Can Protein
Conformers Be Fractionated by Crystallization?
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Abstract
Molecular
crystallization typically singles out a specific conformation,
or a set of conformations that are identical over large parts and
may show some flexibility, from a mixture of equilibrating conformations
in solution. To critically evaluate the selectivity of this process,
human lactate dehydrogenase isozyme 1 (LDH-1) microcrystals were separately
dissolved and subsequently assayed inside capillaries with electrophoretically
mediated microanalysis (EMMA) at both the ensemble and the single-molecule
level. While fragments from the same crystal exhibited identical enzyme
activities, different crystals, even when grown from the same drop
of mother liquor, showed markedly different activities. Activities
of individual molecules from a crystal were found to be essentially
identical, whereas molecules obtained directly from solution showed
a 4-fold variation in activity. Furthermore, after storage at 37 °C,
the distribution of single-molecule LDH activities from solutions
of individual crystals broadened and approached that of LDH obtained
from the original solution. X-ray crystallography also showed distinct
conformations for single microcrystals and confirms that crystallization
properly selects even small conformational variants of proteins and
that the slow equilibration to multiple stable conformations in solution
is responsible for the observed single-molecule heterogeneity