Several model systems have been used to evaluate the alpha-helical propensities of different amino acids. In contrast, experimental quantitation of beta-sheet preferences has been addressed in only one model system, a zinc-finger peptide. Here we measure the relative propensity for beta-sheet formation of the twenty naturally occurring amino acids in a variant of the small, monomeric, beta-sheet-rich, IgG-binding domain from protein G. Amino-acid substitutions were made at a guest site on the solvent-exposed surface of the beta-sheet. Several criteria were used to establish that the mutations did not cause significant structural changes: binding to the Fc domain of IgG, calorimetric unfolding and NMR spectroscopy. Characterization of the terminal stabilities of these proteins leads to a thermodynamic scale for beta-sheet propensities that spans a range of approximately 2 kcal mol-1 for the naturally occurring amino acids, excluding proline. The magnitude of the differences suggests that beta-sheet preferences can be important determinants of protein stability.

Voltage-gated calcium channels (Ca V s) govern muscle contraction, hormone and neurotransmitter release, neuronal migration, activation of calcium-dependent signalling cascades, and synaptic input integration 1 . An essential Ca V intracellular protein, the β-subunit (Ca V β)1 ,2 , binds a conserved domain (the α-interaction domain, AID) between transmembrane domains I and II of the pore-forming α 1 subunit 3 and profoundly affects multiple channel properties such as voltagedependent activation 2 , inactivation rates 2 , G-protein modulation 4 , drug sensitivity 5 and cell surface expression 6,7 . Here, we report the high-resolution crystal structures of the Ca V β 2a conserved core, alone and in complex with the AID. Previous work suggested that a conserved region, the β-interaction domain (BID), formed the AID-binding site 3, 8; however, this region is largely buried in the Ca V β core and is unavailable for protein-protein interactions. The structure of the AID-Ca V β 2a complex shows instead that Ca V β 2a engages the AID through an extensive, conserved hydrophobic cleft (named the α-binding pocket, ABP). The ABP-AID interaction positions one end of the Ca V β near the intracellular end of a pore-lining segment, called IS6, that has a critical role in Ca V inactivation 9,10 . Together, these data suggest that Ca V βs influence Ca V gating by direct modulation of IS6 movement within the channel pore. The 1.97 Å resolution structure of the Ca V β 2a core shows that Ca V βs comprise two wellconserved domains (Fig. 1a). The first, an SH3 fold, contains five antiparallel β-strands (β1-β5), a 3 10 helix (η1), and two α-helices (α1 and α2) that lie amino-terminal to β1 and carboxy-terminal to β4, respectively. The strand that completes the SH3 fold, β5 (residues 217-224), is separated in the primary structure from the core of the SH3 domain by approximately 70 residues (variable domain 2, V2, a site of splice variation and amino acid insertions and deletions2) that are absent from the structure (Fig. 1b). The second conserved domain consists of a five-stranded parallel β-sheet (β6-β10), surrounded by six α-helices (α3-α8) and two 3 10 helices (η2 and η3), and is related to the core of nucleotide kinase enzymes.Ca V βs share structural features with membrane-associated guanylate kinases (MAGUKs), a protein scaffold family that organizes signalling components near membranes 11 Comparison of Ca V β 2a with a representative MAGUK, 13), reveals other differences. Superposition of the nucleotide kinase domains shows that the relative orientations of the SH3 and nucleotide kinase domains differ by approximately 90°, an arrangement that makes Ca V β 2a a more elongated structure (Fig. 2a). The nucleotide kinase domain of MAGUKs is homologous to guanylate kinases and retains guanosine monophosphate (GMP) binding, but key residues for enzymatic function are missing 12 . The four-stranded β-sheet nucleotide kinase subdomain that binds GMP in MAGUKs is absent in Ca v β 2a (Fig. 2a). Furthermore, two Ca V β 2a loops (b...

Changes in activity-dependent calcium flux through voltage-gated calcium channels (Ca V s) drive two self-regulatory calcium-dependent feedback processes that require interaction between Ca 2+ / calmodulin (Ca 2+ /CaM) and a Ca V channel consensus isoleucine-glutamine (IQ) motif: calciumdependent inactivation (CDI) and calcium-dependent facilitation (CDF). Here, we report the highresolution structure of the Ca 2+ /CaM-Ca V 1.2 IQ domain complex. The IQ domain engages hydrophobic pockets in the N-terminal and C-terminal Ca 2+ /CaM lobes through sets of conserved 'aromatic anchors.' Ca 2+ /N lobe adopts two conformations that suggest inherent conformational plasticity at the Ca 2+ /N lobe-IQ domain interface. Titration calorimetry experiments reveal competition between the lobes for IQ domain sites. Electrophysiological examination of Ca 2+ /N lobe aromatic anchors uncovers their role in Ca V 1.2 CDF. Together, our data suggest that Ca V subtype differences in CDI and CDF are tuned by changes in IQ domain anchoring positions and establish a framework for understanding CaM lobe-specific regulation of Ca V s.Voltage-gated calcium channels are the ion channels that define excitable cells 1 . These channels control cellular calcium entry in response to changes in membrane potential and are pivotal in the generation of cardiac action potentials, excitation-contraction coupling, hormone and neurotransmitter release and activity-dependent transcription initiation 1,2 . Ca V s are multisubunit complexes composed of three essential channel subunits 2 , Ca V α 1 , Ca V β and Ca V α 2 δ, plus the ubiquitous intracellular calcium sensor calmodulin (CaM) 3 . An additional subunit, Ca V γ, is associated with skeletal muscle channels, but its general importance in other tissues is unsettled 4 .The Ca V α 1 subunits are single polypeptide chains of ∼1,800-2,200 residues in which the ion-conducting pore is formed from four homologous repeats that each bear six transmembrane segments 2 . There are three Ca V subfamilies, which have diverse physiological and pharmacological properties that depend largely on the Ca V α 1 -subunit: Ca V 1.x (L-type), Ca V 2.x (2.1, P/Q-type; 2.2, N-type; 2.3, R-type) and Ca V 3.x (T-type) 1 . Large interdomain intracellular loops bridge the four transmembrane repeats of the Ca V α 1 subunit and serve as docking sites for auxiliary subunits and regulatory molecules that Competing Interests Statement:The authors declare that they have no competing financial interests.Reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions/ NIH Public Access control channel activity and connect Ca V channels to larger macromolecular complexes and cellular signaling pathways 5,6 .Calcium influx is a potent activator of intracellular signaling pathways but is toxic in excess 1,7 . Because Ca V s are major sources of calcium influx, Ca V activity is strongly controlled by both self-regulatory and extrinsic mechanisms that tune channel action in response to electrical...

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