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Title
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Ancestral lysozymes reconstructed, neutrality tested, and thermostability linked to hydrocarbon packing.
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Authors
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B.A.Malcolm,
K.P.Wilson,
B.W.Matthews,
J.F.Kirsch,
A.C.Wilson.
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Ref.
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Nature, 1990,
345,
86-89.
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PubMed id
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Abstract
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The controversy surrounding the idea that neutral mutations dominate protein
evolution is attributable in part to the inadequacy of the tools available to
evolutionary investigators. With a few exceptions, most investigations into the
force driving protein evolution have relied on indirect criteria for
distinguishing neutral and non-neutral variants. To investigate a particular
pathway of molecular evolution, we have reconstructed by site-directed
mutagenesis likely ancestral variants of the lysozymes of modern game birds
(order Galliformes), tested their activity and thermostability and determined
their three-dimensional structure. We focused on amino acids at three positions
that are occupied in all known game birds either by the triplet Thr 40, Ile 55,
Ser 91, or by the triplet Ser 40, Val 55, Thr 91. We have synthesized proteins
representing intermediates along the possible three-step evolutionary pathways
between these triplets. Although all of these are active and stable, none of
these intermediates is found in known lysozymes. A comparison of the structures
and thermostabilities of the variants reveals a linear correlation between the
side-chain volume of the triplet and the thermostability of the protein. Each
pathway connecting the two extant triplet sequences includes a variant with a
thermostability outside the range of the extant proteins. This observation is
consistent with a non-neutral evolutionary pathway. The existence of variants
that are more stable than the extant proteins suggests that selection for
maximum thermostability may not have been an important factor in the evolution
of this enzyme.
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