80 research outputs found
In silico prediction of mutant HIV-1 proteases cleaving a target sequence
HIV-1 protease represents an appealing system for directed enzyme re-design,
since it has various different endogenous targets, a relatively simple
structure and it is well studied. Recently Chaudhury and Gray (Structure (2009)
17: 1636 -- 1648) published a computational algorithm to discern the
specificity determining residues of HIV-1 protease. In this paper we present
two computational tools aimed at re-designing HIV-1 protease, derived from the
algorithm of Chaudhuri and Gray. First, we present an energy-only based
methodology to discriminate cleavable and non cleavable peptides for HIV-1
proteases, both wild type and mutant. Secondly, we show an algorithm we
developed to predict mutant HIV-1 proteases capable of cleaving a new target
substrate peptide, different from the natural targets of HIV-1 protease. The
obtained in silico mutant enzymes were analyzed in terms of cleavability and
specificity towards the target peptide using the energy-only methodology. We
found two mutant proteases as best candidates for specificity and cleavability
towards the target sequence
Structure of the dimeric form of CTP synthase from Sulfolobus solfataricus
CTP synthase catalyzes the last committed step in de novo pyrimidine-nucleotide biosynthesis. Active CTP synthase is a tetrameric enzyme composed of a dimer of dimers. The tetramer is favoured in the presence of the substrate nucleotides ATP and UTP; when saturated with nucleotide, the tetramer completely dominates the oligomeric state of the enzyme. Furthermore, phosphorylation has been shown to regulate the oligomeric states of the enzymes from yeast and human. The crystal structure of a dimeric form of CTP synthase from Sulfolobus solfataricus has been determined at 2.5 Å resolution. A comparison of the dimeric interface with the intermolecular interfaces in the tetrameric structures of Thermus thermophilus CTP synthase and Escherichia coli CTP synthase shows that the dimeric interfaces are almost identical in the three systems. Residues that are involved in the tetramerization of S. solfataricus CTP synthase according to a structural alignment with the E. coli enzyme all have large thermal parameters in the dimeric form. Furthermore, they are seen to undergo substantial movement upon tetramerization
Characterization of different crystal forms of the α-glucosidase MalA from \u3ci\u3eSulfolobus solfataricus\u3c/i\u3e
MalA is an _-glucosidase from the hyperthermophilic archaeon Sulfolobus
solfataricus. It belongs to glycoside hydrolase family 31, which includes several
medically interesting α-glucosidases. MalA and its selenomethionine derivative
have been overproduced in Escherichia coli and crystallized in four different
crystal forms. Microseeding was essential for the formation of good-quality
crystals of forms 2 and 4. For three of the crystal forms (2, 3 and 4) full data sets
could be collected. The most suitable crystals for structure determination are the
monoclinic form 4 crystals, belonging to space group P21, from which data sets
extending to 2.5 Å resolution have been collected. Self-rotation functions
calculated for this form and for the orthorhombic (P212121) form 2 indicate the
presence of six molecules in the asymmetric unit related by 32 symmetry
Characterization of different crystal forms of the α-glucosidase MalA from \u3ci\u3eSulfolobus solfataricus\u3c/i\u3e
MalA is an _-glucosidase from the hyperthermophilic archaeon Sulfolobus
solfataricus. It belongs to glycoside hydrolase family 31, which includes several
medically interesting α-glucosidases. MalA and its selenomethionine derivative
have been overproduced in Escherichia coli and crystallized in four different
crystal forms. Microseeding was essential for the formation of good-quality
crystals of forms 2 and 4. For three of the crystal forms (2, 3 and 4) full data sets
could be collected. The most suitable crystals for structure determination are the
monoclinic form 4 crystals, belonging to space group P21, from which data sets
extending to 2.5 Å resolution have been collected. Self-rotation functions
calculated for this form and for the orthorhombic (P212121) form 2 indicate the
presence of six molecules in the asymmetric unit related by 32 symmetry
Computational redesign of thioredoxin is hypersensitive towards minor conformational changes in the backbone template
Despite the development of powerful computational tools, the full-sequence design of proteins still remains a challenging task. To investigate the limits and capabilities of computational tools, we conducted a study of the ability of the program Rosetta to predict sequences that recreate the authentic fold of thioredoxin. Focusing on the influence of conformational details in the template structures, we based our study on 8 experimentally determined template structures and generated 120 designs from each. For experimental evaluation, we chose six sequences from each of the eight templates by objective criteria. The 48 selected sequences were evaluated based on their progressive ability to (1) produce soluble protein in Escherichia coli and (2) yield stable monomeric protein, and (3) on the ability of the stable, soluble proteins to adopt the target fold. Of the 48 designs, we were able to synthesize 32, 20 of which resulted in soluble protein. Of these, only two were sufficiently stable to be purified. An X-ray crystal structure was solved for one of the designs, revealing a close resemblance to the target structure. We found a significant difference among the eight template structures to realize the above three criteria despite their high structural similarity. Thus, in order to improve the success rate of computational full-sequence design methods, we recommend that multiple template structures are used. Furthermore, this study shows that special care should be taken when optimizing the geometry of a structure prior to computational design when using a method that is based on rigid conformations
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