directed mutagenesis.
This study shows a useful approach to improve the catalytic activity and alkaline pH stability of T. fusca xylanase A toward the hydrolysis of xylan. 相似文献
8.
Charles M. Matthews Cassandra J. Vandenberg Clive N.A. Trotman 《Journal of molecular evolution》1998,46(6):729-733
The Artemia hemoglobin is a dimer comprising two nine-domain covalent polymers in quaternary association. Each polymer is encoded by
a gene representing nine successive globin domains which have different sequences and are presumed to have been copied originally
from a single-domain gene. Two different polymers exist as the result of a complete duplication of the nine-domain gene, allowing
the formation of either homodimers or the heterodimer. The total population size of 18 domains comprising nine corresponding
pairs, coupled with the probability that they reflect several hundred million years of evolution in the same lineage, provides
a unique model in which the process of gene multiplication can be analyzed. The outcome has important implications for the
reliability of local molecular clocks.
The two polymers differ from each other at 11.7% of amino acid sites; however when corresponding individual domains are compared
between polymers, amino acid substitution fluctuates by a factor of 2.7-fold from lowest to highest. This variation is not
obvious at the DNA level: Domain pair identity values fluctuate by 1.3-fold. Identity values are, however, uncorrected for
multiple substitutions, and both silent and nonsilent changes are pooled. Therefore, to determine the variability in relative
substitution rates at the DNA level, we have used the method of Li (1993, J Mol Evol 36:96–99) to determine estimates of nonsynonymous (K
A
) and synonymous (K
S
) substitutions per site for the nine pairs of domains. As expected, the overall level of silent substitutions (K
S
of 56.9%) far exceeded nonsilent substitutions (K
A
of 6.7%); however, for corresponding domain pairs, K
A
fluctuates by 2.3-fold and K
S
by 1.7-fold. The large discrepancies reflected in the expressed protein have accrued within a single lineage and the implication
is that divergence dates of different genera based on amino acid sequences, even with well-studied proteins of reasonable
size, can be wrong by a factor well in excess of 2.
Received: 4 June 1997 / Accepted: 17 December 1997 相似文献
9.
M. Wallis 《Journal of molecular evolution》1994,38(6):619-627
In mammals pituitary growth hormone (GH) shows a slow basal rate of evolution (0.22 ± 0.03 × 10–9 substitutions/amino acid site/year) which appears to have increased by at least 25–50-fold on two occasions, during the evolution of primates (to at least 10.8 ± 1.3 X 10–9 substitutions/amino acid site/year) and artiodactyl ruminants (to at least 5.6 ± 1.3 X 10–9 substitutions/amino acid site/year). That these rate increases are real, and not due to inadvertent comparison of nonorthologous genes, was established by showing that features of the GH gene sequences that are not expressed as mature hormone do not show corresponding changes in evolutionary rate. Thus, analysis of nonsynonymous substitutions in the coding sequence for the mature protein confirmed the rate increases seen in the primate and ruminant GHs, but analysis of nonsynonymous substitutions in the signal peptide sequence, synonymous substitutions in the coding sequence for signal peptide or mature protein, and 5 and 3 untranslated sequences showed no statistically significant changes in evolutionary rate. Evidence that the increases in evolutionary rate are probably due to positive selection is provided by the observation that in the cases of both ruminant and primate GHs the periods of rapid evolution were followed by a return to a slow rate similar to the basal rate seen in other mammalian GHs. Differences between the biological properties of GHs have been identified which may relate to these periods of rapid adaptive molecular evolution. On the basis of sequence data currently available (but excluding rodent GHs which show an intermediate rate, the basis of which is not clear) for most (90%) of evolutionary time mammalian GHs have been in the slow phase of evolution, with possibly most of the few amino acid substitutions that have occurred being neutral in nature. But most (80%) of the amino acid substitutions that have been introduced into GH during the course of mammalian evolution have been accepted during the rapid phases and were adaptive in nature. 相似文献
10.
The thermal and alkaline pH stability of Streptomyces lividans xylanase B was improved greatly by random mutagenesis using DNA shuffling. Positive clones with improved thermal stability
in an alkaline buffer were screened on a solid agar plate containing RBB-xylan (blue). Three rounds of directed evolution
resulted in the best mutant enzyme 3SlxB6 with a significantly improved stability. The recombinant enzyme exhibited significant
thermostability at 70°C for 360 min, while the wild-type lost 50% of its activity after only 3 min. In addition, mutant enzyme
3SlxB6 shows increased stability to treatment with pH 9.0 alkaline buffer. The K
m value of 3SlxB6 was estimated to be similar to that of wild-type enzyme; however k
cat was slightly decreased, leading to a slightly reduced value of k
cat/K
m, compared with wild-type enzyme. DNA sequence analysis revealed that eight amino acid residues were changed in 3SlxB6 and
substitutions included V3A, T6S, S23A, Q24P, M31L, S33P, G65A, and N93S. The stabilizing effects of each amino acid residue
were investigated by incorporating mutations individually into wild-type enzyme. Our results suggest that DNA shuffling is
an effective approach for simultaneous improvement of thermal and alkaline pH stability of Streptomyces lividans xylanase B even without structural information. 相似文献
11.
On the rate of molecular evolution 总被引:3,自引:0,他引:3
Summary There are at least two outstanding features that characterize the rate of evolution at the molecular level as compared with that at the phenotypic level. They are; (1) remarkable uniformity for each molecule, and (2) very high overall rate when extrapolated to the whole DNA content.The population dynamics for the rate of mutant substitution was developed, and it was shown that if mutant substitutions in the population are carried out mainly by natural selection, the rate of substitution is given byk = 4 N
e
s
1
v, whereN
e
is the effective population number,s
1 is the selective advantage of the mutants, andv is the mutation rate per gamete for such advantageous mutants (assuming that 4N
e
s
1 1). On the other hand, if the substitutions are mainly carried out by random fixation of selectively neutral or nearly neutral mutants, we havek = v, wherev is the mutation rate per gamete for such mutants.Reasons were presented for the view that evolutionary change of amino acids in proteins has been mainly caused by random fixation of neutral mutants rather than by natural selection.It was concluded that if this view is correct, we should expect that genes of living fossils have undergone almost as many DNA base replacements as the corresponding genes of more rapidly evolving species.Contribution No. 789 from the National Institute of Genetics, Mishima, Shizuokaken 411 Japan. Aided in part by a grant-in-aid from the Ministry of Education, Japan. 相似文献
12.
C. Balocco G. Bogé H. Roche 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1993,163(4):340-347
This work was devoted to the study of the structure-affinity relationships in neutral amino acid transport by intestinal brush border of marine fish (Dicentrarchus labrax). The effects of the length of the side chain on kinetics of glycine, alanine, methionine and amino isobutyric acid were investigated. In the presence of K+ two components were characterized: one is saturable by increased substrate concentrations, whereas the other can be described by simple diffusion mechanism. Simple diffusion, a passive, non-saturable, Na+-independent route, contributes largely to the transport of methionine and to a much lesser extend to alanine, glycine or alphaaminoisobutyric acid uptakes. If a branched chain is present, as in the case of amino isobutyric acid, diffusion is low. A Na+-independent, saturable system has been fully characterized for methionine, but not for branched amino acids such as amino isobutyric acid. In the presence of Na+ saturable components were shown. Two distinct Na+-dependent pathways have been characterized for glycine uptake, with low and high affinities. For alanine and methionine only one Na+-dependent high affinity system exists with the same half-saturation concentration and the same maximum uptake at saturable concentrations. Glycine high affinity system has the same half-saturation concentration as methionine or alanine uptake, whereas maximum uptake is lower. The substitution of the hydrogen by a methyl group results in a severe decrease of uptake (aminoisobutyric acid). Mutual inhibition experiments indicate that the same carriers could be responsible for methionine and alanine uptakes and probably glycine Na+-dependent uptake. The influence of Na+ concentrations (100-1 mol·l-1) on amino acid uptake was examined. Glycine, alanine, methionine and amino isobutyric acid transport can be described by a hyperbolic function, with a saturation uptake which is highly increased for methionine. However, the half-saturation concentration does not seem to be strongly affected by the amino acid structure. The effect of Na+ concentration (25 and 100 mmol·l-1) on the kinetics of methionine uptake have been also examined. The maximum uptake of the saturable system clearly shows a typical relationship with concentration.Abbreviations [AA]
amino acid concentration
- AIB
aminoisobutyric acid
- [I]
Inhibitor amino acid concentration
-
J
i
uptake in the presence of inhibitor
-
J
o
uptake without inhibitor
-
K
d
passive diffusion constant
-
K
i
inhibitor constant
-
K
t
concentration of test amino acid for half-maximal flux
- MES
2[N-morpholino]ethanesulphonic acid
-
V
max
maximum uptake at saturable amino acid concentrations
-
V
tot
total amino acid uptake 相似文献
13.
Recently, we screened several KV channels for possible dependence on plasma membrane phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). The channels were expressed in tsA-201 cells and the PI(4,5)P2 was depleted by several manipulations in whole-cell experiments with parallel measurements of channel activity. In contrast to reports on excised-patches using Xenopus laevis oocytes, we found only KV7, but none of the other tested KV channels, to be strongly dependent on PI(4,5)P2. We now have extended our study to KV1.2 channels, a KV channel we had not previously tested, because a new published study on excised patches showed regulation of the voltage-dependence of activation by PI(4,5)P2. In full agreement with those published results, we found a reduction of current amplitude by ~20% after depletion of PI(4,5)P2 and a small left shift in the activation curve of KV1.2 channels. We also found a small reduction of KV11.1 (hERG) currents that was not accompanied by a gating shift. In conclusion, our whole-cell methods yield a PI(4,5)P2-dependence of KV1.2 currents in tsA-201 cells that is comparable to findings from excised patches of Xenopus laevis oocytes. We discuss possible physiological rationales for PI(4,5)P2 sensitivity of some ion channels and insensitivity of others. 相似文献
14.
The entropy of the amino acid sequences coded by DNA is considered as a measure of diversity or variety of proteins, and is taken as a measure of evolution. The DNA or m-RNA sequence is corsidered as a stationary second-order Markov chain composed of four kinds of bases. Because of the biased nature of the genetic code table, increase of entropy of amino acid sequences is possible with biased nucleotide sequence. Thus the biased DNA base composition and the extreme rarity of the base doubletC
p
G of higher organisms are explained. It is expected that the amino acid composition was highly biased at the days of the origin of the genetic code table, and the more frequent amino acids have tended to get rarer, and the rarer ones more frequent. This tendency is observed in the evolution of hemoglobin, cytochrome C, fibrinopeptide, immunoglobulin and lysozyme, and protein as a whole. 相似文献
15.
Evolutionary dynamics of Rh2 opsins in birds demonstrate an episode of accelerated evolution in the New World warblers (Setophaga)
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Low rates of sequence evolution associated with purifying selection can be interrupted by episodic changes in selective regimes. Visual pigments are a unique system in which we can investigate the functional consequences of genetic changes, therefore connecting genotype to phenotype in the context of natural and sexual selection pressures. We study the RH2 and RH1 visual pigments (opsins) across 22 bird species belonging to two ecologically convergent clades, the New World warblers (Parulidae) and Old World warblers (Phylloscopidae) and evaluate rates of evolution in these clades along with data from 21 additional species. We demonstrate generally slow evolution of these opsins: both Rh1 and Rh2 are highly conserved across Old World and New World warblers. However, Rh2 underwent a burst of evolution within the New World genus Setophaga, where it accumulated substitutions at 6 amino acid sites across the species we studied. Evolutionary analyses revealed a significant increase in dN/dS in Setophaga, implying relatively strong selective pressures to overcome long‐standing purifying selection. We studied the effects of each substitution on spectral tuning and found they do not cause large spectral shifts. Thus, substitutions may reflect other aspects of opsin function, such as those affecting photosensitivity and/or dark–light adaptation. Although it is unclear what these alterations mean for colour perception, we suggest that rapid evolution is linked to sexual selection, given the exceptional plumage colour diversification in Setophaga. 相似文献
16.
Summary The ionic dependencies of the transepithelial and intracellular electrical parameters were measured in the isolated frog cornea. In NaCl Ringer's the intracellular potential differenceV
sc measured under short-circuit conditions depolarized by nearly the same amount after either increasing the stromal-side KCl concentration from 2.5 to 25mm or exposure to 2mm BaCl2 (K+ channel blocker). With Ba2+ the depolarization of theV
sc by 25mm K+ was reduced to one-quarter of the control change. If the Cl-permselective apical membrane resistanceR
o remained unchanged, the relative basolateral membrane resistanceR
i, which includes the lateral intercellular space, increased at the most by less than twofold after Ba2+. These effects in conjunction with the depolarization of theV
sc by 62 mV after increasing the stromal-side K+ from 2.5 to 100mm in Cl-free Ringer's as well as the increase of the apparent ratio of membrane resistances (a=R
o/Ri) from 13 to 32 are all indicative of an appreciable basolateral membrane K+ conductance. This ratio decreased significantly after exposure to either 25mm K+ or Ba2+. The decline ofR
o/Ri with 25mm K+ appears to be anomalous since this decrease is not consistent with just an increase of basolateral membrane conductance by 25mm K+, but rather perhaps a larger decrease ofR
o thanR
iAlso an increase of lateral space resistance may offset the effect of decreasingR
i with 25mm K+. In contrast,R
o/Ri did transiently increase during voltage clamping of the apical membrane potential differenceV
o and exposure to 25mm K+ on the stromal side. This increase and subsequent decrease ofR
o/Ri supports the idea that increases in stromal K+ concentration may produce secondary membrane resistance changes. These effects onR
o/Ri show that the presence of asymmetric ionic conductance properties in the apical and basolateral membranes can limit the interpretative value of this parameter. The complete substitution of Na+ withn-methyl-glucamine in Cl-free Ringer's on the stromal side hyperpolarized theV
sc by 6 mV whereas 10–4
m ouabain depolarized theV
sc by 7 mV. Thus the basolateral membrane contains K+, Na+ and perhaps Cl– pathways in parallel with the Na/K pump component. 相似文献
17.
Buchwald, P., Bodor, N. Octanol-Water Partition of Nonzwitterionic Peptides: Predictive Power of a Molecular Size-Based Model. Proteins 30:86–99, 1998. Equation 2 should read: P = (Cin − Cfin) Vw/Cfin Vo. In the printed version, the volume ratio (Vw/Vo) incorrectly divides, and not multiplies, the concentration ratio. The publisher apologizes for this error. 相似文献
18.
19.
The usefulness of Lilly's kinetic equation to describe penicillin G hydrolysis performed by immobilized penicillin acylase onto the acrylic carrier has been shown. Based on the experimental results characteristic kinetic constants have been estimated. The effect of noncompetitive inhibition of 6-amino penicillanic acid has not been found. Five components of reaction resistance have been defined. These components were also estimated for the reaction of the native enzyme as well as the Boehringer preparation.List of Symbols
C
E g/m3
enzyme concentration
-
C
P,C
Q mol/m3
product concentrations
-
C
S mol/m3
substrate concentration
-
C
SO mol/m3
initial substrate concentration
-
K
A mol/m3
constant which defines the affinity of a substrate to the enzyme
-
K
iS mol/m3
substrate inhibitory constant
-
K
iP mol/m3
PhAA inhibitory constant
-
K
iQ mol/m3
6-APA inhibitory constant
-
k
3 mol/g/min
constant rate of dissociation of the active complex
-
R(1)
concentrational component of reaction resistance
-
R(2)
resistance component derived from substrate affinity
-
R(3)
resistance component due to the inhibition of the enzyme by substrate
-
R(4)
resistance component due to the inhibition of the enzyme by PhAA
-
R(5)
resistance component due to inhibition of the enzyme by 6-APA
-
r = dCs/dt mol/m3 min
rate of reaction
-
t
min reaction time
- (i)
relative resistance of reaction 相似文献
20.
Double Asymmetric Induction During the Addition of (RP)‐Menthyl Phenyl Phosphine Oxide to Chiral Aldimines
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P,C‐Stereogenic α‐amino phosphine oxides were prepared from the addition of (RP)‐menthyl phenyl phosphine oxide to chiral aldimines under neat condition at 80 °C in up to 91:9 drC and 99% yields. The diastereoselectivity was mainly induced by chiral phosphorus that showed matched or mismatched induction with (S)‐ or (R)‐aldimines, respectively. Chirality 28:132–135, 2016. © 2015 Wiley Periodicals, Inc. 相似文献