Intein-mediated cyclization of bacterial acyl carrier protein stabilizes its folded conformation but does not abolish function.

Bacterial acyl carrier protein (ACP) is essential for the synthesis of fatty acids and serves as the major acyl donor for the formation of phospholipids and other lipid products. Acyl-ACP encloses attached fatty acyl groups in a hydrophobic pocket within a four-helix bundle, but must at least partially unfold to present the acyl chain to the active sites of its multiple enzyme partners. To further examine the constraints of ACP structure and function, we have constructed a cyclic version of Vibrio harveyi ACP, using split-intein technology to covalently join its closely apposed N and C termini. Cyclization stabilized ACP in a folded helical conformation as indicated by gel electrophoresis, circular dichroism, fluorescence, and mass spectrometry. Molecular dynamics simulations also indicated overall decreased polypeptide chain mobility in cyclic ACP, although no major conformational rearrangements over a 10-ns period were noted. In vivo complementation assays revealed that cyclic ACP can functionally replace the linear wild-type protein and support growth of an Escherichia coli ACP-null mutant strain. Cyclization of a folding-deficient ACP mutant (F50A) both restored its ability to adopt a folded conformation and enhanced complementation of growth. Our results thus suggest that ACP must be able to adopt a folded conformation for biological activity, and that its function does not require complete unfolding of the protein.

Remodeling of ryanodine receptor complex causes "leaky" channels: a molecular mechanism for decreased exercise capacity.

During exercise, defects in calcium (Ca2+) release have been proposed to impair muscle function. Here, we show that during exercise in mice and humans, the major Ca2+ release channel required for excitation-contraction coupling (ECC) in skeletal muscle, the ryanodine receptor (RyR1), is progressively PKA-hyperphosphorylated, S-nitrosylated, and depleted of the phosphodiesterase PDE4D3 and the RyR1 stabilizing subunit calstabin1 (FKBP12), resulting in "leaky" channels that cause decreased exercise tolerance in mice. Mice with skeletal muscle-specific calstabin1 deletion or PDE4D deficiency exhibited significantly impaired exercise capacity. A small molecule (S107) that prevents depletion of calstabin1 from the RyR1 complex improved force generation and exercise capacity, reduced Ca2+-dependent neutral protease calpain activity and plasma creatine kinase levels. Taken together, these data suggest a possible mechanism by which Ca2+ leak via calstabin1-depleted RyR1 channels leads to defective Ca2+ signaling, muscle damage, and impaired exercise capacity.

Tryptophan fluorescence reveals induced folding of Vibrio harveyi acyl carrier protein upon interaction with partner enzymes.

We have introduced tryptophan as a local fluorescent probe to monitor the conformation of Vibrio harveyi acyl carrier protein (ACP), a small flexible protein that is unfolded at neutral pH but must undergo reversible conformational change during the synthesis and delivery of bacterial fatty acids. Consistent with known 3D structures of ACP, steady-state fluorescence and quenching experiments indicated that Trp at positions 46, 50, and 72 are buried in the hydrophobic core upon Mg(2+)-induced ACP folding, whereas residues 25 and 45 remain in a hydrophilic environment on the protein surface. Attachment of fatty acids to the phosphopantetheine prosthetic group progressively stabilized the folded conformation of all Trp-substituted ACPs, but longer chains (14:0) were less effective than medium chains (8:0) in shielding Trp from acrylamide quenching in the L46W protein. Interaction with ACP-dependent enzymes LpxA and holo-ACP synthase also caused folding of L46W; fluorescence quenching indicated proximity of Trp-45 in helix II of ACP in LpxA binding. Our results suggest that divalent cations and fatty acylation produce differing environments in the ACP core and also reveal enzyme partner-induced folding of ACP, a key feature of "natively unfolded" proteins.

Electrospray ionization mass spectra of acyl carrier protein are insensitive to its solution phase conformation.

Electrospray ionization mass spectrometry (ESI-MS) can be used to monitor conformational changes of proteins in solution based on the charge state distribution (CSD) of the corresponding gas-phase ions, although relatively few studies of acidic proteins have been reported. Here, we have compared the CSD and solution structure of recombinant Vibrio harveyi acyl carrier protein (rACP), a small acidic protein whose secondary and tertiary structure can be manipulated by pH, fatty acylation, and site-directed mutagenesis. Circular dichroism and intrinsic fluorescence demonstrated that apo-rACP adopts a folded helical conformation in aqueous solution below pH 6 or in 50% acetonitrile/0.1% formic acid, but is unfolded at neutral and basic pH values. A rACP mutant, in which seven conserved acidic residues were replaced with their corresponding neutral amides, was folded over the entire pH range of 5 to 9. However, under the same solvent conditions, both wild type and mutant ACPs exhibited similar CSDs (6(+)-9(+) species) at all pH values. Covalent attachment of myristic acid to the phosphopantetheine prosthetic group of rACP, which is known to stabilize a folded conformation in solution, also had little influence on its CSD in either positive or negative ion modes. Overall, our results are consistent with ACP as a "natively unfolded" protein in a dynamic conformational equilibrium, which allows access to (de)protonation events during the electrospray process.

Acquired inhibitors to factor VIII and fibrinogen in the setting of T-cell large granular lymphocyte leukemia: a case report and review of the literature.

Large granular lymphocyte (LGL) leukemia is an indolent lymphoproliferative malignancy which dysregulates humoral immunity and underlies the myriad autoimmune phenomena. We describe a 62-year-old woman with Felty's syndrome who developed a severe bleeding diathesis. Laboratory evaluation demonstrated acquired inhibitors to both factor VIII (FVIII) and fibrinogen, likely secondary to T-cell LGL leukemia. After a complicated course, the patient's inhibitors were extinguished with rituximab and high-dose corticosteroids. Bleeding was controlled with alternating FEIBA (factor eight inhibitor bypassing activity) and recombinant activated FVII. This report reviews the literature comparing the efficacy of various treatment modalities for both disorders. To our knowledge, this is the first reported case of a patient with LGL leukemia acquiring an inhibitor to FVIII or fibrinogen.