A model-independent approach to infer hierarchical codon substitution dynamics

Background  

Codon substitution constitutes a fundamental process in molecular biology that has been studied extensively. However, prior studies rely on various assumptions, e.g. regarding the relevance of specific biochemical properties, or on conservation criteria for defining substitution groups. Ideally, one would instead like to analyze the substitution process in terms of raw dynamics, independently of underlying system specifics. In this paper we propose a method for doing this by identifying groups of codons and amino acids such that these groups imply closed dynamics. The approach relies on recently developed spectral and agglomerative techniques for identifying hierarchical organization in dynamical systems.     

Syntheses of amino nitrones. Potential intramolecular traps for radical intermediates in monoamine oxidase-catalyzed reactions

Monoamine oxidase (MAO) is a flavin-dependent enzyme that catalyzes the oxidative deamination of a variety of amine neurotransmitters and toxic amines. Although there have been several studies that support the intermediacy of an amine radical cation and an -radical during enzyme catalysis, there is no direct, i.e. EPR, evidence for these species as they are formed. Amino nitrones have been designed which, upon radical formation would produce an intermediate that is a resonance structure of the corresponding nitroxyl radical, which should be observable by EPR spectroscopy. Syntheses of seven different amino nitrones, three acyclic, and four cyclic analogues were attempted. The protected amino nitrones were stable, but all three of the acyclic amino nitrones were unstable. One of the cyclic analogues was very stable (39), one was stable only in organic solvents (40), one was stable only in aqueous medium below pH 6.5 (41), and the other (42) was stable for just a short time at room temperature, decomposing to a stable free radical. None of these analogues produced a MAO-catalyzed radical, yet 41 is a poor substrate (K_m = 0.2 mM; k_cat = 0.034 min⁻¹) and 39 is a mixed inhibitor (K_i = 26.5 mM). Although this approach does not appear to be applicable to amino nitrones, it should be a valuable approach for other enzymes where radical intermediates are suspected and nonamine nitrones can be utilized.     

Spectroscopic characterization of the photocycle intermediates of photoactive yellow protein.

The absorption spectra of photocycle intermediates of photoactive yellow protein mutants were compared with those of the corresponding intermediates of wild type to probe which amino acid residues interact with the chromophore in the intermediate states. B and H intermediates were produced by irradiation and trapped at 80 K, and L intermediates at 193 K. The absorption spectra of these intermediates produced from R52Q were identical to those from wild type, whereas those from E46Q and T50V were 7-15 nm red-shifted as those in the dark states. The absorption spectra of M intermediates were measured by flash photolysis at room temperature. Those of Y42F, T50V, and R52Q were identical to that of wild type, whereas that of E46Q was 11 nm red-shifted. Assuming that the intermediates of mutants have a structure comparable to that of wild type, these findings suggest the following: Glu46 interacts with the chromophore throughout the photocycle, interaction between the chromophore and Thr50 as well as Tyr42 is lost upon the formation of M intermediate, and Arg52 never interacts with the chromophore directly. The hydrogen-bonding network around the phenolic oxygen of the chromophore would be thus maintained until L intermediate decays, and the global conformational change would take place by the loss of the hydrogen bond between the chromophore and Tyr42. This model conflicts with some of the results of previous crystallographic studies, suggesting that the reaction mechanism in the crystal may be different from that in solution.     

Low-temperature photoreactions of halorhodopsin. 2. Description of the photocycle and its intermediates

Photostationary states of halorhodopsin (HR, a retinal protein in the halobacterial membrane) and their further thermal conversions were investigated at 140-230 K by absorption spectroscopy in the visible. The difference spectra confirm several steps of the all-trans-HR photocycle, in the presence of chloride, proposed earlier on the basis of room temperature flash spectroscopy. Thus, at 140 K, the spectra reveal the HR600----HR520 reaction, and at 170-230 K the HR640----HR578 and the HR520----HR578 reactions can be seen. No evidence for the expected HR520 in equilibrium HR640 process was found, however. From the difference spectra at various temperatures, exact absorption spectra of HR600 and HR520 were calculated, and an estimate of the HR640 spectrum in a mixture also containing HR520 was obtained. The low-temperature absorption maxima of HR578 and its photointermediates relate to the room temperature maxima differently from what is expected from the spectra of the corresponding intermediates in the bacteriorhodopsin photocycle.     

Analytical bayesian approach for assigning individuals to populations

We propose a general formulation of the Bayesian method for assigning individuals to a population among a predetermined set of reference populations using molecular marker information. Compared to previously published methods, ours allows us to consider different types of prior information about allele frequencies by using a Dirichlet prior probability distribution. It also makes it possible to assign a set of individuals assumed to belong to the same population with increased accuracy using their pooled genotype data. The efficiency of the method is illustrated by application to a group of closely related coconut populations. An interesting feature of the Bayesian procedure is the way it handles imprecise information. With a poor or even incomplete dataset, assignment is still be possible and gives valid results: poor data quality is reflected in an ambiguous result rather than in a false conclusion.     

Thermal equilibration between the M and N intermediates in the photocycle of bacteriorhodopsin.

The stages in the photocycle of bacteriorhodopsin (BR) involving the M and N intermediates are investigated using a double pulse excitation method. A first (cycling) pulse at 532 nm is followed, with an appropriate time delay, by a second pulse (337, 406, 446, or 470 nm) which induces the M-->BR back-photoreaction. After depletion by the second pulse a repopulation of M in the millisecond range is observed which is interpreted in terms of a thermal N-->M relaxation. It is thus concluded that a (thermal) M<-->N equilibrium accounts for the biphasic decay of M in the BR photocycle. Other models for this stage of the light-driven proton-pump are therefore unnecessary.     

A low temperature investigation of the intermediates of the photocycle of light-adapted bacteriorhodopsin. Optical absorption and fluorescence measurements.

Optical absorption and emission measurements have been made on samples of light-adapted purple membrane of Halobacterium halobium at temperatures ranging from 77 K to room temperature. As a result of these experiments a set of equations is given which described thermal and photochemical reactions interrelating various intermediates of the reaction cycle of the chromophore of light-adapted bacteriorhodopsin (BR). Further some specific problems connected to these intermediates have been investigated. Thus the room temperature emission spectrum of bacteriorhodopsin has been found to exhibit a Stokes shift of 3430 cm-1 only, if low excitation intensities are used. The recently detected intermiediate P-BR can be shown to convert thermally into bacteriorhodopsin following a first-order decay with the activation energy delta E = 2.4 +/- 0.2 kcal/mol. The thermal decay of K-BR consists of two exponentials if measured on purple membrane suspensions in a mixture of H2O and glycerol (1 : 1, v/v). A simple procedure is given for trapping the intermediate L-BR at 170 K in a very pure form. M-BR is shown to consist of two species, MI-BR and MII-BR. They are characterized by similar optical absorption spectra but different thermal stability. Further the oscillator strengths corresponding to the long wavelength absorption bands of the intermediates bacteriorhodopsin, K-, L, MI- and MII-BR have been calculated. They have been discussed with respect to the question which of the corresponding absorption spectra show the characteristics of isomerism of the chromophore or simply solvatochromism.     

The photocycle of the chloride pump halorhodopsin. I: Azide-catalyzed deprotonation of the chromophore is a side reaction of photocycle intermediates inactivating the pump

Halorhodopsin, the light-driven chloride pump of halobacteria, undergoes a photochemical cycle in the 10 ms range. Two intermediates, HR640 and HR520, accumulate in the photosteady state after short times (within 100 ms) of illumination. Upon prolonged illumination a third species, HRL410 accumulates, which is formed from HR520/HR640 by deprotonation of the chromophore in a side reaction of the photocycle. In the dark, HRL410 requires several minutes to reconvert thermally to HR478. Thus, molecules in the HRL410 state must be inactive pumps since their maximal turnover number could only be a few per hour. Inorganic bases, such as azide, catalyze the deprotonation of HR520/HR640 as well as the reprotonation of HRL410. Both reactions are accelerated several hundred times by azide but the photosteady-state concentration of HRL410 remains unchanged.     

Tyrosine and carboxyl protonation changes in the bacteriorhodopsin photocycle. 1. M412 and L550 intermediates

The role of tyrosines in the bacteriorhodopsin (bR) photocycle has been investigated by using Fourier transform infrared (FTIR) and UV difference spectroscopies. Tyrosine contributions to the BR570----M412 FTIR difference spectra recorded at several temperatures and pH's were identified by isotopically labelling tyrosine residues in bacteriorhodopsin. The frequencies and deuterium/hydrogen exchange sensitivities of these peaks and of peaks in spectra of model compounds in several environments suggest that at least two different tyrosine groups participate in the bR photocycle during the formation of M412. One group undergoes a tyrosinate----tyrosine conversion during the BR570----K630 transition. A second tyrosine group deprotonates between L550 and M412. Low-temperature UV difference spectra in the 220--350-nm region of both purple membrane suspensions and rehydrated films support these conclusions. The UV spectra also indicate perturbation(s) of one or more tryptophan group(s). Several carboxyl groups appear to undergo a series of protonation changes between BR570 and M412, as indicated by infrared absorption changes in the 1770--1720-cm-1 region. These results are consistent with the existence of a proton wire in bacteriorhodopsin that involves both tyrosine and carboxyl groups.     

A model-independent approach to the theory of experimental designs and a special discussion of bib. II. properties and construction of bib

In addition to the first part we prove independently of the model some theorems on properties of balanced block designs. Then we propose nine methods of construction of BIB and prove theorems in connection with these methods. In a table we give a survey on block designs with v$25 and possibilities of their construction with these methods. Further follow theorems with the number of nonisomorphic BIB for some fixed v and k.     

On-resin macrocyclization of peptides via intramolecular SnAr reactions.

On-resin macrocyclization via an SNAr reaction is employed in the synthesis of tocinoic acid analogs. Specifically, an N-terminal nitrofluorobenzene is attacked by a nucleophilic C-terminal sidechain. The remaining nitro group can be reduced and acylated. NMR is used to compare the conformation of the new macrocyclic peptides to tocinoic acid.     

The energetics of intramolecular reactions and enzyme catalysis.

The relative rates of reactions should always be examined by an awareness of differential effects. The magnitude and variation of the relative rates of intramolecular reactions can be rationalized by the differences in entropy and strain energy. The relative rates of enzyme-catalysed reactions are sometimes due to groundstate effects. The beta-lactamase-catalysed hydrolysis of beta-lactam antibiotics may require a unique disposition of catalytic groups owing to an unusual process of bond fission in the four membered ring.     

New photocycle intermediates in the photoactive yellow protein from Ectothiorhodospira halophila: picosecond transient absorption spectroscopy.

Previous studies have shown that the room temperature photocycle of the photoactive yellow protein (PYP) from Ectothiorhodospira halophila involves at least two intermediate species: I1, which forms in <10 ns and decays with a 200-micros lifetime to I2, which itself subsequently returns to the ground state with a 140-ms time constant at pH 7 (Genick et al. 1997. Biochemistry. 36:8-14). Picosecond transient absorption spectroscopy has been used here to reveal a photophysical relaxation process (stimulated emission) and photochemical intermediates in the PYP photocycle that have not been reported previously. The first new intermediate (I0) exhibits maximum absorption at approximately 510 nm and appears in </=3 ps after 452 nm excitation (5 ps pulse width) of PYP. Kinetic analysis shows that I0 decays with a 220 +/- 20 ps lifetime, forming another intermediate (Idouble dagger0) that has a similar difference wavelength maximum, but with lower absorptivity. Idouble dagger0 decays with a 3 +/- 0.15 ns time constant to form I1. Stimulated emission from an excited electronic state of PYP is observed both within the 4-6-ps cross-correlation times used in this work, and with a 16-ps delay for all probe wavelengths throughout the 426-525-nm region studied. These transient absorption and emission data provide a more detailed understanding of the mechanistic dynamics occurring during the PYP photocycle.     

Femtosecond spectroscopic observations of initial intermediates in the photocycle of the photoactive yellow protein from Ectothiorhodospira halophila.

Femtosecond time-resolved absorbance measurements were used to probe the subpicosecond primary events of the photoactive yellow protein (PYP), a 14-kD soluble photoreceptor from Ectothiorhodospira halophila. Previous picosecond absorption studies from our laboratory have revealed the presence of two new early photochemical intermediates in the PYP photocycle, I(0), which appears in 相似文献   

Kinetics of and intermediates in a photocycle branching reaction of the photoactive yellow protein from Ectothiorhodospira halophila.

We have studied the kinetics of the blue light-induced branching reaction in the photocycle of photoactive yellow protein (PYP) from Ectothiorhodospira halophila, by nanosecond time-resolved UV/Vis spectroscopy. As compared to the parallel dark recovery reaction of the presumed blue-shifted signaling state pB, the light-induced branching reaction showed a 1000-fold higher rate. In addition, a new intermediate was detected in this branching pathway, which, compared to pB, showed a larger extinction coefficient and a blue-shifted absorption maximum. This substantiates the conclusion that isomerization of the chromophore is the rate-controlling step in the thermal photocycle reactions of PYP and implies that absorption of a blue photon leads to cis-->trans isomerization of the 4-hydroxy-cinnamyl chromophore of PYP in its pB state.     

A mass spectrometry-based probe of equilibrium intermediates in protein-folding reactions

Described here is a mass spectrometry- and H/D exchange-based approach for the detection of equilibrium intermediate state(s) in protein-folding reactions. The approach utilizes the stability of unpurified proteins from rates of H/D exchange (SUPREX) technique to measure the m value (i.e., delta DeltaG/delta [denaturant] value) associated with the folding reaction of a protein. Such SUPREX m-value analyses can be made over a wide range of denaturant concentrations. Thus, the described approach is well-suited for the detection of high-energy intermediates that might be populated at low denaturant concentrations and hard to detect in conventional chemical denaturation experiments using spectroscopic probes. The approach is demonstrated on four known non-two-state folding proteins, including alpha-lactalbumin, cytochrome c, intestinal fatty acid binding protein (IFABP), and myoglobin. The non-two-state folding behavior of each model protein system was detected by the described method. The cytochrome c, myoglobin, and IFABP systems each had high-energy intermediate states that were undetected in conventional optical spectroscopy-based studies and previously required other more specialized biophysical approaches (e.g., nuclear magnetic resonance spectroscopy-based methods and protease protection assays) for their detection. The SUPREX-based approach outlined here offers an attractive alternative to these other approaches, because it has the advantage of speed and the ability to analyze both purified and unpurified protein samples in either concentrated or dilute solution.     

Fourier transform infrared double-flash experiments resolve bacteriorhodopsin''s M1 to M2 transition.

The orientation of the central proton-binding site, the protonated Schiff base, away from the proton release side to the proton uptake side is crucial for the directionality of the proton pump bacteriorhodopsin. It has been proposed that this movement, called the reprotonation switch, takes place in the M1 to M2 transition. To resolve the molecular events in this M1 to M2 transition, we performed double-flash experiments. In these experiments a first pulse initiates the photocycle and a second pulse selectively drives bR molecules in the M intermediate back into the BR ground state. For short delay times between initiating and resetting pulses, most of the M molecules being reset are in the M1 intermediate, and for longer delay times most of the reset M molecules are in the M2 intermediate. The BR-M1 and BR-M2 difference spectra are monitored with nanosecond step-scan Fourier transform infrared spectroscopy. Because the Schiff base reprotonation rate is kM1 = 0.8 x 10(7) s(-1) in the light-induced M1 back-reaction and kM2 = 0.36 x 10(7) s(-1) in the M2 back-reaction, the two different M intermediates represent two different proton accessibility configurations of the Schiff base. The results show only a minute movement of one or two peptide bonds in the M1 to M2 transition that changes the interaction of the Schiff base with Y185. This backbone movement is distinct from the larger one in the subsequent M to N transition. No evidence of a chromophore isomerization is seen in the M1 to M2 transition. Furthermore, the results show time-resolved reprotonation of the Schiff base from D85 in the M photo-back-reaction, instead of from D96, as in the conventional cycle.     

On the structure of flavin-oxygen intermediates involved in enzymatic reactions.

During the catalytic reactions of flavoprotein hydroxylases and bacterial luciferase, flavin peroxides are formed as intermediates [see Massey, V. and Hemmerich, P. (1976) in The Enzymes, 3rd edn (P. Boyer, ed.) pp. 421--505, Academic Press, New York]. These intermediates have been postulated to be C(4a) derivatives of the flavin coenzyme. To test this hypothesis, modified flavin coenzymes carrying an oxygen substituent at position C(4a) of the isoalloxazine ring were synthesized. They are tightly bound by the apoenzymes of D-amino acid oxidase, p-hydroxybenzoate hydroxylase and lactate oxidase; the resulting complexes show spectral properties closely similar to those of the transient oxygen adducts of the hydroxylases. The optical spectra of the lumiflavin model compounds were found to be highly dependent on the solvent environment and nature of the subsituents. Under appropriate conditions they simulate satisfactorily the spectra of the transient enzymatic oxygen adducts. The results support the proposal that the primary oxygen adducts formed with these flavoproteins on reaction of the reduced enzymes with oxygen are flavin C(4a) peroxides.     

A simplified approach to derive Cleland model for enzymatic reactions

Metabolic modeling can suggest which is the key enzyme activity that needs to be controlled or its activity enhanced for the required production of a metabolite in a pathway. It also helps to find possible drug targets (enzymes to be inhibited). In metabolic modeling, knowing the kinetics of the enzymes involved in a pathway is mandatory. Most enzymatic reactions involve multi-substrates and follow an ordered sequential or ping–pong mechanism. The kinetic parameters involved in the model are obtained by fitting experimental data using a model based on the mechanism. The Cleland model has been used for some years. The grouping of parameters, such as dissociation constant and Michaelis–Menten constant, makes the strategy meaningful and hence the Cleland model is still in use. Although other alternate methods, e.g., the King-Altman method, are available, derivation by determinants can be used to derive a rate expression for the sequential or ping–pong mechanism, they are tedious. Hence, a meaningful modification is suggested in this communication for deriving the enzyme mechanism which is based on Thilakavathi et al. (Biotech Lett 28:1889–1894, 2006) to obtain the Cleland model in an easier way.