3DM: Systematic analysis of heterogeneous superfamily data to discover protein functionalities

Abstract

Ten years of experience with molecular class-specific information systems (MCSIS) such as with the hand-curated G protein-coupled receptor database (GPCRDB) or the semiautomatically generated nuclear receptor database has made clear that a wide variety of questions can be answered when protein-related data from many different origins can be flexibly combined. MCSISes revolve around a multiple sequence alignment (MSA) that includes "all" available sequences from the entire superfamily, and it has been shown at many occasions that the quality of these alignments is the most crucial aspect of the MCSIS approach. We describe here a system called 3DM that can automatically build an entire MCSIS. 3DM bases the MSA on a multiple structure alignment, which implies that the availability of a large number of superfamily members with a known three-dimensional structure is a requirement for 3DM to succeed well. Thirteen MCSISes were constructed and placed on the Internet for examination. These systems have been instrumental in a large series of research projects related to enzyme activity or the understanding and engineering of specificity, protein stability engineering, DNA-diagnostics, drug design, and so forth.

Directed evolution 2.0: improving and deciphering enzyme properties

“…Broadly and successfully applied software packages are among others 3DM 48 and HotSpot Wizard 49 . A further interesting structure-sequenced based approach was to alter the pH profile of proteases through surface charge engineering.…”

Molecular Modeling in Enzyme Design, Toward In Silico Guided Directed Evolution

“…At the present stage, such techniques are mostly used to restrict mutagenesis to relevant protein regions [72][73][74][75]77,78] or to guide directed evolution "on-the-fly" [199]. A second filtering, for example, could then be performed with fast molecular modeling techniques such as FoldX or Rosetta.…”

“…For this reason, sequence-based strategies that use evolutionary information or statistical data from previous DE rounds will not be explored. These often use phylogenetic analyses and multiple sequence alignments for exploring the amino acid conservation and relationships between homologs protein sequences [67,[72][73][74][75][76][77][78]. Instead we will center our attention on how computations and structural information may aid and focus mainly in physics-based methods to assist in the improvement of three major aspects in enzyme design: catalytic rate constants, protein stability and protein-ligand binding processes.…”

Site-Directed Mutagenesis as Applied to Biocatalysts

“…This approach was used to improve the activity and enantioselectivity of an esterase from Pseudomonas fluorescens (PFE). The amino acid distribution at four positions near the active site of PFE, previously reported to influence the enantioselectivity of the enzyme, was determined by a structure-guided multiple-sequence alignment of 171 esterases generated by the 3DM database (Kuipers et al, 2010). A library was created by site-directed mutagenesis of the coding regions for the four active site positions in PFE.…”