Characterization of the cholesteric phase of filamentous bacteriophage fd for molecular alignment.

Residual dipolar couplings arise from small degrees of alignment of molecules in a magnetic field and have proven to provide valuable structural information. Colloidal suspensions of rod-shaped viruses and bacteriophages constitute a frequently employed medium for imparting such alignment onto biomolecules. The stability and behavior of the liquid crystalline phases with respect to solution conditions such as pH, ionic strength, and temperature vary, and characterization should benefit practical applications as well as theoretical understanding. In this Communication we describe the pH dependence of the cholesteric liquid crystalline phase of the filamentous bacteriophage fd and demonstrate that the alignment tensor of the solute protein is modulated by pH. We also report the interesting observation that the relative sign of the residual dipolar coupling changes at low pH values. In addition, we demonstrate that the degree of alignment inversely scales with the lengths of the phage particles for phages with identical mass and charge per unit length.

Characterization of surfactant liquid crystal phases suitable for molecular alignment and measurement of dipolar couplings.

Media employed for imparting partial alignment onto solute molecules have recently attracted considerable attention, since they permit the measurement of NMR parameters for solute biomolecules commonly associated with solid state NMR. Here we characterize a medium which is based on a quasi-ternary surfactant system comprising cetylpyridinium bromide/hexanol/sodium bromide. We demonstrate that dilute solutions of this system can exist in liquid crystalline phases which orient in the magnetic field and allow the measurement of residual dipolar couplings under a variety of conditions. The present system is extremely versatile and robust, tolerating different buffer conditions, temperature ranges and concentrations.

Domain organization of the 39-kDa receptor-associated protein.

The 39-kDa receptor-associated protein (RAP) is an endoplasmic reticulum resident protein that binds to the low density lipoprotein receptor-related protein (LRP) as well as certain members of the low density lipoprotein receptor superfamily and antagonizes ligand binding. In order to identify important functional regions of RAP, studies were performed to define the domain organization and domain boundaries of this molecule. Differential scanning calorimetry (DSC) experiments revealed that the process of thermal denaturation of RAP is highly reversible and occurs in a broad temperature range with two well resolved heat absorption peaks. A good fit of the endotherm was obtained with four two-state transitions suggesting these many cooperative domains in the molecule. A number of recombinant fragments of RAP were expressed in bacteria, and their domain composition and stability were characterized by DSC, circular dichroism, and fluorescence spectroscopy. The results confirmed that RAP is composed of four independently folded domains, D1, D2, D3, and D4, that encompass residues 1-92, 93-163, 164-216, and 217-323, respectively. The first and the fourth domains preserved their structure and stability when isolated, whereas the compact structure of the fragment corresponding to D2 seems to be altered when isolated from the parent molecule. Isolated D3 was partially degraded during isolation from bacterial lysates. The isolated D4 was capable of binding with high affinity to LRP whereas neither D1 nor D2 bound. At the same time a fragment containing both D1 and D2 exhibited high affinity binding to LRP. These facts combined with the thermodynamic analysis of the melting process of the fragments containing D1 and D2 indicate that these two domains interact with each other and that the proper folding of the second domain into a native-like active conformation requires presence of the first domain.

Selective inhibition by kringle 5 of human plasminogen on endothelial cell migration, an important process in angiogenesis.

Angiogenesis is a multi-step process that includes endothelial cell proliferation, migration, basement membrane degradation, and new lumen organization. Angiostatin, an internal fragment of plasminogen comprising the first four triple disulfide-linked kringle structures, is one of the most potent endogenous angiogenesis inhibitors described to date. The kringle 5 domain of plasminogen, which shares high sequence homology with the four kringles of angiostatin, was previously shown to antagonize endothelial cell growth. We now describe that the recombinant kringle 5 of human plasminogen inhibits endothelial cell migration with an IC50 (concentration for half maximal inhibition) of approximately 500 nM. We demonstrate that the lysine-binding sites of kringle 5 may not be involved in its anti-migratory activities. The anti-migratory activity of kringle 5 is similar to that of angiostatin. Kringle 5 also shows selective inhibition on endothelial cells as opposed to other cell types. Relative to its native form, reduced kringle 5 displays a significant increase in anti-migratory activity, implying that the kringle conformation may shield kringle 5 from effectively interacting with endothelial cells. This report thus constitutes the first demonstration that kringle 5 of plasminogen is a selective inhibitor for endothelial cell migration.

Conformational studies of myo-inositol phosphates.

The discovery of the second messenger role of myo-inositol 1,4,5-trisdihydrogenphosphate [Ins(1,4,5)P3] has triggered tremendous interest in investigating the structure, metabolism, and biological roles of inositol phosphates. Although the conformation of phytic acid [(myo-inositol hexakisdihydrogenphosphate), Ins P6] has been the subject of much study, the conformations of lower inositol phosphates such as inositol-pentakis-, tetrakis-, and tris-dihydrogenphosphates have not been investigated. We investigated, by 1H NMR spectroscopy, the conformations of inositol phosphates (Ins P5, Ins P4, Ins P3, Ins P2, and Ins P1) and monitored the influence of pH on conformational preferences. Ins P6 adopts the sterically stable 1ax/5eq (one phosphate in the axial position and five phosphates in the equatorial position) conformation in the pH range 0.5-9.0, and the sterically hindered 5ax/1eq (five phosphates in the axial position and one phosphate in the equatorial position) conformation above pH 9.5. At pH 9.5, both conformations are in dynamic equilibrium. Ins(1,2,3,4,6)P5 and Ins(1,2,3,5,6)P5 adopt the 1ax/5eq form in the pH range 1.0-9.0; in the pH range 9.5-13.0, the 1ax/5eq and 5ax/1eq conformations are in dynamic equilibrium. In contrast to Ins P6 and Ins P5, all the lower inositol phosphates (Ins P4 to Ins P1) investigated adopt the 1ax/5eq conformation over the entire pH range, 1.0-13.0. Preference for the 5ax/1eq conformation by Ins P6 and Ins P5 is probably due to decreased electrostatic repulsion between negatively charged vicinal equatorial phosphates in the 1ax/5eq conformation and stabilization of the sterically hindered 5ax/1eq conformation by hydrogen bonding and/or sodium counter-ions bonding between the syn-oriented phosphates. On the basis of conformations adopted by the inositol phosphates (Ins P6 to Ins P1) at different pH, we conclude that the presence of four or five equatorial phosphates on the inositol ring induces a change in the conformation from the sterically unhindered 1ax/5eq structure to the sterically hindered 5ax/1eq conformation, at high pH. This investigation illustrates that the conformational preferences of inositol phosphates at different pH is unique to the particular isomer and does not parallel the behaviour of phytic acid.

Application of two-dimensional total correlation spectroscopy for structure determination of individual inositol phosphates in a mixture.

The discovery of the second messenger role of myoinositol (1,4,5)trisphosphate has triggered tremendous interest in investigating the structures and metabolism of inositol phosphates. The structures of these compounds are established by first purifying the compounds of interest and then identifying the structures through chemical degradation or NMR analysis. In this paper we describe a method that allows simultaneous determination, without separation, of the structures of multiple inositol phosphates in a mixture. The use of two-dimensional total correlation spectroscopy (2D TOCSY) experiments together with subspectra extracted from 2D TOCSY data allowed us to determine, in 3 to 4 h, the structures of five compounds in a mixture, without prior separation.