The plasma membrane Na+/Ca2+ exchanger (NCX) is almost certainly the major Ca2+ extrusion mechanism in cardiac myocytes. Binding of Na+ and Ca2+ ions to its large cytosolic loop regulates ion transport of the exchanger. We determined the solution structures of two Ca2+ binding domains (CBD1 and CBD2) that, together with an alpha-catenin-like domain (CLD), form the regulatory exchanger loop. CBD1 and CBD2 are very similar in the Ca2+ bound state and describe the Calx-beta motif. Strikingly, in the absence of Ca2+, the upper half of CBD1 unfolds while CBD2 maintains its structural integrity. Together with a 7-fold higher affinity for Ca2+, this suggests that CBD1 is the primary Ca2+ sensor. Specific point mutations in either domain largely allow the interchange of their functionality and uncover the mechanism underlying Ca2+ sensing in NCX.

High-resolution NMR and UV-melting experiments have been used to study the hairpin formation of partly self-complementary DNA fragments in an attempt to derive rules that describe the folding in these molecules. Earlier experiments on the hexadecanucleotide d(ATCCTA-TTTT-TAGGAT) had indicated that within the loop of four thymidines a wobble T-T pair is formed (Blommers et al., 1987). In the present paper it is shown that if the first and the last thymines of the intervening sequence are replaced by complementary bases, sometimes base pairs can be formed. Thus for the intervening sequences -CTTG- and -TTTA- with the pyrimidine in the 5'-position and the purine in the 3'-position, a base pair is formed leading to a loop consisting of two residues. For the intervening sequences -GTTC- and -ATTT- with the purine in the 5'-position and the pyrimidine in the 3'-position, this turns out not to be the case. It was found that it made no difference when the four-membered sequence was closed by a G-C base pair or an A-T base pair. Replacement of the two central thymidine residues by the more bulky adenine residues limits the hairpin to a four-membered loop scheme. Very surprisingly, it was found from 2D NOE experiments that the T-A base pair, formed in the loop consisting of the -TTTA- sequence, is a Hoogsteen pair. It is argued that the pairing of the bases in this scheme may facilitate the formation of a loop of two residues, since the distance of the C1' atoms in this base pair is 8.6 A instead of 10.4 A found in the canonical Watson-Crick base pair. Combination of the data obtained for the series of DNA fragments studied shows that the results can be explained by a simple, earlier proposed, loop folding principle which assumes that the folding of the four-membered loop is dictated by the stacking of the double-helical stem of the hairpin.

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PDZ domains represent a large family of protein-interaction modules associated with a variety of unrelated proteins with different functions. We report a complete characterization of the kinetic folding mechanism of a fluorescent variant of PDZ2 from PTP-BL, investigated under a variety of different experimental conditions. For this purpose, we engineered a fluorescent variant of this protein Y43W (called pseudo-wild-type, pWT43). The results suggest the presence of a high-energy intermediate in the folding of PDZ2, as revealed by a pronounced non-linear dependence of the unfolding rate constant on denaturant concentration. Such an intermediate may or may not be detectable depending on the experimental conditions, giving rise to apparent two-state folding under stabilizing conditions (e.g. in the presence of sodium sulfate). Interestingly, even under these conditions, three-state folding can be restored by selectively destabilizing the native-like rate-limiting barrier by one specific mutation (V44A). Finally, we show that data taken on pWT43 under different experimental conditions (e.g. different pH values from 2.1 to 8.0 or in the presence of a stabilizing salt) and also data on a site-directed conservative mutant can be rationalized in terms of a simple reaction scheme involving a single set of intermediates and transition states.

CBD1. We propose that the electrostatic switch in CBD1 and the associated conformational change are necessary for exchanger activation. The response of the CBD1 switch to intracellular Ca 2؉ is influenced by the closely located cassette exons. We further propose that Ca 2؉ -binding to CBD2 induces a second electrostatic switch, required to alleviate Na ؉ -dependent inactivation of Na ؉ /Ca 2؉ exchange. In contrast to CBD1, the electrostatic switch in CBD2 is isoform-and splice variant-specific and allows for tailored exchange activities.alternative splicing ͉ Ca 2ϩ -binding domain ͉ homeostasis ͉ Ca 2ϩ sensor