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Functional Redesign of an EF-hand Ca2+-binding
Protein
Principal Investigator: Walter J. Chazin
A comprehensive
program in Ca site design is proposed to directly address the primary
objective of this program project: bridging the gap between
description of structures and comprehension of activity of
metalloproteins. The broad, long-term objective of our research is to
develop the ability to control Ca2+ binding properties in
protein and thereby generate the potential to design Ca2+
sites for specific biological activities and therapeutic strategies.
To achieve this goal, we seek an understanding of how the EF-hand
family of Ca2+-binding proteins (CaBPs) work a the
molecular level. These proteins have been selected because they have
central roles in nearly all Ca2+ signaling pathways and
consequently, are associate with a wide-range of effects on health
and disease, e.g. the cell cycle and cancer. This proposal is
directed at determining how EF-hand CaBP sequence and structure
specify the response to Ca2+ binding. Three highly
integrated specific aims are proposed. In Aim 1, a database about
EF-hand CaBPs will be constructed to gather, organize, and provide
tools to analyze the extensive body of information on sequences,
three-dimensional structures and biochemical studies. In Aim 2,
in-depth comparative analyses of EF-hand CaBPs will be utilized to
develop hypotheses about what structural factors and amino acid
properties control the response to Ca2+ binding. Aim 3
involves pooling the information available from Aims 1 and 2 to
ultimately design, produce and characterize calbindomodulin, a
calbindin D9k remodeled to adopt a calmodulin-like open
conformation upon Ca2+ binding. This will involve an
initial phase of site-directed mutagenesis experiments to test
Hypotheses about interactions that are important for maintaining the
stability of the open and closed conformations. Then a subset or all
of these sites will be mutated to preferentially stabilize an open
conformation for Ca2+-bound calbindin D9k. The
mutants will be characterized by their stability, Ca2+
affinity, and a structural screen using NMR. The three-dimensional
structure of Ca2+-loaded calbindomodulin will be
determined by NMR. This project makes important contributions to the
overall goals of the program by integrating knowledge and expertise
on the structural/regulatory class of metalloproteins, and by
focusing on binding-induced conformational changes in metal sites.