| 1. |
- Emrén, Lars O., et al.
(författare)
-
Functionally diverging molecular quasi-species evolve by crossing two enzymes
- 2006
-
Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 103:29, s. 10866-10870
-
Tidskriftsartikel (refereegranskat)abstract
- Molecular evolution is frequently portrayed by structural relationships, but delineation of separate functional species is more elusive. We have generated enzyme variants by stochastic recombinations of DNA encoding two homologous detoxication enzymes, human glutathione transferases M1-1 and M2-2, and explored their catalytic versatilities. Sampled mutants were screened for activities with eight alternative substrates, and the activity fingerprints were subjected to principal component analysis. This phenotype characterization clearly identified at least three distributions of substrate selectivity, where one was orthogonal to those of the parent-like distributions. This approach to evolutionary data mining serves to identify emerging molecular quasi-species and indicates potential trajectories available for further protein evolution.
|
|
| 2. |
- Fedulova, Natalia, 1980-, et al.
(författare)
-
Expression and purification of catalytically active human PHD3 in Escherichia coli.
- 2007
-
Ingår i: Protein expression and purification. - : Elsevier BV. - 1046-5928 .- 1096-0279. ; 54:1, s. 1-10
-
Tidskriftsartikel (refereegranskat)abstract
- Transcription factor HIF-1 is a key regulator in cellular adaptation to hypoxia. HIF prolyl hydroxylases (PHDs) control HIF-1 accumulation by hydroxylation dependent on molecular oxygen. Due to this regulation, PHDs have been pointed out as potential drug targets. We have purified catalytically active human PHD3 after heterologous expression in Escherichia coli. Histidine-tagged enzyme was isolated as monomer by immobilized Ni-affinity chromatography followed by gel filtration. Overexpression of bacterial chaperonins GroEL/ES at 30 degrees C substantially increased the yield of soluble PHD3. High concentrations of salt and reducing agent during purification prevented protein aggregation. The enzyme activity with peptide derived from HIF-1alpha was inhibited by Zn(2+), desferrioxamine and imidazole. The hydroxylation activity was verified by mass spectrometry, and Pro567 in HIF-1alpha was discovered as a new site of hydroxylation.
|
|
| 3. |
|
|
| 4. |
|
|
| 5. |
- Kurtovic, Sanela, et al.
(författare)
-
Multivariate-activity mining for molecular quasi-species in a glutathione transferase mutant library
- 2007
-
Ingår i: Protein Engineering Design & Selection. - : Oxford University Press (OUP). - 1741-0126 .- 1741-0134. ; 20:5, s. 243-256
-
Tidskriftsartikel (refereegranskat)abstract
- A library of recombinant glutathione transferases (GSTs) generated by shuffling of DNA encoding human GST M1-1 and GST M2-2 was screened with eight alternative substrates, and the activities were subjected to multivariate analysis. Assays were made in lysates of bacteria in which the GST variants had been expressed. The primary data showed clustering of the activities in eight-dimensional substrate-activity space. For an incisive analysis, the rows of the data matrix, corresponding to the different enzyme variants, were individually scaled to unit length, thus accounting for different expression levels of the enzymes. The columns representing the activities with alternative substrates were subsequently individually normalized to unit variance and a zero mean. By this standardization, the data were adjusted to comparable orders of magnitude. Three molecular quasi-species were recognized by multivariate K-means and principal component analyses. Two of them encompassed the parental GST M1-1 and GST M2-2. A third one diverged functionally by displaying enhanced activities with some substrates and suppressed activities with signature substrates for GST M1-1 and GST M2-2. A fourth cluster contained mutants with impaired functions and was not regarded as a quasi-species. Sequence analysis of representatives of the mutant clusters demonstrated that the majority of the variants in the diverging novel quasi-species were structurally similar to the M1-like GSTs, but distinguished themselves from GST M1-1 by a Ser to Thr substitution in the active site. The data show that multivariate analysis of functional profiles can identify small structural changes influencing the evolution of enzymes with novel substrate-activity profiles.
|
|
| 6. |
|
|
| 7. |
|
|
