scholarly journals FireProtASR: A Web Server for Fully Automated Ancestral Sequence Reconstruction

2020 ◽  
Author(s):  
Milos Musil ◽  
Rayyan Tariq Khan ◽  
Andy Beier ◽  
Jan Stourac ◽  
Hannes Konegger ◽  
...  
Keyword(s):  
Ancestral Sequence ◽  
Reconstruction Algorithms ◽  
Ancestral Reconstruction ◽  
Web Interface ◽  
Computational Tools ◽  
Ancestral Sequence Reconstruction ◽  
Ancestral Sequences ◽  
Native Proteins ◽  
Sequence Reconstruction ◽  
History Of

Abstract There is a great interest in increasing proteins’ stability to widen their usability in numerous biomedical and biotechnological applications. However, native proteins cannot usually withstand the harsh industrial environment, since they are evolved to function under mild conditions. Ancestral sequence reconstruction is a well-established method for deducing the evolutionary history of genes. Besides its applicability to discover the most probable evolutionary ancestors of the modern proteins, ancestral sequence reconstruction has proven to be a useful approach for the design of highly stable proteins. Recently, several computational tools were developed, which make the ancestral reconstruction algorithms accessible to the community, while leaving the most crucial steps of the preparation of the input data on users’ side. FireProtASR aims to overcome this obstacle by constructing a fully automated workflow, allowing even the unexperienced users to obtain ancestral sequences based on a sequence query as the only input. FireProtASR is complemented with an interactive, easy-to-use web interface and is freely available at https://loschmidt.chemi.muni.cz/fireprotasr/.

scholarly journals Ancestral sequence reconstruction produces thermally stable enzymes with mesophilic enzyme-like catalytic properties

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Ryutaro Furukawa ◽  
Wakako Toma ◽  
Koji Yamazaki ◽  
Satoshi Akanuma
Keyword(s):  
Low Temperature ◽  
Catalytic Properties ◽  
Amino Acid Sequences ◽  
Ancestral Sequence ◽  
Kinetic Properties ◽  
Reconstruction Technique ◽  
Thermally Stable ◽  
Ancestral Sequence Reconstruction ◽  
Ancestral Sequences ◽  
Sequence Reconstruction

Abstract Enzymes have high catalytic efficiency and low environmental impact, and are therefore potentially useful tools for various industrial processes. Crucially, however, natural enzymes do not always have the properties required for specific processes. It may be necessary, therefore, to design, engineer, and evolve enzymes with properties that are not found in natural enzymes. In particular, the creation of enzymes that are thermally stable and catalytically active at low temperature is desirable for processes involving both high and low temperatures. In the current study, we designed two ancestral sequences of 3-isopropylmalate dehydrogenase by an ancestral sequence reconstruction technique based on a phylogenetic analysis of extant homologous amino acid sequences. Genes encoding the designed sequences were artificially synthesized and expressed in Escherichia coli. The reconstructed enzymes were found to be slightly more thermally stable than the extant thermophilic homologue from Thermus thermophilus. Moreover, they had considerably higher low-temperature catalytic activity as compared with the T. thermophilus enzyme. Detailed analyses of their temperature-dependent specific activities and kinetic properties showed that the reconstructed enzymes have catalytic properties similar to those of mesophilic homologues. Collectively, our study demonstrates that ancestral sequence reconstruction can produce a thermally stable enzyme with catalytic properties adapted to low-temperature reactions.

scholarly journals Ancestral Sequence Reconstructions of MotB Are Proton-Motile and Require MotA for Motility

2020 ◽  
Vol 11 ◽  
Author(s):  
Md Imtiazul Islam ◽  
Angela Lin ◽  
Yu-Wen Lai ◽  
Nicholas J. Matzke ◽  
Matthew A. B. Baker
Keyword(s):  
Ion Selectivity ◽  
Power Source ◽  
Ancestral Sequence ◽  
Ancestral Reconstruction ◽  
Aquifex Aeolicus ◽  
Independent Manner ◽  
B Subunit ◽  
Ancestral Sequences ◽  
Sequence Reconstruction ◽  
A Subunit

The bacterial flagellar motor (BFM) is a nanomachine that rotates the flagellum to propel many known bacteria. The BFM is powered by ion transit across the cell membrane through the stator complex, a membrane protein. Different bacteria use various ions to run their BFM, but the majority of BFMs are powered by either proton (H+) or sodium (Na+) ions. The transmembrane (TM) domain of the B-subunit of the stator complex is crucial for ion selectivity, as it forms the ion channel in complex with TM3 and TM4 of the A-subunit. In this study, we reconstructed and engineered thirteen ancestral sequences of the stator B-subunit to evaluate the functional properties and ionic power source of the stator proteins at reconstruction nodes to evaluate the potential of ancestral sequence reconstruction (ASR) methods for stator engineering and to test specific motifs previously hypothesized to be involved in ion-selectivity. We found that all thirteen of our reconstructed ancient B-subunit proteins could assemble into functional stator complexes in combination with the contemporary Escherichia coli MotA-subunit to restore motility in stator deleted E. coli strains. The flagellar rotation of the thirteen ancestral MotBs was found to be Na+ independent which suggested that the F30/Y30 residue was not significantly correlated with sodium/proton phenotype, in contrast to what we had reported previously. Additionally, four among the thirteen reconstructed B-subunits were compatible with the A-subunit of Aquifex aeolicus and able to function in a sodium-independent manner. Overall, this work demonstrates the use of ancestral reconstruction to generate novel stators and quantify which residues are correlated with which ionic power source.

Exploring the past and the future of protein evolution with ancestral sequence reconstruction: the ‘retro’ approach to protein engineering

2016 ◽  
Vol 474 (1) ◽  
pp. 1-19 ◽  
Author(s):  
Yosephine Gumulya ◽  
Elizabeth M.J. Gillam
Keyword(s):  
Protein Engineering ◽  
Protein Evolution ◽  
Phylogenetic Trees ◽  
Ancestral Sequence ◽  
Novel Proteins ◽  
Ancestral Sequence Reconstruction ◽  
Ancestral Sequences ◽  
Sequence Reconstruction ◽  
Ancient Proteins ◽  
Life On Earth

A central goal in molecular evolution is to understand the ways in which genes and proteins evolve in response to changing environments. In the absence of intact DNA from fossils, ancestral sequence reconstruction (ASR) can be used to infer the evolutionary precursors of extant proteins. To date, ancestral proteins belonging to eubacteria, archaea, yeast and vertebrates have been inferred that have been hypothesized to date from between several million to over 3 billion years ago. ASR has yielded insights into the early history of life on Earth and the evolution of proteins and macromolecular complexes. Recently, however, ASR has developed from a tool for testing hypotheses about protein evolution to a useful means for designing novel proteins. The strength of this approach lies in the ability to infer ancestral sequences encoding proteins that have desirable properties compared with contemporary forms, particularly thermostability and broad substrate range, making them good starting points for laboratory evolution. Developments in technologies for DNA sequencing and synthesis and computational phylogenetic analysis have led to an escalation in the number of ancient proteins resurrected in the last decade and greatly facilitated the use of ASR in the burgeoning field of synthetic biology. However, the primary challenge of ASR remains in accurately inferring ancestral states, despite the uncertainty arising from evolutionary models, incomplete sequences and limited phylogenetic trees. This review will focus, firstly, on the use of ASR to uncover links between sequence and phenotype and, secondly, on the practical application of ASR in protein engineering.

Ancestral sequence reconstruction: accounting for structural information by averaging over replacement matrices

2018 ◽  
Vol 35 (15) ◽  
pp. 2562-2568
Author(s):  
Asher Moshe ◽  
Tal Pupko
Keyword(s):  
Structural Information ◽  
Solvent Accessibility ◽  
3D Structure ◽  
Three Dimensional ◽  
Ancestral Sequence ◽  
Supplementary Information ◽  
Ancestral Sequence Reconstruction ◽  
Ancestral Sequences ◽  
Sequence Reconstruction ◽  
And Function

Abstract Motivation Ancestral sequence reconstruction (ASR) is widely used to understand protein evolution, structure and function. Current ASR methodologies do not fully consider differences in evolutionary constraints among positions imposed by the three-dimensional (3D) structure of the protein. Here, we developed an ASR algorithm that allows different protein sites to evolve according to different mixtures of replacement matrices. We show that assigning replacement matrices to protein positions based on their solvent accessibility leads to ASR with higher log-likelihoods compared to naïve models that assume a single replacement matrix for all sites. Improved ASR log-likelihoods are also demonstrated when solvent accessibility is predicted from protein sequences rather than inferred from a known 3D structure. Finally, we show that using such structure-aware mixture models results in substantial differences in the inferred ancestral sequences. Availability and implementation http://fastml.tau.ac.il. Supplementary information Supplementary data are available at Bioinformatics online.

scholarly journals Ancestral Sequence Reconstruction Reveals the Evolutionary History of the Folding Pathway and Landscape of Ribonucleases H

2016 ◽  
Vol 110 (3) ◽  
pp. 391a
Author(s):  
Shion A. Lim ◽  
Eric R. Bolin ◽  
Michael J. Harms ◽  
Kathryn M. Hart ◽  
Joseph W. Thornton ◽  
...  
Keyword(s):  
Evolutionary History ◽  
Ancestral Sequence ◽  
Folding Pathway ◽  
Ancestral Sequence Reconstruction ◽  
Sequence Reconstruction ◽  
History Of ◽  
Ribonucleases H

scholarly journals Revenant: a database of resurrected proteins

Database ◽  
2020 ◽  
Vol 2020 ◽  
Author(s):  
Matias Sebastian Carletti ◽  
Alexander Miguel Monzon ◽  
Emilio Garcia-Rios ◽  
Guillermo Benitez ◽  
Layla Hirsh ◽  
...  
Keyword(s):  
Protein Stability ◽  
Ancestral Sequence ◽  
Computational Tools ◽  
Noticeable Increase ◽  
The Past ◽  
Ancestral Sequence Reconstruction ◽  
Bibliographic Data ◽  
Sequence Reconstruction ◽  
Ancient Proteins ◽  
Laboratory Synthesis

Abstract Revenant is a database of resurrected proteins coming from extinct organisms. Currently, it contains a manually curated collection of 84 resurrected proteins derived from bibliographic data. Each protein is extensively annotated, including structural, biochemical and biophysical information. Revenant contains a browse capability designed as a timeline from where the different proteins can be accessed. The oldest Revenant entries are between 4200 and 3500 million years ago, while the younger entries are between 8.8 and 6.3 million years ago. These proteins have been resurrected using computational tools called ancestral sequence reconstruction techniques combined with wet-laboratory synthesis and expression. Resurrected proteins are commonly used, with a noticeable increase during the past years, to explore and test different evolutionary hypotheses such as protein stability, to explore the origin of new functions, to get biochemical insights into past metabolisms and to explore specificity and promiscuous behaviour of ancient proteins.

scholarly journals Highly thermostable carboxylic acid reductases generated by ancestral sequence reconstruction

2019 ◽  
Vol 2 (1) ◽  
Author(s):  
Adam Thomas ◽  
Rhys Cutlan ◽  
William Finnigan ◽  
Mark van der Giezen ◽  
Nicholas Harmer
Keyword(s):  
High Temperature ◽  
Carboxylic Acid ◽  
Ancestral Sequence ◽  
Ancestral Reconstruction ◽  
Ancestral Sequence Reconstruction ◽  
Sequence Reconstruction ◽  
Neutral Mutations ◽  
New Applications ◽  
Poor Stability

AbstractCarboxylic acid reductases (CARs) are biocatalysts of industrial importance. Their properties, especially their poor stability, render them sub-optimal for use in a bioindustrial pipeline. Here, we employed ancestral sequence reconstruction (ASR) – a burgeoning engineering tool that can identify stabilizing but enzymatically neutral mutations throughout a protein. We used a three-algorithm approach to reconstruct functional ancestors of the Mycobacterial and Nocardial CAR1 orthologues. Ancestral CARs (AncCARs) were confirmed to be CAR enzymes with a preference for aromatic carboxylic acids. Ancestors also showed varied tolerances to solvents, pH and in vivo-like salt concentrations. Compared to well-studied extant CARs, AncCARs had a Tm up to 35 °C higher, with half-lives up to nine times longer than the greatest previously observed. Using ancestral reconstruction we have expanded the existing CAR toolbox with three new thermostable CAR enzymes, providing access to the high temperature biosynthesis of aldehydes to drive new applications in biocatalysis.

Ancestral sequence reconstruction for protein engineers

2021 ◽  
Vol 69 ◽  
pp. 131-141
Author(s):  
Matthew A. Spence ◽  
Joe A. Kaczmarski ◽  
Jake W. Saunders ◽  
Colin J. Jackson
Keyword(s):  
Ancestral Sequence ◽  
Ancestral Sequence Reconstruction ◽  
Sequence Reconstruction

scholarly journals Alignment Modulates Ancestral Sequence Reconstruction Accuracy

2018 ◽  
Vol 35 (7) ◽  
pp. 1783-1797 ◽  
Author(s):  
Ricardo Assunção Vialle ◽  
Asif U Tamuri ◽  
Nick Goldman
Keyword(s):  
Ancestral Sequence ◽  
Reconstruction Accuracy ◽  
Ancestral Sequence Reconstruction ◽  
Sequence Reconstruction
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