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The results from characterization of the conformational change and secondary chemical shift analysis of endosulfine-[alpha] (ENSA), a 121-residue cAMP-regulated phosphoprotein, are presented. ENSA, originally identified as an endogenous regulator of ATP-sensitive potassium channels, is implicated in the regulation of insulin secretion, decrease of expression of ENSA in brains of both Alzheimer's disease (AD) and Down's syndrome patients.

The potential capability of magic-angle spinning (MAs) solid-state NMR (SSNMR) to study the formulation chemistry of industrial and therapeutic proteins is demonstrated. The method could also be utilized to understand the phenomenon of single-crystal growth of proteins.

The through-bond correlation in solids is implemented in an efficient constant time manner, with the indirect evolution and transfer periods combined into a single constant time interval, which provides increased spectral resolution without compromising sensitivity. In the beta1 immunoglobulin binding domain of protein G (GB1), the method has allowed to resolve peaks that are otherwise unresolved and to make assignments in the absence of multibond transfers.

ä-Synuclein (AS) is an intrinsically unstructured protein in aqueous solution but is capable of forming-sheet-rich fibrils that accumulate as intracytoplasmic inclusions in Parkinson disease and certain other neurological disorders. However, AS binding to phospholipid membranes leads to a distinct change in protein conformation, stabilizing an extended amphipathic ä-helical domain reminiscent of the exchangeable apolipoproteins. To better understand the significance of this conformational change, we devised a novel bacteriophage display screen to identify protein binding partners of helical AS and have identified 20 proteins with roles in diverse cellular processes related to membrane trafficking, ion channel modulation, redox metabolism, and gene regulation. To verify that the screen identifies proteins with specificity for helical AS, we further characterized one of these candidates, endosulfine a (ENSA), a small cAMP-regulated phosphoprotein implicated in the regulation of insulin secretion but also expressed abundantly in the brain. We used solution NMR to probe the interaction between ENSA and AS on the surface of SDS micelles. Chemical shift perturbation mapping experiments indicate that ENSA interacts specifically with residues in the N-terminal helical domain of AS in the presence of SDS but not in aqueous buffer lacking SDS. The ENSA-related protein ARPP-19 (cAMP-regulated phosphoprotein 19) also displays specific interactions with helical AS, These results confirm that the helical N terminus of AS can mediate specific interactions with other proteins and suggest that membrane binding may regulate the physiological activity of AS in vivo. [ABSTRACT FROM AUTHOR]

The article focuses on the advantages of using constant-time correlation spectroscopy for assigning protein resonances with solid-state NMR spectroscopy. According to the authors, superior resolution is offered by the constant-time correlation spectroscopy as compared to that of directly evolved experiments. Moreover, the sensitivity is also increased and the sequence performs better than the commonly used dipolar methods.

Alpha-synuclein (AS) is an intrinsically unstructured protein in aqueous solution but is capable of forming beta-sheet-rich fibrils that accumulate as intracytoplasmic inclusions in Parkinson disease and certain other neurological disorders. However, AS binding to phospholipid membranes leads to a distinct change in protein conformation, stabilizing an extended amphipathic alpha-helical domain reminiscent of the exchangeable apolipoproteins. To better understand the significance of this conformational change, we devised a novel bacteriophage display screen to identify protein binding partners of helical AS and have identified 20 proteins with roles in diverse cellular processes related to membrane trafficking, ion channel modulation, redox metabolism, and gene regulation. To verify that the screen identifies proteins with specificity for helical AS, we further characterized one of these candidates, endosulfine alpha (ENSA), a small cAMP-regulated phosphoprotein implicated in the regulation of insulin secretion but also expressed abundantly in the brain. We used solution NMR to probe the interaction between ENSA and AS on the surface of SDS micelles. Chemical shift perturbation mapping experiments indicate that ENSA interacts specifically with residues in the N-terminal helical domain of AS in the presence of SDS but not in aqueous buffer lacking SDS. The ENSA-related protein ARPP-19 (cAMP-regulated phosphoprotein 19) also displays specific interactions with helical AS. These results confirm that the helical N terminus of AS can mediate specific interactions with other proteins and suggest that membrane binding may regulate the physiological activity of AS in vivo.