Electronic structure of neighboring extein residue modulates intein C-terminal cleavage activity.

Abstract:

:Protein splicing is an autocatalytic reaction where an intervening element (intein) is excised and the remaining two flanking sequences (exteins) are joined. The reaction requires specific conserved residues, and activity may be affected by both the intein and the extein sequence. Predicting how sequence will affect activity is a challenging task. Based on first-principles density functional theory and multiscale quantum mechanics/molecular mechanics, we report C-terminal cleavage reaction rates for five mutations at the first residue of the C-extein (+1), and describe molecular properties that may be used as predictors for future mutations. Independently, we report on experimental characterization of the same set of mutations at the +1 residue resulting in a wide range of C-terminal cleavage activities. With some exceptions, there is general agreement between computational rates and experimental cleavage, giving molecular insight into previous claims that the +1 extein residue affects intein catalysis. These data suggest utilization of attenuating +1 mutants for intein-mediated protein manipulations because they facilitate precursor accumulation in vivo for standard purification schemes. A more detailed analysis of the "+1 effect" will also help to predict sequence-defined effects on insertion points of the intein into proteins of interest.

journal_name

Biophys J

journal_title

Biophysical journal

authors

Shemella PT,Topilina NI,Soga I,Pereira B,Belfort G,Belfort M,Nayak SK

doi

10.1016/j.bpj.2011.02.037

subject

Has Abstract

pub_date

2011-05-04 00:00:00

pages

2217-25

issue

9

eissn

0006-3495

issn

1542-0086

pii

S0006-3495(11)00255-4

journal_volume

100

pub_type

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