Splitting the BLOSUM Score into Numbers of Biological Significance

Mathematical tools developed in the context of Shannon information theory were used to analyze the meaning of the BLOSUM score, which was split into three components termed as the BLOSUM spectrum (or BLOSpectrum). These relate respectively to the sequence convergence (the stochastic similarity of the two protein sequences), to the background frequency divergence (typicality of the amino acid probability distribution in each sequence), and to the target frequency divergence (compliance of the amino acid variations between the two sequences to the protein model implicit in the BLOCKS database). This treatment sharpens the protein sequence comparison, providing a rationale for the biological significance of the obtained score, and helps to identify weakly related sequences. Moreover, the BLOSpectrum can guide the choice of the most appropriate scoring matrix, tailoring it to the evolutionary divergence associated with the two sequences, or indicate if a compositionally adjusted matrix could perform better.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Surf1, Associated with Leigh Syndrome in Humans, Is a Heme-binding Protein in Bacterial Oxidase Biogenesis

Biogenesis of mitochondrial cytochrome c oxidase (COX) relies on a large number of assembly factors, among them the transmembrane protein Surf1. The loss of human Surf1 function is associated with Leigh syndrome, a fatal neurodegenerative disorder caused by severe COX deficiency. In the bacterium Paracoccus denitrificans, two homologous proteins, Surf1c and Surf1q, were identified, which we characterize in the present study. When coexpressed in Escherichia coli together with enzymes for heme a synthesis, the bacterial Surf1 proteins bind heme a in vivo. Using redox difference spectroscopy and isothermal titration calorimetry, the binding of the heme cofactor to purified apo-Surf1c and apo-Surf1q is quantified: Each of the Paracoccus proteins binds heme a in a 1:1 stoichiometry and with K_d values in the submicromolar range. In addition, we identify a conserved histidine as a residue crucial for heme binding. Contrary to most earlier concepts, these data support a direct role of Surf1 in heme a cofactor insertion into COX subunit I by providing a protein-bound heme a pool.Leigh syndrome (LS)³ is an autosomal recessive inherited neurodegenerative disorder characterized by focal, bilateral lesions in one or more areas of the central nervous system (1). Symptoms start in early childhood, and the disease usually progresses rapidly. Although mutations in various mitochondrial enzymes can result in LS, its most frequent trigger is deficiency of cytochrome c oxidase (COX) caused by mutations in the SURF1 gene, as identified in LS patients (2, 3). Human SURF1, the first gene of the SURFEIT gene locus on chromosome 9, encodes a 30-kDa protein related to COX assembly (2, 3).Mitochondrial COX consists of up to 13 subunits (SU). The three core SU encoded by the mitochondrial genome carry all of the redox-active cofactors, two heme a moieties, and three copper ions. These three SU are highly conserved among different organisms and represent the main components of bacterial oxidase complexes as well (4, 5). The assembly process of mitochondrial COX is only marginally understood, involving the interplay of a large number of auxiliary proteins (6–9).Despite intensive efforts over more than a decade to unravel Surf1 function, its exact role in COX assembly still remains unclear. Surf1 is not strictly essential for COX assembly because patients with LS have residuals of assembled oxidase with remaining activity of approximately 10–20% in all tissues (2, 3). Located in the inner mitochondrial membrane, Surf1 is predicted to form two transmembrane helices connected by a long loop facing the intermembrane space (10, 11). Sequence alignments confirm the presence of Surf1 homologs in many eukaryotes and prokaryotes (12).One of the best studied Surf1 proteins is the yeast homolog Shy1p, which has been discovered and characterized in the context of pet mutants (10). Deletion of the gene leads to a strongly decreased COX level, although the residual enzyme appears fully functional. This points to a role of Shy1p in assembly or stabilization of COX (13), most likely during the formation of an early assembly intermediate consisting of the highly conserved core SU I and II (14).So far, only three bacterial homologs have been inspected in closer detail (15, 16). In Paracoccus denitrificans, two Surf1 homologs were identified and named Surf1c and Surf1q for their specific role in serving a heme aa₃-type COX and a related heme ba₃-type quinol oxidase, respectively (15). With the function of Surf1 in COX assembly still being speculative, a role in heme a insertion into COX SU I seemed conceivable (15, 16).Here we show that P. denitrificans Surf1c and Surf1q are able to bind heme a both in vivo and in vitro. This novel finding suggests that Surf1 proteins promote heme a insertion into SU I of either cytochrome c oxidase or quinol oxidase. In addition, Surf1 may modulate heme a synthase activity and provide a heme a cofactor pool in a safe, chelated form for COX SU I biogenesis.     

In Vivo Mutational Analysis of YtvA from Bacillus subtilis: MECHANISM OF LIGHT ACTIVATION OF THE GENERAL STRESS RESPONSE

The general stress response of Bacillus subtilis can be activated by stimuli such as the addition of salt or ethanol and with blue light. In the latter response, YtvA activates σ^B through a cascade of Rsb proteins, organized in stressosomes. YtvA is composed of an N-terminal LOV (light, oxygen, and voltage) domain and a C-terminal STAS (sulfate transporter and anti-sigma factor) domain and shows light-modulated GTP binding in vitro. Here, we examine the mechanism of YtvA-mediated activation of σ^B in vivo with site-directed mutagenesis. Constitutive off and constitutive on mutations have been identified. Disruption of GTP binding in the STAS domain eliminates light activation of σ^B. In contrast, modification of sites relevant for phosphorylation of STAS domains does not affect the stress response significantly. The data obtained are integrated into a model for the structure of full-length YtvA, which presumably functions as a dimer.LOV² domains (1), members of the superfamily of PAS domains (2, 3), are abundant in all domains of life and were first identified in plant phototropins (4). These photoreceptors regulate stomatal opening, phototropism, etc. and contain two N-terminal LOV domains that confer light regulation on the C-terminal Ser/Thr kinase domain (4). They also occur in bacteria, in which YtvA from Bacillus subtilis has been best characterized (for a review, see e.g. Ref. 5). Its N-terminal LOV domain binds FMN and shows the typical LOV photochemistry (6, 7): covalent adduct formation between a cysteine and the FMN chromophore. A linker helix, denoted Jα (7), connects the LOV domain to a STAS domain. The latter domain is present in many regulators of the general stress response of B. subtilis (8, 9). Stress via the addition of salt or ethanol (for a review, see Ref. 10) and blue light (11, 12) activates the general stress response via the environmental pathway, which integrates various signals via a large multiprotein complex, called the stressosome (13, 14). YtvA, which mediates light activation of σ^B (11, 12, 15), co-purifies with other STAS domain proteins in the stressosomes (16).When cells are stressed, STAS domains of several stressosome proteins (e.g. RsbS and RsbR) are phosphorylated by another intrinsic stressosome component, the serine/threonine kinase RsbT (9, 14, 17, 18). Next, RsbT is released from the complex to trigger RsbU, a protein phosphatase, thus (indirectly) activating σ^B (19). Phosphorylation of YtvA, however, has never been detected. Rather, it has been demonstrated in vitro that YtvA shows light-dependent GTP binding, presumably at its NTP-binding site in the STAS domain (20).Little is known about the mechanism of signal transmission in and by YtvA, except that in the C62A mutant, photochemistry in vitro (12) and light activation of σ^B in vivo (12, 15) are abolished. More detailed information is available for LOV domains of phototropins. A conserved glutamine, which is in hydrogen-bonding contact with the isoalloxazine ring of FMN, rotates its side chain by 180° upon covalent adduct formation (21). Replacement of this residue by leucine in the LOV2 domain of Phy3 from Adiantum results in a considerable reduction of the light-induced structural change (22). The corresponding mutation in phototropin 1 from Arabidopsis impairs autophosphorylation activity (23). The signal generated in the LOV2 domain is transmitted to the downstream kinase domain of phototropin 1 of Avena sativa through disruption of the interaction between its central β-sheet and the C-terminal linker region, the Jα-helix (24).Here, we study the mechanism of activation of YtvA in vivo, i.e. light-induced activation of the σ^B response, with site-directed mutagenesis. We focus on three regions of the protein, the flavin-binding pocket, the β-sheet of the LOV domain, and the GTP-binding site, and on potential phosphorylation sites of the STAS domain. We demonstrate that light-activated GTP binding is crucial for functional YtvA. A computational approach was used to model the structure of full-length YtvA. The model suggests that light modulates accessibility of the GTP-binding site of the STAS domain of YtvA.     

Algorithms for Finding Small Attractors in Boolean Networks

A Boolean network is a model used to study the interactions between different genes in genetic regulatory networks. In this paper, we present several algorithms using gene ordering and feedback vertex sets to identify singleton attractors and small attractors in Boolean networks. We analyze the average case time complexities of some of the proposed algorithms. For instance, it is shown that the outdegree-based ordering algorithm for finding singleton attractors works in time for , which is much faster than the naive time algorithm, where is the number of genes and is the maximum indegree. We performed extensive computational experiments on these algorithms, which resulted in good agreement with theoretical results. In contrast, we give a simple and complete proof for showing that finding an attractor with the shortest period is NP-hard.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]