An intensively investigated intermediate state of protein folding is the molten globule (MG) state, which contains secondary but hardly any tertiary structure. In previous work, we have determined the distances between interacting spins within maltose binding protein (MBP) in its native state using continuous wave and double electron-electron resonance (DEER) electron paramagnetic resonance (EPR) spectroscopy. Seven double mutants had been employed to investigate the structure within the two domains of MBP. DEER data nicely corroborated the previously available X-ray data. Even in its MG state, MBP is known to still bind its ligand maltose. We therefore hypothesized that there must be a defined structure around the binding pocket of MBP already in the absence of tertiary structure. Here we have investigated the functional and structural difference between native and MG state in the open and closed form with a new set of MBP mutants. In these, the spin-label positions were placed near the active site. Binding of its ligands leads to a conformational change from open to closed state, where the two domains are more closely together. The complete set of MBP mutants was analyzed at pH 3.2 (MG) and pH 7.4 (native state) using double-quantum coherence EPR. The values were compared with theoretical predictions of distances between the labels in biradicals constructed by molecular modeling from the crystal structures of MBP in open and closed form and were found to be in excellent agreement. Measurements show a defined structure around the binding pocket of MBP in MG, which explains maltose binding. A new and important finding is that in both states ligand-free MBP can be found in open and closed form, while ligand-bound MBP appears only in closed form because of maltose binding.
Mouse islets lacking TALK-1 (KO) exhibit accelerated Ca 2þ c oscillations and enhanced insulin secretion relative to controls (WT). The Ca 2þ c oscillation frequency is affected by K slow , a repolarizing Ca 2þ -dependent K þ current. K slow is reduced in KO b-cells, possibly contributing to the accelerated Ca 2þ c oscillations of KO islets. Since TALK-1 activity is not regulated by Ca 2þ c , we assessed whether TALK-1 impacts Ca 2þ release from endoplasmic reticulum (ER) stores (Ca 2þ ER ), which modulates K slow . We find lower basal Ca 2þ c in KO b-cells, and application of the SERCA inhibitor CPA causes a greater increase in Ca 2þ c in KO versus WT b-cells. These observations suggest that TALK-1 regulates Ca 2þ ER . Therefore, we tested how heterologous expression of TALK-1 in HEK293 cells affects Ca 2þ ER . TALK-1 overexpression significantly increases basal Ca 2þ c and reduces the CPA-induced Ca 2þ c response. However, expression of the K2P channels TREK-1 or TREK-2 does not affect Ca 2þ ER . Furthermore, K þ -impermeable mutant TALK-1 channels do not recapitulate Ca 2þ ER defects, thus, TALK-1 K þ channel function presumably modulates Ca 2þ ER . Consistent with an intracellular role for TALK-1, fluorescence microscopy reveals expression of TALK-1 in the b-cell ER. Additionally, nuclear patch-clamp experiments demonstrate the presence of functional TALK-1 channels on the outer nuclear membrane, which is continuous with the ER. Moreover, specific inhibition of endogenous TALK-1 channels with expression of a K þ -impermeable mutant increased human b-cell Ca 2þER . Together, our data suggest that TALK-1 channel activity augments islet-cell Ca 2þ ER ''leak.'' Altered TALK-1 channel function may perturb islet-cell Ca 2þ ER , which can affect Ca 2þ c handling, hormone release, and contribute to disease pathogenesis.
Employing site-directed spin labeling (SDSL), the structure of maltose-binding protein (MBP) had previously been studied in the native state by electron paramagnetic resonance (EPR) spectroscopy. Several spin-labeled double cysteine mutants were distributed all over the structure of this cysteine-free protein and revealed distance information between the nitroxide residues from double electron–electron resonance (DEER). The results were in good agreement with the known X-ray structure. We have now extended these studies to the molten globule (MG) state, a folding intermediate, which can be stabilized around pH 3 and that is characterized by secondary but hardly any tertiary structure. Instead of clearly defined distance features as found in the native state, several additional characteristics indicate that the MG structure of MBP contains different polypeptide chain and domain orientations. MBP is also known to bind its substrate maltose even in MG state although with lower affinity. Additionally, we have now created new mutants allowing for spin labeling at or near the active site. Our data confirm an already preformed ligand site structure in the MG explaining its substrate binding capability and thus most probably serving as a nucleation center for the final native structure.
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