An approach to calculating molecular electronic structures of active-site clusters in the presence of protein environments has been developed. The active-site cluster is treated by density functional theory. The protein field, together with the reaction field arising mainly from solvent, is obtained from a finite-difference solution to the Poisson−Boltzmann equation with three dielectric regions, and then these are coupled to the density functional calculation by a self-consistent iterative procedure. The method is applied to compute redox potentials of ferredoxin from Anabaena 7120 and phthalate dioxygenase reductase (PDR) from Pseudomonas cepacia, both having similar [Fe2S2(SR)4] active-site clusters. The calculated redox potentials, −1.007 V and −0.812 V in 0.05 M ionic strength for ferredoxin and PDR, respectively, deviate significantly from experimental values of −0.440 and −0.174 V. However, the calculated data reproduce the experimental trend fairly well. The calculated redox potential for PDR is 195 mV more positive than that for ferredoxin, comparing very well with the experimental value of 266 mV. The energy decomposition scheme reveals that the protein field plays a key role in differentiating the redox potentials of these two proteins.

Calcium sensor proteins translate transient increases in intracellular calcium levels into metabolic or mechanical responses, by undergoing dramatic conformational changes upon Ca2+ binding. A detailed analysis of the calcium binding-induced conformational changes in the representative calcium sensors calmodulin (CaM) and troponin C was performed to obtain insights into the underlying molecular basis for their response to the binding of calcium. Distance difference matrices, analysis of interresidue contacts, comparisons of interhelical angles, and inspection of structures using molecular graphics were used to make unbiased comparisons of the various structures. The calcium-induced conformational changes in these proteins are dominated by reorganization of the packing of the four helices within each domain. Comparison of the closed and open conformations confirms that calcium binding causes opening within each of the EF-hands. A secondary analysis of the conformation of the C-terminal domain of CaM (Ca"C) clearly shows that Ca"C occupies a closed conformation in the absence of calcium that is distinct from the semi-open conformation observed in the C-terminal EF-hand domains of myosin light chains. These studies provide insight into the structural basis for these changes and into the differential response to calcium binding of various members of the EF-hand calcium-binding protein family. Factors contributing to the stability of the Ca2+-loaded open conformation are discussed, including a new hypothesis that critical hydrophobic interactions stabilize the open conformation in Ca2+ sensors, but are absent in "non-sensor" proteins that remain closed upon Ca2+ binding. A role for methionine residues in stabilizing the open conformation is also proposed.

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EF-hand Ca2+ -binding proteins participate in both modulation of Ca 2+ signals and direct transduction of the ionic signal into downstream biochemical events. The range of biochemical functions of these proteins is correlated with differences in the way in which they respond to the binding of Ca 2+. The EF-hand domains of calbindin D 9k and calmodulin are homologous, yet they respond to the binding of calcium ions in a drastically different manner. A series of comparative analyses of their structures enabled the development of hypotheses about which residues in these proteins control the calcium-induced changes in conformation. To test our understanding of the relationship between protein sequence and structure, we specifically designed the F36G mutation of the EF-hand protein calbindin D 9k to alter the packing of helices I and II in the apoprotein. The three-dimensional structure of apo F36G was determined in solution by nuclear magnetic resonance spectroscopy and showed that the design was successful. Surprisingly, significant structural perturbations also were found to extend far from the site of mutation. The observation of such long-range effects provides clear evidence that four-helix EF-hand domains should be treated as a single globally cooperative unit. A hypothetical mechanism for how the long-range effects are transmitted is described. Our results support the concept of energetic and structural coupling of the key residues that are crucial for a protein's fold and function.Keywords: Calcium-binding protein; conformational change; EF-hand; mutagenesis; NMR spectroscopy; protein engineeringThe EF-hand family of Ca 2+ -binding proteins (CaBPs) provides a rich framework for investigating fundamental relationships between protein sequence and biochemical function. The EF-hand motif is among the most common in animal cells (Henikoff et al. 1997); more than 1000 have been identified from their unique sequence signatures. These motifs are organized into structural units/domains containing two or more EF hands that form highly stable helical bundles (Nelson and Chazin 1998a). Despite the similarities in their sequences and structures, EF-hand proteins perform a diverse range of functions. There are two primary classes of EF-hand proteins: Ca 2+ sensors, which transduce Ca 2+ signals, and Ca 2+ signal modulators, which modulate the shape and/or duration of Ca 2+ signals or participate in Ca 2+ homeostasis. Here, we describe research on representative sensor and signal modulator proteins, calmodulin (CaM) and calbindin D 9k (calbindin), which is directed toward understanding how differences in amino acid sequence specify differences in how EF-hand domains re-

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