Channel adaptation is a fundamental feature of sarcoplasmic reticulum calcium release channels (called ryanodine receptors, RyRs). It permits successive increases in the intracellular concentration of calcium (Ca 2+ ) to repeatedly but transiently activate channels. Adaptation of RyRs in the absence of magnesium (Mg 2+ ) and adenosine triphosphate is an extremely slow process (taking seconds). Photorelease of Ca 2+ from nitrophenyl-EGTA, a photolabile Ca 2+ chelator, demonstrated that RyR adaptation is rapid (milliseconds) in canine heart muscle when physiological Mg 2+ concentrations are present. Phosphorylation of the RyR by protein kinase A increased the responsiveness of the channel to Ca 2+ and accelerated the kinetics of adaptation. These properties of the RyR from heart may also be relevant to other cells in which multiple agonist-dependent triggering events regulate cellular functions.Control of intracellular Ca 2+ homeostasis is fundamental to the contraction of cardiac muscle. Entry of extracellular Ca 2+ through voltage-sensitive Ca2+ channels triggers the release of Ca 2+ from the sarcoplasmic reticulum (SR) (1-3). This process, Ca 2+ -induced Ca 2+ release (CICR), is mediated by the Ca 2+ -gated Ca 2+ release channel called the ryanodine receptor (RyR) (4). Reconstitution of RyRs in planar lipid bilayers indicates that individual channels are modulated by Ca 2+ (5, 6), Mg2 (6-8), adenine nucleotides (9), and several protein kinases (10-12) under steady-state conditions. However, in the presence of physiological concentrations of Mg 2+ and adenosine triphosphate (ATP), unphysiologically high concentrations of free Ca 2+ are required to activate the channel (7, 13). This suggests either that a regulatory factor that alters Ca 2+ sensitivity is lost during RyR reconstitution or that steady-state experiments do not reveal key functional properties of the channel.The RyR channels have a regulatory mechanism termed adaptation that is triggered when the concentration of Ca 2+ ([Ca 2+ ]) is increased quickly by flash photolysis of caged Ca 2+ (14). Successive increases in [Ca 2+ ] repeatedly open the RyRs which then close (adapt) even though the increased [Ca 2+ ] is maintained. The multiple cycles of opening and closing as the agonist concentration is increased in steps is not predicted by traditional gating models. Adaptation may be the negative feedback mechanism that counters the inherent positive feedback of CICR. However, the rate constant of adaptation in vitro (τ = 1.3s) is much * to whom correspondence should be addressed. Individual canine cardiac RyR channels were reconstituted in planar lipid bilayers (16), and the concentration of free Ca 2+ of the solution surrounding the cytosolic face of the channel was buffered to 1 piM by mixing 1 mM NP-EGTA with 0.96 mM CaCI 2 (17). Under these conditions, long (ca. 2 to 5 ms) and short (≤ 1 ms) channel openings were evident (Fig. 1A). The probability of the channel being open (P O ) was 0.21 and remained constant throughout the recording perio...

The de novo design of catalytic proteins provides a stringent test of our understanding of enzyme function, while simultaneously laying the groundwork for the design of novel catalysts. Here we describe the design of an O2-dependent phenol oxidase whose structure, sequence, and activity are designed from first principles. The protein catalyzes the two-electron oxidation of 4-aminophenol (kcat͞KM ‫؍‬ 1,500 M ؊1 ⅐min ؊1 ) to the corresponding quinone monoimine by using a diiron cofactor. The catalytic efficiency is sensitive to changes of the size of a methyl group in the protein, illustrating the specificity of the design.T he de novo design of proteins provides a stringent test of our understanding of their molecular mechanisms of action (1-3). Recently, it has become possible to design proteins with novel three-dimensional structures (4), which has laid the groundwork for the elaboration of function. Catalysis provides a particularly challenging function to achieve, because a successfully designed protein catalyst must bind and precisely orient substrates, transition states, and intermediates adjacent to catalytic groups such as metal ions, general acids, and͞or general bases. Two approaches to the design of catalytic proteins include automated sequence design, in which a novel catalytic site is engineered into a natural protein by mutating a subset of its side chains (5-7), and de novo protein design, which requires the simultaneous design of the entire backbone structure and sequence (2). The first method has the advantage of separating the problem of protein design and folding from the more restricted problem of designing an active site. The second approach has the potential advantage of greater applicability in terms of the sizes and shapes of substrates that can be accommodated. Furthermore, de novo protein design critically tests our understanding of how an amino acid sequence dictates both the folding as well as the activity of a protein.To date, most work on the design of catalytic proteins has focused on hydrolysis of activated 4-nitrophenyl esters, using an active site histidine side chain as a nucleophilic catalyst. Automated sequence design methods have been used to design a variant of thioredoxin that hydrolyses 4-ntirophenyl acetate with a rate enhancement of Ϸ25-fold (7), when the value of k cat ͞K M was compared to the second order rate constant for hydrolysis of 4-methyl imidazole. Proteins with similar or greater catalytic efficiencies were observed frequently in a library of four-helix bundle proteins, whose polar exterior residues were randomly selected from Lys, His, Glu, Gln, Asp, and Asn (8). Baltzer and Nilsson (1) have designed a series of helical bundles that employ His residues to promote hydrolysis and intrapeptide acyl transfer reactions. It has also been possible to design helical bundles that catalyze decarboxylation of oxaloacetate (9), in one case by recognizing a key aldamine intermediate in the reaction (10).Automated sequence design has also been used to design catalytic meta...

DF1 is a small, idealized model for carboxylate-bridged diiron proteins. This protein was designed to form a dimeric four-helix bundle with a dimetal ion-binding site near the center of the structure, and its crystal structure has confirmed that it adopts the intended conformation. However, the protein showed limited solubility in aqueous buffer, and access to its active site was blocked by two hydrophobic side chains. The sequence of DF1 has now been modified to provide a very soluble protein (DF2) that binds metal ions in a rapid and reversible manner. Furthermore, the DF2 protein shows significant ferroxidase activity, suggesting that its dimetal center is accessible to oxygen. The affinity of DF2 for various first-row divalent cations deviates from the Irving-Willliams series, suggesting that its structure imparts significant geometric preferences on the metal ion-binding site. Furthermore, in the absence of metal ions, the protein folds into a dimer with concomitant binding of two protons. The uptake of two protons is expected if the structure of the apo-protein is similar to that of the crystal structure of dizinc DF1. Thus, this result suggests that the active site of DF2 is retained in the absence of metal ions. Keywords:De novo protein design; four-helix bundle; diiron; metalloprotein design Proteins use a limited set of metal ions to mediate a variety of processes. Often the same metal center can serve a number of roles; for example, binuclear iron centers engage in reversible binding of oxygen, electron transfer, and the catalysis of hydroxylation reactions (Feig and

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