Arne Gericke

Importance of Phosphorylation for Osteopontin Regulation of Biomineralization

Previous in vitro and in vivo studies demonstrated that osteopontin (OPN) is an inhibitor of the formation and growth of hydroxyapatite (HA) and other biominerals. The present study tests the hypotheses that the interaction of OPN with HA is determined by the extent of protein phosphorylation and that this interaction regulates the mineralization process. Bone OPN as previously reported inhibited HA formation and HA-seeded growth in a gelatin-gel system. A transglutaminase-linked OPN polymer had similar effects. Recombinant, nonphosphorylated OPN and chemically dephosphorylated OPN, had no effect on HA formation or growth in this system. In contrast, highly phosphorylated milk OPN (mOPN) promoted HA formation. The mOPN stabilized the conversion of amorphous calcium phosphate (a noncrystalline constituent of milk) to HA, whereas bone OPN had a lesser effect on this conversion. Mixtures of OPN and osteocalcin known to form a complex in vitro, unexpectedly promoted HA formation. To test the hypothesis that small alterations in protein conformation caused by phosphorylation account for the differences in the observed ability of OPN to interact with HA, the conformation of bone OPN and mOPN in the presence and absence of crystalline HA was determined by attenuated total reflection (ATR) infrared (IR) spectroscopy. Both proteins exhibited a predominantly random coil structure, which was unaffected by the addition of Ca2+. Binding to HA did not alter the secondary structure of bone OPN, but induced a small increase of β-sheet (few percent) in mOPN. These data taken together suggest that the phosphorylation of OPN is an important factor in regulating the OPN-mediated mineralization process.

External Infrared Reflection Absorption Spectrometry of Monolayer Films at the Air-Water Interface

The theory and practice of external infrared reflection absorption spectrometry (IRRAS) as applied to monomolecular films at the air-water interface are reviewed. The observed IR frequencies for films of amphiphilic species provide information about the conformational states of the hydrocarbon chains and the hydrogen bonding and ionization states of the polar head groups, under conditions of controlled surface pressure. Determination of molecular orientation is also feasible and requires detailed consideration of the reflection-absorption properties of the three- phase (air-monolayer-water) system. Current theoretical approaches are described. Applications of IRRAS to the study of single- and double-chain amphiphiles and proteins are reviewed, and initial excursions into biochemistry (interfacial enzyme catalysis) and physiology (pulmonary surfactant function) are reported.

Ionization properties of phosphatidylinositol polyphosphates in mixed model membranes.

Phosphatidylinositol polyphosphate lipids (phosphoinositides) form only a minor pool of membrane phospholipids but are involved in many intracellular signaling processes, including membrane trafficking, cytoskeletal remodeling, and receptor signal transduction. Phosphoinositide properties are largely determined by the characteristics of their headgroup, which at physiological pH is highly charged but also capable of forming hydrogen bonds. Many proteins have developed special binding domains that facilitate specific binding to particular phosphoinositides, while other proteins interact with phosphoinositides via nonspecific electrostatic interactions. Despite its importance, only limited information is available about the ionization properties of phosphoinositides. We have investigated the pH-dependent ionization behavior of all three naturally occurring phosphatidylinositol bisphosphates as well as of phosphatidylinositol 3,4,5-trisphosphate in mixed phosphoinositide/phosphatidylcholine vesicles using magic angle spinning (31)P NMR spectroscopy. For phosphatidylinositol 3,5-bisphosphate, where the two phosphomonoester groups are separated by a hydroxyl group at the 4-position, the pH-dependent chemical shift variation can be fitted with a Henderson-Hasselbalch-type formalism, yielding pK(a)(2) values of 6.96 +/- 0.04 and 6.58 +/- 0.04 for the 3- and 5-phosphates, respectively. In contrast, phosphatidylinositol 3,4-bisphosphate [PI(3,4)P(2)] as well as phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] show a biphasic pH-dependent ionization behavior that cannot be explained by a Henderson-Hasselbalch-type formalism. This biphasic behavior can be attributed to the sharing of the last remaining proton between the vicinal phosphomonoester groups. At pH 7.0, the overall charge (including the phosphodiester group charge) is found to be -3.96 +/- 0.10 for PI(3,4)P(2) and -3.99 +/- 0.10 for PI(4,5)P(2). While for PI(3,5)P(2) and PI(4,5)P(2) the charges of the individual phosphate groups in the molecule differ, they are equal for PI(3,4)P(2). Differences in the charges of the phosphomonoester groups can be rationalized on the basis of the ability of the respective phosphomonoester group to form intramolecular hydrogen bonds with adjacent hydroxyl groups. Phosphatidylinositol 3,4,5-trisphosphate shows an extraordinary complex ionization behavior. While at pH 4 the (31)P NMR peak of the 4-phosphate is found downfield from the other two phosphomonoester group peaks, an increase in pH leads to a crossover of the 4-phosphate, which positions this peak eventually upfield from the other two peaks. As a result, the 4-phosphate group shows a significantly lower charge at pH values between 7 and 9.5 than the other two phosphomonoester groups. The charge of the respective phosphomonoester group in PI(3,4,5)P(3) is lower than the corresponding charge of the phosphatidylinositol bisphosphate phosphomonoester groups, leading to an overall charge of PI(3,4,5)P(3) of -5.05 +/- 0.15 at pH 7.0. The charge of all investigated phosphoinositides at pH 7.0 is equal or higher than the corresponding charge of soluble inositol polyphosphate headgroup analogues, which is the opposite of what is expected on the basis of simple electrostatic considerations. This higher than expected headgroup charge can be rationalized with mutual intermolecular hydrogen bond formation. Measurements using different concentrations of PI(4,5)P(2) in the lipid vesicles (1, 5, and 20 mol %) did not reveal any significant concentration-dependent shift of the two phosphomonoester peaks, suggesting that PI(4,5)P(2) is clustered even at 1 mol %.

PTEN phosphatase selectively binds phosphoinositides and undergoes structural changes.

PTEN (phosphatase and tensin homologue deleted on chromosome 10) is a tumor suppressor that is mutated or deleted in a variety of human tumors, and even loss of only one PTEN gene profoundly affects carcinogenesis. PTEN encodes a phosphatidylinositol phosphate phosphatase specific for the 3-position of the inositol ring. Despite its importance, we are just beginning to understand the regulatory circuits that maintain the correct levels of PTEN phosphatase activity. Several independent studies reported that PI(4,5)P2 enhances PTEN phosphatase activity, but the reasons for this enhancement are currently being debated. In this study, PTEN bound to PI(4,5)P2-bearing vesicles has increased alpha-helicity, providing direct spectroscopic proof of a conformational change. Neither PI(3,5)P2 nor PI(3,4,5)P3 induced this conformational change. On the basis of experiments with two mutant PTEN proteins, it is shown that PI(4,5)P2 induces this conformational change by binding to the PTEN N-terminal domain. Using PTEN protein and a 21-amino acid peptide based on the PTEN N-terminus, we tested all natural phosphatidylinositol phosphates and found preferential binding of PI(4,5)P2. PTEN also binds to phosphatidylserine-bearing vesicles, resulting in a slight increase in beta-sheet content. In addition, PTEN binds synergistically to PI(4,5)P2 and phosphatidylserine, and hence, these anionic lipids do not compete for PTEN binding sites. Collectively, these results demonstrate that PTEN binds to membranes through multiple sites, but only PI(4,5)P2 binding to the N-terminal domain triggers a conformational change with increased alpha-helicity.

Different Forms of DMP1 Play Distinct Roles in Mineralization

Dentin matrix protein-1 (DMP1) is a major synthetic product of hypertrophic chondrocytes and osteocytes. Previous in vitro studies showed full-length DMP1 inhibits hydroxyapatite (HA) formation and growth, while its N-terminal fragment (37K) promotes HA formation. Since there are 3 fragments within the mineralized tissues [N-terminal, C-terminal (57K), and a chondroitin-sulfate-linked N-terminal fragment (DMP1-PG)], we predicted that each would have a distinct effect on mineralization related to its interaction with HA. In a gelatin-gel system, 37K and 57K fragments were both promoters of HA formation and growth; DMP1-PG was an inhibitor. The secondary structures of the 3 fragments and the full-length protein in the presence and absence of Ca2+ and HA determined by FTIR showed that the full-length protein undergoes slight conformational changes on binding to HA, while 37K, 57K, and DMP1-PG do not change conformation. These findings indicate that distinct forms of DMP1 may work collectively in controlling the mineralization process.

The effect of cations on the order of saturated fatty acid monolayers at the air-water interface as determined by infrared reflection-absorption spectrometry

Abstract The influence of the divalent cations cadmium, lead and calcium on the properties of octadecanoic acid monolayers at the air-water interface is studied by infrared reflection-absorption spectrometry. The investigation is carried out for different cation concentrations and pH values of the subphase. It is shown for pH 6 that 1 mM Cd2+ or 1 mM Pb2+ leads to the formation of highly ordered structures even for large areas/molecule, while the ordering effect of Ca2+ at the same pH value is smaller. For the first time, a detailed infrared analysis of the spectral range between 1600 cm−1 and 1400 cm−1, which is of particular importance for carboxylate monolayers at the air-water interface, is presented. For all cations various bands representing the antisymmetric and symmetric carboxylate vibrations are observed indicating the heterogenic character of the monolayer. Ca2+ interacts mainly ionically with the carboxylate, although a small amount is presumably bound covalently. Cd2+ and Pb2+, however, are largely covalently coordinated with the carboxyl group at pH 6, resulting presumably in a chelating bidentate structure. It is proposed that upon reducing the pH value the Pb2+ chelate is broken, i.e. the carboxylate anion is being formed, and that finally the undissociated fatty acid becomes dominant.

Characterization of biological samples by two-dimensional infrared spectroscopy: Simulation of frequency, bandwidth, and intensity changes

Two-dimensional infrared (2D IR) spectroscopy has been shown to be a powerful tool for the analysis of spectra with highly overlapped bands, as often found in IR spectra of biological samples. To date, most 2D IR analyses have focused primarily on intensity changes of the bands under investigation. However, information concerning 2D IR characteristics of bands that change in position or width is sparse. We have thus simulated the effects of frequency and bandwidth changes on 2D IR spectra. In the synchronous plot of a band undergoing a frequency shift, two autopeaks and two crosspeaks are found at the initial and final positions, while the asynchronous plot exhibits two weaker crosspeaks for these positions and a stronger, somewhat elongated feature close to the diagonal. The latter feature is characteristic of a shifting band. Thus, to distinguish a frequency shift in a single band from intensity changes of two overlapped bands it is important to examine the asynchronous plot, since the synchronous plots exhibit comparable characteristics in both cases. A bandwidth change results in a series of crosspeaks. However, when bandwidth changes are coupled with either frequency shifts and/or intensity changes, the effect of the bandwidth change is reduced. Finally, it is shown that the resolution enhancement generally found for the asynchronous plot is accompanied by an error in the positions of the original spectral features as determined from 2D IR peaks. The magnitude of the error increases as the original spectral features approach each other in frequency.

Phosphorylation keeps PTEN phosphatase closed for business

Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a phosphatase that suppresses many tumor types (1, 2). It plays a major role in the development of the nervous system and has been implicated in diseases such as autism (3). Despite its importance, we are only beginning to understand how this important protein is regulated. In 2000 and 2001, Vazquez et al. (4, 5) with great insight proposed that phosphorylation of the C terminus induces PTEN to assume a closed conformation with an inactive phosphatase domain. Upon dephosphorylation, PTEN assumes an open conformation, activating the phosphatase domain and enhancing degradation of PTEN protein. In this view, the phosphorylated closed form acts like a proenzyme, which is stable in the cytoplasm but ready for rapid activation, use, and degradation. Although not fully proven, this model (Fig. 1) has greatly influenced the PTEN field. The study in a recent issue of PNAS by Rahdar et al. (6) provides convincing proof for this model and new exciting insights into the interactions of PTEN with biological membranes.

Polarized external infrared reflection-absorption spectrometry at the air/water interface: Comparison of experimental and theoretical results for different angles of incidence

Abstract Hexadecan-1-ol monolayers were investigated at the air/water interface by external infrared reflection-absorption spectrometry. It is shown that the molecules are oriented vertically, packed in a hexagonal subcell and prefer an all- trans conformation. The symmetrical and anti-symmetrical methylene stretching vibrations are investigated for p- and s-polarization and different angles of incidence. For s-polarization the reflection-absorption decreases with increasing angle of incidence, whereas for p-polarization the band converts its sign from negative to positive near the Brewster angle. An optical model is derived which describes the reflection-absorption for p- and s-polarization at different angles of incidence for an anisotropic, inhomogeneous surface layer on an isotropic low-absorbing substrate. Comparison of the curves calculated by this model with experimental results shows good agreement for s-polarization, whereas for p-polarization a discrepancy between experimental and theoretical data is observed in the range 40–70°. Possible reasons for this deviation are discussed. In particular, evidence is presented that for low-absorbing substrates variations of the components of the real and imaginary parts of the refractive indices are closely related to strong changes of the reflection-absorption of p-polarized radiation near the Brewster angle.

Classification of sea slicks by multifrequency radar techniques: New chemical insights and their geophysical implications

Sea slick experiments with an airborne five-frequency radar scatterometer were performed in the presence of surface active substances that represent different fractions of biogenic slicks (fats, amines, sugar derivatives, and fatty acids). Measurements at water temperatures of 282.2 K (9.0°C) and 290.6 K (17.4°C) showed that temperature effects appear to play a secondary role for slick-induced water wave damping, at least in the temperature range encountered during the present experiments. Different procedures of slick generation, with and without application of a spreading solvent, indicated that the wave-damping effect in the short-gravity/capillary wave range, and thus the modification of backscattered radar signals, is not only dependent on the chemical structure but also on the arrangement and distribution (morphology; formation of domains) of the surface-active compounds. Thus far this aspect, which appears to be of particular importance for biogenic sea slicks, has been completely ignored. External infrared reflection-absorption spectroscopy laboratory measurements with infrared radiation in the wavelength range between 3.3 μm and 7 μm enabled us allowed to form a link between some important elements of the morphological structure of the monolayers and their viscoelastic characteristics, which are closely related to the wave-damping effect of surface active compounds and to the compounds influence on remote sensing signals. Furthermore, the IRAS measurements supplied detailed insight into the relaxation process that occurs during the generation of a sea slick and on a slick-covered undulating water surface. In particular, strong hydration/dehydration effects appear to play an important role.