Effect of linker sequence on the stability of circularly permuted variants of ribonuclease T1.

abstract

• Circularly permuted variants of ribonuclease T1 were constructed with a library of residues covalently linking the original amino and carboxyl terminal ends of the wild-type protein. The library of linking peptides consisted of three amino acids containing any combination of proline, aspartate, asparagine, serine, threonine, tyrosine, alanine, and histidine. Forty two unique linker sequences were isolated and 10 of these mutants were further characterized with regard to catalytic activity and overall thermodynamic stability. The 10 mutants with the different linking sequences (HPD, TPH, DTD, TPD, PYH, PAT, PHP, DSS, SPP, and TPS), in addition to GGG and GPG, were 4.0-6.2 kcal/mol less stable than the wild-type ribonuclease T1. However, these circular permuted variants were only 0.4-2.6 kcal/mol less stable than the direct parent protein that is missing the disulfide bond connecting residues 2 and 10. The most stable linking peptide was HPD.

authors

• Raushel, Frank

published proceedings

• Bioorg Chem

author list (cited authors)

• Garrett, J. B., Mullins, L. S., & Raushel, F. M.

citation count

• 5

complete list of authors

• Garrett, James B||Mullins, Leisha S||Raushel, Frank M

publication date

• October 2003

publisher

• Elsevier  Publisher

published in

• Bioorganic Chemistry  Journal

keywords

• Catalysis
• Circular Dichroism
• Enzyme Stability
• Genetic Engineering
• Models, Molecular
• Protein Conformation
• Protein Folding
• Ribonuclease T1
• Thermodynamics

PubMed Central ID

• 12941293

Digital Object Identifier (DOI)

• 10.1016/s0045-2068(03)00079-8

start page

• 412

end page

• 424

volume

• 31

issue

• 5

URL

• https://doi.org/10.1016/s0045-2068(03)00079-8