The solution structure of human TL was deduced from the position of the emission peaks after site-directed tryptophan fluorescence (SDTF). The fluorescent amino acid tryptophan was sequentially substituted for each native amino acid in the sequence. Characteristic periodicities for eight beta-strands that comprise the beta-barrel and three alpha-helices were identified. The putative beta-strand I was relatively exposed to solvent, suggesting it does not participate in the formation of the beta-barrel. The beta-strands A and F contain beta-bulges. The average lambda(max) of emission maxima reveals that strand D is at the edge of the barrel and beta-strand H interacts with the main alpha-helical domain. On the basis of the SDTF data, a 3D homology model was constructed for TL and compared to the known crystallographic structures of RBP and beta-lactoglobulin. The small size and splayed open configuration of the E-F hairpin facilitate access of ligands into the cavity mouth of TL as compared to that of RBP with a long overhanging loop that restricts access. In the model of TL, four alanine residues are positioned in the binding site as compared to bulkier residues in the corresponding positions of beta-lactoglobulin. Substitution of A51, A66, A86 to Trp results in a 3-4-fold decrease in binding affinity. The data suggest that the smaller side chains of Ala provide more capacity in the cavity of TL than the bulkier side chains (I56, I71, V92) in the cavity of beta-lactoglobulin. The structural features provide an explanation for the promiscuous binding characteristics exhibited by TL. SDTF provides a general approach for determining the solution structure of many proteins and enhances homology modeling in the absence of high sequence identity.

No abstract

Large conductance voltage and Ca 2؉ -dependent K ؉ channels (BKCa) are activated by both membrane depolarization and intracellular Ca 2؉ . Recent studies on bacterial channels have proposed that a Ca 2؉ -induced conformational change within specialized regulators of K ؉ conductance (RCK) domains is responsible for channel gating. Each pore-forming ␣ subunit of the homotetrameric BKCa channel is expected to contain two intracellular RCK domains. The first RCK domain in BKCa channels (RCK1) has been shown to contain residues critical for Ca 2؉ sensitivity, possibly participating in the formation of a Ca 2؉ -binding site. The location and structure of the second RCK domain in the BKCa channel (RCK2) is still being examined, and the presence of a high-affinity Ca 2؉ -binding site within this region is not yet established. Here, we present a structure-based alignment of the C terminus of BK Ca and prokaryotic RCK domains that reveal the location of a second RCK domain in human BK Ca channels (hSloRCK2). hSloRCK2 includes a high-affinity Ca 2؉ -binding site (Ca bowl) and contains similar secondary structural elements as the bacterial RCK domains. Using CD spectroscopy, we provide evidence that hSloRCK2 undergoes a Ca 2؉ -induced change in conformation, associated with an ␣-to-␤ structural transition. We also show that the Ca bowl is an essential element for the Ca 2؉ -induced rearrangement of hSloRCK2. We speculate that the molecular rearrangements of RCK2 likely underlie the Ca 2؉ -dependent gating mechanism of BKCa channels. A structural model of the heterodimeric complex of hSloRCK1 and hSloRCK2 domains is discussed.BK channel ͉ circular dichroism ͉ MaxiK ͉ RCK ͉ structural modeling B K Ca channels are formed by the assembly of four identical pore-forming ␣ subunits. They can couple the membrane potential to the intracellular Ca 2ϩ level (1-4), playing critical roles in cell excitability, for example, by controlling smooth muscle tone and neurotransmitter release (1, 5-7). Each BK Ca ␣ subunit possesses a transmembrane voltage sensor (8-10) and two distinct high-affinity Ca 2ϩ sensors (11-15) located within the large intracellular carboxyl terminus. A well studied Ca 2ϩ -binding site corresponds to a C-terminal region that includes five consecutive negatively charged aspartates (D894-D898), christened the ''Ca bowl'' by the Salkoff laboratory (16,17). The Ca bowl binds Ca 2ϩ with high affinity (18-21) and strongly contributes to the channel's Ca 2ϩ sensitivity (18)(19)(20) [supporting information (SI) Fig. 6]. A second high-affinity Ca 2ϩ -sensing region that is impaired by neutralization of two aspartates (D362/D367) (11, 15) or methionine 513 (22) has been identified Ϸ400 aa upstream the Ca bowl.Most likely, these two high-affinity Ca 2ϩ -binding sites form parts of a complex functional domain that converts the free energy of Ca 2ϩ binding into mechanical work to open the channel. Indeed, specialized intracellular motifs regulating the conductance of K ϩ channels (RCK domains) have been recently described in prok...

No abstract

Tear lipocalin (TL), a major component of human tears, shows pH-dependent endogenous ligand binding. The structural and conformational changes associated with ligand release in the pH range of 7.5-3.0 are monitored by circular dichroism spectroscopy and site-directed tryptophan fluorescence. In the transition from pH 7.5 to pH 5.5, the ligand affinity for 16-(9-anthroyloxy)palmitic acid (16AP) and 8-anilino-1-naphthalenesulfonic acid is reduced. At pH 4.0 these ligands no longer bind within the TL calyx. From pH 7.3 to pH 3.0, the residues on loops CD and EF, which overhang the calyx entrance, show reduced accessibility to acrylamide. In addition resonance energy transfer is enhanced between residues on the two loops; the distance between the loops narrows. These findings suggest that apposition of the loops at low pH excludes the ligand from the intracavitary binding site. The conformational changes observed in transition from pH 7.3 to pH 3.0 for loops CD and EF are quite different. The CD loop shows less population reshuffling than the EF loop with an acidic environment, probably because backbone motion is restrained by the adjacent disulfide bond. The Trp fluorescence wavelength maximum (lambda(max)) reflects internal electrostatic interactions for positions on loops CD and EF. The titration curves of lambda(max) for mutants on the EF loop fit the Hendersen-Hasselbalch equation for two apparent pK(a) values, while the CD loop positions fit satisfactorily with one pK(a) value. Midpoints of transition for the binding affinity of TL tryptophan mutants to 16AP occur at pH 5.5-6.1. Replacement of each amino acid on either loop by single tryptophan mutation does not disrupt the pH-dependent binding affinity to 16AP. Taken together the data suggest that pH-driven ligand release involves ionization changes in several titratable residues associated with CD and EF loop apposition and occlusion of the calyx.