Protein aggregation/crystallization and minor structural changes: universal versus specific aspects

Protein association covers wide interests in biophysics, protein science, and biotechnologies, and it is often viewed as governed by conformation details. More recently, the existence of a universal physical principle governing aggregation/crystallization processes has been suggested by a series of experiments and shown to be linked to the universal scaling properties of concentration fluctuations occurring in the proximity of a phase transition (spinodal demixing in the specific case). Such properties have provided a quantitative basis for capturing kinetic association data on a universal master curve, ruled by the normalized distance of the state of the system from its instability region. Here we report new data on lysozyme crystal nucleation. They strengthen the evidence in favor of universality and show that the system enters the region of universal behavior in a stepwise manner as a result of minor conformation changes. Results also show that the link between conformation details and universal behavior is actuated by interactions mediated by the solvent. Outside the region of universal behavior, nucleation rates become unpredictable and undetectably long.     

Ca2+ channel blockers reverse iron overload by a new mechanism via divalent metal transporter-1

Hereditary hemochromatosis and transfusional iron overload are frequent clinical conditions associated with progressive iron accumulation in parenchymal tissues, leading to eventual organ failure. We have discovered a new mechanism to reverse iron overload-pharmacological modulation of the divalent metal transporter-1 (DMT-1). DMT-1 mediates intracellular iron transport during the transferrin cycle and apical iron absorption in the duodenum. Its additional functions in iron handling in the kidney and liver are less well understood. We show that the L-type calcium channel blocker nifedipine increases DMT-1-mediated cellular iron transport 10- to 100-fold at concentrations between 1 and 100 microM. Mechanistically, nifedipine causes this effect by prolonging the iron-transporting activity of DMT-1. We show that nifedipine mobilizes iron from the liver of mice with primary and secondary iron overload and enhances urinary iron excretion. Modulation of DMT-1 function by L-type calcium channel blockers emerges as a new pharmacological therapy for the treatment of iron overload disorders.     

A minimal transmembrane beta-barrel platform protein studied by nuclear magnetic resonance

In this study, we were concerned with the structural role of the surface-exposed extracellular loops of the N-terminal transmembrane (TM) domain of OmpA. A variant of the TM domain of outer membrane protein A (OmpA) with all four such loops shortened, which we call the beta-barrel platform (BBP), was successfully refolded. This indicates that the removed parts of the surface-exposed loops indeed do not contain amino acid sequences critical for this membrane protein's refolding in vitro. BBP has the potential to be used as a template beta-barrel membrane protein structure for the development of novel functions, although our results also highlight the potential difficulties that can arise when functionality is being engineered into the loop regions of membrane proteins. We have used solution nuclear magnetic resonance spectroscopy to determine the global fold of BBP+EF, BBP with a metal ion-binding EF-hand inserted in one of the shortened loops. BBP and BBP+EF in dihexanoylphosphatidylcholine micelles are eight-stranded antiparallel beta-barrels, and BBP represents the smallest beta-structured integral membrane protein known to date.     

Cellular trafficking of phospholamban and formation of functional sarcoplasmic reticulum during myocyte differentiation

Phospholamban (PLB) associates with the Ca²⁺-ATPase in sarcoplasmic reticulum (SR) membranes to permit the modulation of contraction in response to -adrenergic signaling. To understand how coordinated changes in the abundance and intracellular trafficking of PLB and the Ca²⁺-ATPase contribute to the maturation of functional muscle, we measured changes in abundance, location, and turnover of endogenous and tagged proteins in myoblasts and during their differentiation. We found that PLB is constitutively expressed in both myoblasts and differentiated myotubes, whereas abundance increases of the Ca²⁺-ATPase coincide with the formation of differentiated myotubes. We observed that PLB is primarily present in highly mobile vesicular structures outside the endoplasmic reticulum, irrespective of the expression of the Ca²⁺-ATPase, indicating that PLB targeting is regulated through vesicle trafficking. Moreover, using pulse-chase methods, we observed that in myoblasts, PLB is trafficked through directed transport through the Golgi to the plasma membrane before endosome-mediated internalization. The observed trafficking of PLB to the plasma membrane suggests an important role for PLB during muscle differentiation, which is distinct from its previously recognized role in the regulation of the Ca²⁺-ATPase. sarco(endo)plasmic reticulum calcium-adenosine triphosphatase; differentiation; C2C12 myocytes; vesicle trafficking     

Synthesis of alpha-cyclopiazonic acid by fungi of the genus Aspergillus

The presence of alpha-cyclopiazonic acid has been studied among metabolites of Aspergillus fungi. The study was performed with 138 cultures of 13 species obtained from the All-Russia Collection of Microorganisms and the collection of our institute. alpha-Cyclopiazonic acid was most frequently encountered among the metabolites of the section Flavi (the ability to synthesize alpha-cyclopiazonic acid was expressed in 61% of the strains of A. flavus, 83% of the strains of A. oryzae, and all strains of A. tamarii). This expression index for A. versicolor was less than 5%. We showed for the first time that alpha-cyclopiazonic acid is produced by A. fumigatus and A. phoenicis (expression in 30% of the strains of either species).