Abstract:
The comparative study of protein sequences and structures is traditionally used to
better understand protein fold evolution. The insights we gain from our evolutionary
analyses are applied in protein design projects. At the same time, we engineer proteins
to test evolutionary assumptions; thus, we establish a feedback loop between both
aspects of protein science. First, we compared Profile Hidden Markov Models, state
of the art tools for homology detection, that represent all structures that adopt the
(βα)8-barrel and the flavodoxin-like fold to discover an evolutionary relationship
between these basic structural forms. Moreover, we located the region of the sequence
space where both folds are most closely related. Having found this interface, we
performed remote homologous searches and protein clustering to find sequences with
intermediate features between the (βα)8-barrel and the flavodoxin-like fold.
We determined the x-ray crystal structure of one of these sequences to learn possible
scenarios of fold change during the evolution of these ancestral structures. The
intermediate sequence, named NTM0182, displayed features towards both folds.
Moreover, and by structurally superimposing the three structures, we found classical
evidences of homology among the three folds: high sequence identity over long
aligned fragments. Our approach then starts by using very sensitive novel tools for
homology detection (probability scores), to find intermediary links that provide
classical evidences of common ancestry (high sequence identity).
Next, we extended the Profile Hidden Markov Model comparisons to include all folds
classified as α/β in the Structural Classification of Proteins (SCOP). Our comparisons
showed that high scoring pairwise alignments are correlated with high local structural
similarities between different folds. This observation inspired us to look for
interchangeable sub-domain size protein fragments, related by sequence and structure,
to build chimeric proteins and mimic protein fold evolution. Our global comparisons
revealed that both, the flavodoxin-like and the (βα)8-barrel folds were related to
Periplasmic binding protein-like I proteins.
We then employed sequence comparisons, structural superpositions, homology
modeling and computational assessments to engineer a novel chimeric protein by
fusing a flavodoxin-like fold protein into a PBP-like scaffold. The chimera turned out
to be a well-folded protein with native-like properties. Thus, our research allowed us
to gain evolutionary insights that are applied to protein engineering. In this respect,
we establish a feedback loop between fold evolution and protein engineering. We
finally contribute to an emergent vision where proteins from different folds are
evolutionary related.
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