The contours of evolution: In defence of Darwin's tree of life paradigm.

Both the concept of a Darwinian tree of life (TOL) and the possibility of its accurate reconstruction have been much criticized. Criticisms mostly revolve around the extensive occurrence of lateral gene transfer (LGT), instances of uptake of complete organisms to become organelles (with the associated subsequent gene transfer to the nucleus), as well as the implications of more subtle aspects of the biological species concept. Here we argue that none of these criticisms are sufficient to abandon the valuable TOL concept and the biological realities it captures. Especially important is the need to conceptually distinguish between organismal trees and gene trees, which necessitates incorporating insights of widely occurring LGT into modern evolutionary theory. We demonstrate that all criticisms, while based on important new findings, do not invalidate the TOL. After considering the implications of these new insights, we find that the contours of evolution are best represented by a TOL.

On distinguishing between canonical tRNA genes and tRNA gene fragments in prokaryotes.

Automated genome annotation is essential for extracting biological information from sequence data. The identification and annotation of tRNA genes is frequently performed by the software package tRNAscan-SE, the output of which is listed for selected genomes in the Genomic tRNA database (GtRNAdb). Here, we highlight a pervasive error in prokaryotic tRNA gene sets on GtRNAdb: the mis-categorization of partial, non-canonical tRNA genes as standard, canonical tRNA genes. Firstly, we demonstrate the issue using the tRNA gene sets of 20 organisms from the archaeal taxon Thermococcaceae. According to GtRNAdb, these organisms collectively deviate from the expected set of tRNA genes in 15 instances, including the listing of eleven putative canonical tRNA genes. However, after detailed manual annotation, only one of these eleven remains; the others are either partial, non-canonical tRNA genes resulting from the integration of genetic elements or CRISPR-Cas activity (seven instances), or attributable to ambiguities in input sequences (three instances). Secondly, we show that similar examples of the mis-categorization of predicted tRNA sequences occur throughout the prokaryotic sections of GtRNAdb. While both canonical and non-canonical prokaryotic tRNA gene sequences identified by tRNAscan-SE are biologically interesting, the challenge of reliably distinguishing between them remains. We recommend employing a combination of (i) screening input sequences for the genetic elements typically associated with non-canonical tRNA genes, and ambiguities, (ii) activating the tRNAscan-SE automated pseudogene detection function, and (iii) scrutinizing predicted tRNA genes with low isotype scores. These measures greatly reduce manual annotation efforts, and lead to improved prokaryotic tRNA gene set predictions.

Not All Photoactive Yellow Proteins Are Built Alike: Surprises and Insights into Chromophore Photoisomerization, Protonation, and Thermal Reisomerization of the Photoactive Yellow Protein Isolated from Salinibacter ruber.

We demonstrate that the Halorhodospira halophila (Hhal) photoactive yellow protein (PYP) is not representative of the greater PYP family. The photodynamics of the PYP isolated from Salinibacter ruber (Srub) is characterized with a comprehensive range of spectroscopic techniques including ultrafast transient absorption, photostationary light titrations, Fourier transform infrared, and cryokinetics spectroscopies. We demonstrate that the dark-adapted pG state consists of two subpopulations differing in the protonation state of the chromophore and that both are photoactive, with the protonated species undergoing excited-state proton transfer. However, the primary I0 photoproduct observed in the Hhal PYP photocycle is absent in the Srub PYP photodynamics, which indicates that this intermediate, while important in Hhal photodynamics, is not a critical intermediate in initiating all PYP photocycles. The excited-state lifetime of Srub PYP is the longest of any PYP resolved to date (∼30 ps), which we ascribe to the more constrained chromophore binding pocket of Srub PYP and the absence of the critical Arg52 residue found in Hhal PYP. The final stage of the Srub PYP photocycle involves the slowest known thermal dark reversion of a PYP (∼40 min vs 350 ms in Hhal PYP). This property allowed the characterization of a pH-dependent equilibrium between the light-adapted pB state with a protonated cis chromophore and a newly resolved pG' intermediate with a deprotonated cis chromophore and pG-like protein conformation. This result demonstates that protein conformational changes and chromophore deprotonation precede chromophore reisomerization during the thermal recovery of the PYP photocycle.

Spectroscopic ruler for measuring active-site distortions based on Raman optical activity of a hydrogen out-of-plane vibration.

Photoactive yellow protein (PYP), from the phototrophic bacterium Halorhodospira halophila, is a small water-soluble photoreceptor protein and contains p-coumaric acid (pCA) as a chromophore. PYP has been an attractive model for studying the physical chemistry of protein active sites. Here, we explore how Raman optical activity (ROA) can be used to extract quantitative information on distortions of the pCA chromophore at the active site in PYP. We use 13C8-pCA to assign an intense signal at 826 cm-1 in the ROA spectrum of PYP to a hydrogen out-of-plane vibration of the ethylenic moiety of the chromophore. Quantum-chemical calculations based on density functional theory demonstrate that the sign of this ROA band reports the direction of the distortion in the dihedral angle about the ethylenic C=C bond, while its amplitude is proportional to the dihedral angle. These results document the ability of ROA to quantify structural deformations of a cofactor molecule embedded in a protein moiety.

Detection of Incorporation of p-Coumaric Acid into Photoactive Yellow Protein Variants in Vivo.

We report the design and characterization of photoactive yellow protein (PYP)-blue fluorescent protein (mTagBFP) fusion constructs that permit the direct assay of reconstitution and function of the PYP domain. These constructs allow for in vivo testing of co-expression systems for enzymatic production of the p-coumaric acid-based PYP chromophore, via the action of tyrosine ammonia lyase and p-coumaroyl-CoA ligase (pCL or 4CL). We find that different 4CL enzymes can function to reconstitute PYP, including 4CL from Arabidopsis thaliana that can produce ∼100% holo-PYP protein under optimal conditions. mTagBFP fusion constructs additionally enable rapid analysis of effects of mutations on PYP photocycles. We use this mTagBFP fusion strategy to demonstrate in vivo reconstitution of several PYP-based optogenetic tools in Escherichia coli via a biosynthesized chromophore, an important step for the use of these optogenetic tools in vivo in diverse hosts.

Excitation-Wavelength-Dependent Photocycle Initiation Dynamics Resolve Heterogeneity in the Photoactive Yellow Protein from Halorhodospira halophila.

Photoactive yellow proteins (PYPs) make up a diverse class of blue-light-absorbing bacterial photoreceptors. Electronic excitation of the p-coumaric acid chromophore covalently bound within PYP results in triphasic quenching kinetics; however, the molecular basis of this behavior remains unresolved. Here we explore this question by examining the excitation-wavelength dependence of the photodynamics of the PYP from Halorhodospira halophila via a combined experimental and computational approach. The fluorescence quantum yield, steady-state fluorescence emission maximum, and cryotrapping spectra are demonstrated to depend on excitation wavelength. We also compare the femtosecond photodynamics in PYP at two excitation wavelengths (435 and 475 nm) with a dual-excitation-wavelength-interleaved pump-probe technique. Multicompartment global analysis of these data demonstrates that the excited-state photochemistry of PYP depends subtly, but convincingly, on excitation wavelength with similar kinetics with distinctly different spectral features, including a shifted ground-state beach and altered stimulated emission oscillator strengths and peak positions. Three models involving multiple excited states, vibrationally enhanced barrier crossing, and inhomogeneity are proposed to interpret the observed excitation-wavelength dependence of the data. Conformational heterogeneity was identified as the most probable model, which was supported with molecular mechanics simulations that identified two levels of inhomogeneity involving the orientation of the R52 residue and different hydrogen bonding networks with the p-coumaric acid chromophore. Quantum calculations were used to confirm that these inhomogeneities track to altered spectral properties consistent with the experimental results.

Bifurcation in the Ultrafast Dynamics of the Photoactive Yellow Proteins from Leptospira biflexa and Halorhodospira halophila.

We explored the photoisomerization mechanisms in novel homologues of photoactive yellow protein (PYP) from Leptospira biflexa (Lbif) to identify conserved features and functional diversity in the primary photochemistry of this family of photoreceptors. In close agreement with the prototypical PYP from Halorhodospira halophila (Hhal), we observe excited-state absorbance near 375 nm and stimulated emission near 500 nm, with triphasic excited-state decay. While the excited-state decay for Lbif PYP is the slowest among those of known PYPs due to the redistribution of the amplitudes of the three decay components, the quantum yield for productive photocycle entry is very similar to that of Hhal PYP. Pro68 is highly conserved in PYPs and is important for the high photochemical quantum yield in Hhal PYP, but this residue is Ile in wild-type Lbif PYP. The level of photoproduct formation is slightly increased in I68P Lbif PYP, indicating that this residue regulates the photochemical quantum yield in the entire PYP family. Lbif PYP also exhibited a blue-shifted photoproduct previously undiscovered in ultrafast studies of PYP, which we have named pUV. We posit that pUV is a detour in the PYP photocycle with a twisted protonated pCAH configuration. Cryokinetic experiments with Hhal PYP confirmed the presence of pUV, but the population of this state in room-temperature ultrafast experiments is very small. These results resolve the long-standing inconsistency in the literature regarding the existence of a bifurcation in the room-temperature photocycle of PYP.

Experimental Detection of the Intrinsic Difference in Raman Optical Activity of a Photoreceptor Protein under Preresonance and Resonance Conditions.

Raman optical activity (ROA) is an advanced technique capable of detecting structural deformations of light-absorbing molecules embedded in chromophoric proteins. Resonance Raman (RR) spectroscopy is widely used to enhance the band intensities. However, theoretical work has predicted that under resonance conditions the ROA spectrum resembles the shape of the RR spectrum. Herein, we use photoactive yellow protein (PYP) to measure the first experimental data on the effect of changing the excitation wavelength on the ROA spectra of a protein. We observe a close similarity between the shape of the RR spectrum and the resonance ROA spectrum of PYP. Furthermore, we experimentally verify the theoretical prediction concerning the ratio of the amplitudes of the ROA and Raman spectra. Our data demonstrate that selecting an appropriate excitation wavelength is a key factor for extracting structural information on a protein active site using ROA spectroscopy.

An extremely halophilic proteobacterium combines a highly acidic proteome with a low cytoplasmic potassium content.

Halophilic archaea accumulate molar concentrations of KCl in their cytoplasm as an osmoprotectant and have evolved highly acidic proteomes that function only at high salinity. We examined osmoprotection in the photosynthetic Proteobacteria Halorhodospira halophila and Halorhodospira halochloris. Genome sequencing and isoelectric focusing gel electrophoresis showed that the proteome of H. halophila is acidic. In line with this finding, H. halophila accumulated molar concentrations of KCl when grown in high salt medium as detected by x-ray microanalysis and plasma emission spectrometry. This result extends the taxonomic range of organisms using KCl as a main osmoprotectant to the Proteobacteria. The closely related organism H. halochloris does not exhibit an acidic proteome, matching its inability to accumulate K(+). This observation indicates recent evolutionary changes in the osmoprotection strategy of these organisms. Upon growth of H. halophila in low salt medium, its cytoplasmic K(+) content matches that of Escherichia coli, revealing an acidic proteome that can function in the absence of high cytoplasmic salt concentrations. These findings necessitate a reassessment of two central aspects of theories for understanding extreme halophiles. First, we conclude that proteome acidity is not driven by stabilizing interactions between K(+) ions and acidic side chains but by the need for maintaining sufficient solvation and hydration of the protein surface at high salinity through strongly hydrated carboxylates. Second, we propose that obligate protein halophilicity is a non-adaptive property resulting from genetic drift in which constructive neutral evolution progressively incorporates weakly stabilizing K(+)-binding sites on an increasingly acidic protein surface.

Strong ionic hydrogen bonding causes a spectral isotope effect in photoactive yellow protein.

Standard hydrogen bonds are of great importance for protein structure and function. Ionic hydrogen bonds often are significantly stronger than standard hydrogen bonds and exhibit unique properties, but their role in proteins is not well understood. We report that hydrogen/deuterium exchange causes a redshift in the visible absorbance spectrum of photoactive yellow protein (PYP). We expand the range of interpretable isotope effects by assigning this spectral isotope effect (SIE) to a functionally important hydrogen bond at the active site of PYP. The inverted sign and extent of this SIE is explained by the ionic nature and strength of this hydrogen bond. These results show the relevance of ionic hydrogen bonding for protein active sites, and reveal that the inverted SIE is a novel, to our knowledge, tool to probe ionic hydrogen bonds. Our results support a classification of hydrogen bonds that distinguishes the properties of ionic hydrogen bonds from those of both standard and low barrier hydrogen bonds, and show how this classification helps resolve a recent debate regarding active site hydrogen bonding in PYP.

Total chemical synthesis of fully functional Photoactive Yellow Protein.

The Photoactive Yellow Protein (PYP) is a structural prototype for the PAS superfamily of proteins, which includes hundreds of receptor and regulatory proteins from all three kingdoms of life. PYP itself is a small globular protein that undergoes a photocycle involving a series of conformational changes in response to light excitation of its p-coumaric acid chromophore, making it an excellent model system to study the molecular basis of signaling in the PAS super family. To enable novel chemical approaches to elucidating the structural changes that accompany signaling in PYP, we have chemically synthesized the 125 amino acid residue protein molecule using a combination of Boc chemistry solid phase peptide synthesis and native chemical ligation. Synthetic PYP exhibits the wildtype photocycle, as determined in photobleaching studies. Planned future studies include incorporation of site-specific isotopic labels into specific secondary structural elements to determine which structural elements are involved in signaling state formation using difference FTIR spectroscopy.

Robustness and evolvability in the functional anatomy of a PER-ARNT-SIM (PAS) domain.

The robustness of proteins against point mutations implies that only a small subset of residues determines functional properties. We test this prediction using photoactive yellow protein (PYP), a 125-residue prototype of the PER-ARNT-SIM (PAS) domain superfamily of signaling proteins. PAS domains are defined by a small number of conserved residues of unknown function. We report high-throughput biophysical measurements on a complete Ala scan set of purified PYP mutants. The dataset of 1,193 values on active site properties, functional kinetics, stability, and production level reveals that 124 mutants retain the characteristic photocycle of PYP, but that the majority of substitutions significantly alter functional properties. Only 35% of substitutions that strongly affect function are located at the active site. Unexpectedly, most PAS-conserved residues are required for maintaining protein production. PAS domain activation often involves conformational changes in α-helices linked to the PAS core. However, the mechanism of transmission and kinetic regulation of allosteric structural changes from the PAS domain to these helices is not clear. The Ala scan data reveal interactions governing allosteric switching in PYP. The photocycle kinetics is significantly altered by substitutions at 58 positions and spans a 3,000-fold range. Nine residues that dock the N-terminal α-helices of PYP to its PAS core regulate signaling kinetics. Ile39 and Asn43 are identified as part of a mechanism for regulating allosteric switching that is conserved among PAS domains. These results show that PYP combines robustness with a high degree of evolvability and imply production level as an important factor in protein evolution.

Spectral tuning in photoactive yellow protein by modulation of the shape of the excited state energy surface.

Protein-chromophore interactions in photoreceptors often shift the chromophore absorbance maximum to a biologically relevant spectral region. A fundamental question regarding such spectral tuning effects is how the electronic ground state S(0) and excited state S(1) are modified by the protein. It is widely assumed that changes in energy gap between S(0) and S(1) are the main factor in biological spectral tuning. We report a generally applicable approach to determine if a specific residue modulates the energy gap, or if it alters the equilibrium nuclear geometry or width of the energy surfaces. This approach uses the effects that changes in these three parameters have on the absorbance and fluorescence emission spectra of mutants. We apply this strategy to a set of mutants of photoactive yellow protein (PYP) containing all 20 side chains at active site residue 46. While the mutants exhibit significant variation in both the position and width of their absorbance spectra, the fluorescence emission spectra are largely unchanged. This provides strong evidence against a major role for changes in energy gap in the spectral tuning of these mutants and reveals a change in the width of the S(1) energy surface. We determined the excited state lifetime of selected mutants and the observed correlation between the fluorescence quantum yield and lifetime shows that the fluorescence spectra are representative of the energy surfaces of the mutants. These results reveal that residue 46 tunes the absorbance spectrum of PYP largely by modulating the width of the S(1) energy surface.

Renewing Linnaean taxonomy: a proposal to restructure the highest levels of the Natural System.

During the last century enormous progress has been made in the understanding of biological diversity, involving a dramatic shift from macroscopic to microscopic organisms. The question now arises as to whether the Natural System introduced by Carl Linnaeus, which has served as the central system for organizing biological diversity, can accommodate the great expansion of diversity that has been discovered. Important discoveries regarding biological diversity have not been fully integrated into a formal, coherent taxonomic system. In addition, because of taxonomic challenges and conflicts, various proposals have been made to abandon key aspects of the Linnaean system. We review the current status of taxonomy of the living world, focussing on groups at the taxonomic level of phylum and above. We summarize the main arguments against and in favour of abandoning aspects of the Linnaean system. Based on these considerations, we conclude that retaining the Linnaean Natural System provides important advantages. We propose a relatively small number of amendments for extending this system, particularly to include the named rank of world (Latin alternative mundis) formally to include non-cellular entities (viruses), and the named rank of empire (Latin alternative imperium) to accommodate the depth of diversity in (unicellular) eukaryotes that has been uncovered. We argue that in the case of both the eukaryotic domain and the viruses the cladistic approach intrinsically fails. However, the resulting semi-cladistic system provides a productive way forward that can help resolve taxonomic challenges. The amendments proposed allow us to: (i) retain named taxonomic levels and the three-domain system, (ii) improve understanding of the main eukaryotic lineages, and (iii) incorporate viruses into the Natural System. Of note, the proposal described herein is intended to serve as the starting point for a broad scientific discussion regarding the modernization of the Linnaean system.

Spectroscopic Validation of Crystallographic Structures of a Protein Active Site by Chiroptical Spectroscopy.

Out-of-plane distortions of a cofactor molecule in a protein active site are functionally important, and in photoreceptors, it has been proposed that they are crucial for spectral tuning and energy storage in photocycle intermediates. However, these subtle structural features are often beyond the grasp of structural biology. This issue is strikingly exemplified by photoactive yellow protein: its 14 independently determined crystal structures exhibit considerable differences in the dihedral angles defining the chromophore geometry, even though most of these are at excellent resolution. Here we developed a strategy to verify cofactor distortions in crystal structures by using quantum chemical calculations and chiroptical spectroscopy, particularly Raman optical activity and electronic circular dichroism spectroscopies. Based on this approach, we identify seven crystal structures with the chromophore geometries inconsistent with the experimentally observed data. The strategy implemented here promises to be widely applicable to uncovering cofactor distortions at active sites and to studies of reaction intermediates.

Side-chain specific isotopic labeling of proteins for infrared structural biology: the case of ring-D4-tyrosine isotope labeling of photoactive yellow protein.

An important bottleneck in the use of infrared spectroscopy as a powerful tool for obtaining detailed information on protein structure is the assignment of vibrational modes to specific amino acid residues. Side-chain specific isotopic labeling is a general approach towards obtaining such assignments. We report a method for high yield isotope editing of the bacterial blue light sensor photoactive yellow protein (PYP) containing ring-D(4)-Tyr. PYP was heterologously overproduced in Escherichia coli in minimal media containing ring-D(4)-Tyr in the presence of glyphosate, which inhibits endogenous biosynthesis of aromatic amino acids (Phe, Trp, and Tyr). Mass spectrometry of the intact protein and of tryptic peptides unambiguously demonstrated highly specific labeling of all five Tyr residues in PYP with 98% incorporation and undetectable isotopic scrambling. FTIR spectroscopy of the protein reveals a characteristic Tyr ring vibrational mode at 1515 cm(-1) that is shifted to 1436 cm(-1), consistent with that from ab initio calculations. PYP is a model system for protein structural dynamics and for receptor activation in biological signaling. The results described here open the way to the analysis of PYP using isotope-edited FTIR spectroscopy with side-chain specific labeling.

Modulating native-like residual structure in the fully denatured state of photoactive yellow protein affects its refolding.

Residual structure in the fully unfolded state is a key element for understanding protein folding. We show that the residual structure in fully denatured photoactive yellow protein (PYP) is affected by isomerization of its p-coumaric acid (pCA) chromophore. The exposure of total surface area and hydrophobic surface area upon unfolding was quantified by denaturant m values and heat capacity changes (DeltaC(p)), respectively. The exposure of the buried surface area upon the unfolding of the acid-denatured state of PYP containing trans-pCA is approximately 20% smaller than that of the native state. In contrast, for the partially unfolded pB photocycle intermediate containing cis-pCA, unfolding-induced exposure of the surface area is not decreased. These results show that pCA photoisomerization reduces residual structure in the fully unfolded state. Thus, residual structure in the fully unfolded state of PYP is under direct experimental control by photoexcitation. The sensitivity of the unfolded state to pCA isomerization provides a novel criterion that residual structure in the unfolded state of PYP is native-like, involving native-like protein-chromophore interactions. A largely untested prediction is that native-like residual structure facilitates the conformational search during folding. In the case of PYP, refolding from the less disordered fully unfolded state containing trans-pCA indeed is substantially accelerated. The burial of hydrophobic surface area in the fully unfolded state suggests that a significant part of the hydrophobic collapse process already has occurred in the denatured state.

Unassigned codons, nonsense suppression, and anticodon modifications in the evolution of the genetic code.

The origin of the genetic code is a central open problem regarding the early evolution of life. Here, we consider two undeveloped but important aspects of possible scenarios for the evolutionary pathway of the translation machinery: the role of unassigned codons in early stages of the code and the incorporation of tRNA anticodon modifications. As the first codons started to encode amino acids, the translation machinery likely was faced with a large number of unassigned codons. Current molecular scenarios for the evolution of the code usually assume the very rapid assignment of all codons before all 20 amino acids became encoded. We show that the phenomenon of nonsense suppression as observed in current organisms allows for a scenario in which many unassigned codons persisted throughout most of the evolutionary development of the code. In addition, we demonstrate that incorporation of anticodon modifications at a late stage is feasible. The wobble rules allow a set of 20 tRNAs fully lacking anticodon modifications to encode all 20 canonical amino acids. These observations have implications for the biochemical plausibility of early stages in the evolution of the genetic code predating tRNA anticodon modifications and allow for effective translation by a relatively small and simple early tRNA set.

Health Functionalities of Betaine in Patients With Homocystinuria.

Homocystinuria is a medical condition that can have widespread and harmful effects on multiple organ systems within the body. This disease is caused by a deficiency in one of the enzymes involved in the methionine metabolism pathway. One example would be a deficiency in cystathionine-ß-synthase (CBS), which is seen in classical homocystinuria. A deficiency in CBS can lead to elevated levels of homocysteine (HCY) and possible depletion of methionine and/or cysteine. There are several different treatment options for patients with this condition, one of which is the administration of the drug betaine. Here we review the use of betaine to decrease these elevated levels of homocysteine back to within normal ranges. Published literature indicates that the use of this choline derivative is most beneficial to patients who are either not compliant with the recommended low methionine and low protein diet or wish to consume a less restricted diet.

A conserved helical capping hydrogen bond in PAS domains controls signaling kinetics in the superfamily prototype photoactive yellow protein.

PAS domains form a divergent protein superfamily with more than 20 000 members that perform a wide array of sensing and regulatory functions in all three domains of life. Only nine residues are well-conserved in PAS domains, with an Asn residue at the start of α-helix 3 showing the strongest conservation. The molecular functions of these nine conserved residues are unknown. We use static and time-resolved visible and FTIR spectroscopy to investigate receptor activation in the photosensor photoactive yellow protein (PYP), a PAS domain prototype. The N43A and N43S mutants allow an investigation of the role of side-chain hydrogen bonding at this conserved position. The mutants exhibit a blue-shifted visible absorbance maximum and up-shifted chromophore pK(a). Disruption of the hydrogen bonds in N43A PYP causes both a reduction in protein stability and a 3400-fold increase in the lifetime of the signaling state of this photoreceptor. A significant part of this increase in lifetime can be attributed to the helical capping interaction of Asn43. This extends the known importance of helical capping for protein structure to regulating functional protein kinetics. A model for PYP activation has been proposed in which side-chain hydrogen bonding of Asn43 is critical for relaying light-induced conformational changes. However, FTIR spectroscopy shows that both Asn43 mutants retain full allosteric transmission of structural changes. Analysis of 30 available high-resolution structures of PAS domains reveals that the side-chain hydrogen bonding of residue 43 but not residue identity is highly conserved and suggests that its helical cap affects signaling kinetics in other PAS domains.