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]     

Vinyl Sulfones as Antiparasitic Agents and a Structural Basis for Drug Design

Cysteine proteases of the papain superfamily are implicated in a number of cellular processes and are important virulence factors in the pathogenesis of parasitic disease. These enzymes have therefore emerged as promising targets for antiparasitic drugs. We report the crystal structures of three major parasite cysteine proteases, cruzain, falcipain-3, and the first reported structure of rhodesain, in complex with a class of potent, small molecule, cysteine protease inhibitors, the vinyl sulfones. These data, in conjunction with comparative inhibition kinetics, provide insight into the molecular mechanisms that drive cysteine protease inhibition by vinyl sulfones, the binding specificity of these important proteases and the potential of vinyl sulfones as antiparasitic drugs.Sleeping sickness (African trypanosomiasis), caused by Trypanosoma brucei, and malaria, caused by Plasmodium falciparum, are significant, parasitic diseases of sub-Saharan Africa (1). Chagas'' disease (South American trypanosomiasis), caused by Trypanosoma cruzi, affects approximately, 16–18 million people in South and Central America. For all three of these protozoan diseases, resistance and toxicity to current therapies makes treatment increasingly problematic, and thus the development of new drugs is an important priority (2–4).T. cruzi, T. brucei, and P. falciparum produce an array of potential target enzymes implicated in pathogenesis and host cell invasion, including a number of essential and closely related papain-family cysteine proteases (5, 6). Inhibitors of cruzain and rhodesain, major cathepsin L-like papain-family cysteine proteases of T. cruzi and T. brucei rhodesiense (7–10) display considerable antitrypanosomal activity (11, 12), and some classes have been shown to cure T. cruzi infection in mouse models (11, 13, 14).In P. falciparum, the papain-family cysteine proteases falcipain-2 (FP-2)⁶ and falcipain-3 (FP-3) are known to catalyze the proteolysis of host hemoglobin, a process that is essential for the development of erythrocytic parasites (15–17). Specific inhibitors, targeted to both enzymes, display antiplasmodial activity (18). However, although the abnormal phenotype of FP-2 knock-outs is “rescued” during later stages of trophozoite development (17), FP-3 has proved recalcitrant to gene knock-out (16) suggesting a critical function for this enzyme and underscoring its potential as a drug target.Sequence analyses and substrate profiling identify cruzain, rhodesain, and FP-3 as cathepsin L-like, and several studies describe classes of small molecule inhibitors that target multiple cathepsin L-like cysteine proteases, some with overlapping antiparasitic activity (19–22). Among these small molecules, vinyl sulfones have been shown to be effective inhibitors of a number of papain family-like cysteine proteases (19, 23–27). Vinyl sulfones have many desirable attributes, including selectivity for cysteine proteases over serine proteases, stable inactivation of the target enzyme, and relative inertness in the absence of the protease target active site (25). This class has also been shown to have desirable pharmacokinetic and safety profiles in rodents, dogs, and primates (28, 29). We have determined the crystal structures of cruzain, rhodesain, and FP-3 bound to vinyl sulfone inhibitors and performed inhibition kinetics for each enzyme. Our results highlight key areas of interaction between proteases and inhibitors. These results help validate the vinyl sulfones as a class of antiparasitic drugs and provide structural insights to facilitate the design or modification of other small molecule inhibitor scaffolds.     

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]     

Exploring the Role of Integral Membrane Proteins in ATP-Binding Cassette Transporters: Analysis of a Collection of MalG Insertion Mutants

The maltose transport complex of Escherichia coli is a well-studied example of an ATP-binding cassette transporter. The complex, containing one copy each of the integral membrane proteins MalG and MalF and two copies of the peripheral cytoplasmic membrane protein MalK, interacts with the periplasmic maltose-binding protein to efficiently translocate maltose and maltodextrins across the bacterial cytoplasmic membrane. To investigate the role of MalG both in MalFGK₂ assembly interactions and in subsequent transport interactions, we isolated and characterized 18 different MalG mutants, each containing a 31-residue insertion in the protein. Eight insertions mapping to distinct hydrophilic regions of MalG permitted either assembly or both assembly and transport interactions to occur. In particular, we isolated two insertions mapping to extracytoplasmic (periplasmic) regions of MalG which preserved both assembly and transport abilities, suggesting that these are permissive sites in the protein. Another periplasmic insertion seems to affect only transport-specific interactions between MalG and maltose-binding protein, defining a novel class of MalG mutants. Finally, four MalG mutant proteins, although stably expressed, are unable to assemble into the MalFGK₂ complex. These mutants contain insertions in only two different hydrophilic regions of MalG, consistent with the notion that a restricted number of domains in this protein are critical complex assembly determinants. These MalG mutants will allow us to further explore the intermolecular interactions of this model transporter.Integral membrane proteins play a central role in the ATP-binding cassette (ABC) transporter superfamily, whose prokaryotic and eukaryotic members traffic a variety of substrates such as ions, sugars, amino acids, peptides, and proteins (15). This large family of transporters is defined by a conserved cytoplasmic ATPase component and integral membrane domains which interact to carry out the specific transport process (4, 15). Among the eukaryotic members are such medically relevant proteins as the P-glycoprotein implicated in multidrug-resistant cancer cells, the cystic fibrosis transmembrane regulator protein, and the human peroxisomal adrenoleukodystrophy protein (2, 34, 35). Among the prokaryotic members of the ABC superfamily are the periplasmic binding protein-dependent transporters. These family members are characterized by a conserved region of the integral membrane component(s) in addition to the conserved cytoplasmic ATPase (4). One member of this prokaryotic subgroup, the maltose transport complex of Escherichia coli, presents a useful model for the integral membrane folding and assembly interactions required for ABC transporters. The maltose transport complex consists of the integral membrane proteins MalF and MalG and a peripheral cytoplasmic membrane ATPase, MalK (reviewed in reference 24). These three proteins copurify (11), forming a MalFGK₂ tetrameric complex which acts in concert with the periplasmic maltose-binding protein (MBP), the product of malE, to efficiently translocate maltose and maltodextrins across the bacterial cytoplasmic membrane.MalF has been shown to have eight transmembrane (TM) domains (5), whereas MalG possesses six TM domains (6, 10). Following independent insertion of these proteins into the membrane (22a, 31), assembly of the MalFGK₂ complex is likely mediated by interactions among discrete domains of MalF, MalG, and MalK, resulting in tetramerization (20, 26).Although the specifics of these interactions are unknown, a combination of biochemistry and genetics has allowed for a partial characterization of the complex. Shuman and colleagues isolated and characterized MalF and MalG mutants which enable the MalFGK₂ complex to transport maltose in the absence of MBP (7, 32). These analyses have pointed toward a direct interaction between MBP and periplasmic portions of MalG and MalF (16), between MalG and MalF themselves (7), and between MalK and both MalF and MalG (12). Davidson and Nikaido purified the MalFGK₂ complex and demonstrated extensive chemical cross-linking between MalG and MalF and among MalG, MalF, and MalK (11). Traxler and Beckwith observed that periplasmic loops of MalF become protease resistant only in the presence of MalG and MalK, also suggesting that specific interactions occur among the proteins in the context of an assembled complex (31). Finally, a potentially important MalG-MalK protein interaction signal has been identified in the hydrophilic cytoplasmic loop between the fourth and fifth TM domains of MalG (reference 9; Fig. ​Fig.1).1). This motif is conserved in MalF and in other binding protein-dependent transporters of the ABC superfamily (9, 28) and has been hypothesized to mediate interactions with the conserved ATPase subunit of the complex (17, 22). Open in a separate windowFIG. 1Topology model of MalG. Hydropathy plots and fusion protein analyses (6, 10) suggest that the N and C termini of the 296-residue protein are cytoplasmically localized. The shaded boxes represent putative TM domains, and the shaded amino acids are conserved in integral membrane proteins of periplasmic binding protein-dependent ABC transporters (9, 28). The location of each 31-residue insertion is shown by an arrowhead. The black arrowhead represents an insertion which did not significantly affect MalG transport function, the gray arrowhead depicts partial transport function, and the white arrowheads represent loss of transport ability for the corresponding insertion mutants. Each numbered disc shows the mutant classification of the adjacent insertion mutant (see Discussion for details).Recently, a transposon-mediated insertion mutagenesis technique was developed and used to characterize both permissive and nonpermissive regions of the integral membrane protein LacY (19), as well as the cytoplasmic MalK and LacI proteins (18, 23). These analyses not only identified tolerant hydrophilic regions of each protein but also defined several distinct mutant classes (18, 19, 23). In particular, the phenotypes attributable to the lacI insertion mutations that we isolated were strikingly similar to those of previously characterized amino acid substitutions mapping to the same sites in lacI. Here, we describe the results of this insertion mutagenesis on the MalG protein. This analysis provides a unique in vivo view of the requirements for proper MalG protein folding and of the interactions necessary for MalFGK₂ assembly and maltose transport.