Quantitative measurement of water diffusion lifetimes at a protein/DNA interface by NMR

Hydration site lifetimes of slowly diffusing water molecules at the protein/DNA interface of the vnd/NK-2 homeodomain DNA complex were determined using novel three-dimensional NMR techniques. The lifetimes were calculated using the ratios of ROE and NOE cross-relaxation rates between the water and the protein backbone and side chain amides. This calculation of the lifetimes is based on a model of the spectral density function of the water-protein interaction consisting of three timescales of motion: fast vibrational/rotational motion, diffusion into/out of the hydration site, and overall macromolecular tumbling. The lifetimes measured ranged from approximately 400 ps to more than 5 ns, and nearly all the slowly diffusing water molecules detected lie at the protein/DNA interface. A quantitative analysis of relayed water cross-relaxation indicated that even at very short mixing times, 5 ms for ROESY and 12 ms for NOESY, relay of magnetization can make a small but detectable contribution to the measured rates. The temperature dependences of the NOE rates were measured to help discriminate direct dipolar cross-relaxation from chemical exchange. Comparison with several X-ray structures of homeodomain/DNA complexes reveals a strong correspondence between water molecules in conserved locations and the slowly diffusing water molecules detected by NMR. A homology model based on the X-ray structures was created to visualize the conserved water molecules detected at the vnd/NK-2 homeodomain DNA interface. Two chains of water molecules are seen at the right and left sides of the major groove, adjacent to the third helix of the homeodomain. Two water-mediated hydrogen bond bridges spanning the protein/DNA interface are present in the model, one between the backbone of Phe8 and a DNA phosphate, and one between the side chain of Asn51 and a DNA phosphate. The hydrogen bond bridge between Asn51 and the DNA might be especially important since the DNA contact made by the invariant Asn51 residue, seen in all known homeodomain/DNA structures, is critical for binding affinity and specificity.     

X-ray diffraction of a protein crystal anchored at the air/water interface.

We report the first successful in situ x-ray diffraction experiment with a 2D protein array at the lipid/water interface and demonstrate that the order can be controlled via lateral pressure or density. A protein (streptavidin) was bound to a monolayer of biotinylated lipid at the air/water interface, and diffraction of the protein layer could be measured to many orders. Compression of the monolayer changed the diffraction pattern drastically, indicating that the protein structure can be strongly influenced by external parameters like lateral pressure or density. From the width of the peaks, we find that aggregates consisting of as few as 100 monomers contribute to the diffraction. This indicates that the structure of even low order aggregates can be studied in situ. Grazing incidence diffraction can become a strong new method to study the crystallization and the interactions between proteins free from artifacts by staining or sample preparation.     

Humicola lanuginosa lipase hydrolysis of mono-oleoyl-rac-glycerol at the lipid-water interface observed by atomic force microscopy

A new type of planar lipid substrate for Humicola lanuginosa lipase (HLL) has been prepared by depositing a monolayer of 1-mono-oleoyl-rac-glycerol (MOG) on top of a monolayer of dipalmitoyl-phosphatidylcholine (DPPC) on mica by the Langmuir-Blodgett (LB) technique. The bilayer was subsequently exposed to HLL in a liquid cell of an atomic force microscope (AFM) allowing the time course of the lipolytic degradation to be observed. By analysing a series of AFM images, we find that enzymes are preferentially activated at the edge of nano-scale defects present in the bilayer prior to enzyme injection, while defect-free areas of the substrate are surprisingly stable towards enzyme degradation. The initial rate of hydrolysis is found to be proportional to the perimeter length, P, of the initial nano-scale defects as well as the bulk enzyme concentration, c(HLL); d(lipid)/dt=k P c(HLL). We estimate the specific rate of MOG hydrolysis by HLL to be 2.5x10(4) MOG molecules/(minute x molecule of HLL).     

An analysis of the interaction of protein with lipid monolayers at the air/water interface

1. Measurements have been made of the interaction of cytochrome c, bovine serum albumin and synthetic oxytocin with low-pressure (2dyn/cm) monolayers of stearic acid, phosphatidylcholine and phosphatidylethanolamine. 2. [(14)C]Carboxymethylation of the cytochrome c and albumin followed by surface-radioactivity determinations have shown that only a proportion of the protein added to the subphase is bound to the monolayers and that initially the degree of binding is dependent on the protein concentration. The binding is irreversible in the sense that the adsorbed protein cannot be removed by transferring the film containing the interacted protein to a fresh subphase containing no protein. 3. Three successive types of interaction can usually be recognized. (a) Initially, whole molecules of protein penetrate the lipid film and occupy the same area as those of the protein spread at the air/water interface. (b) Above certain film pressures a part of each protein molecule, probably hydrophobic side chains, penetrates the film. The change in surface pressure per unit of bound protein is much smaller than in (a). (c) At higher film pressures, adsorption without penetration occurs. With cytochrome c this is initially dependent on a favourable electrostatic interaction.     

Adsorption properties of proteins at and near the air/water interface from IRRAS spectra of protein solutions

A detailed study is performed using infrared reflection absorption spectroscopy (IRRAS) to characterize the molecular behaviour of proteins at and near the air/water interface of protein solutions. IRRAS spectra of beta-casein solutions in H2O and D2O show spectral shifts and derivative-like features not commonly observed in monomolecular layer systems. They can be fully understood using optical theory. Fair agreement between experimental and simulated IRRAS spectra over a broad spectral range (4000-1000 cm(-1)) is obtained using a stratified layer model. An attenuated total reflection and transmission spectrum is used to represent the protein extinction coefficient in H2O and D2O, respectively. It is shown that the derivative-like features observed result from the reflective properties of the proteins themselves. Furthermore, both concentration and film thickness could be fitted. At high protein concentrations (100 mg/mL) the spectrum is that of a single homogeneous protein solution. At 0.1 mg/mL, beta-casein is accumulated at the surface in a thin layer of approximately 10 nm thickness, with a concentration about 2500 times higher than in the sub-phase. At an initial concentration of 10 mg/mL, the concentration in the surface layer is about 15 times higher than in the subphase, while the thickness is about 30 nm.     

Structure of poly(ethylene glycol)-modified horseradish peroxidase in organic solvents: infrared amide I spectral changes upon protein dehydration are largely caused by protein structural changes and not by water removal per se

Fourier transform infrared (FTIR) spectroscopy has emerged as a powerful tool to guide the development of stable lyophilized protein formulations by providing information on the structure of proteins in amorphous solids. The underlying assumption is that IR spectral changes in the amide I and III region upon protein dehydration are caused by protein structural changes. However, it has been claimed that amide I IR spectral changes could be the result of water removal per se. Here, we investigated whether such claims hold true. The structure of horseradish peroxidase (HRP) and poly(ethylene glycol)-modified HRP (HRP-PEG) has been investigated under various conditions (in aqueous solution, the amorphous dehydrated state, and dissolved/suspended in toluene and benzene) by UV-visible (UV-Vis), FTIR, and resonance Raman spectroscopy. The resonance Raman and UV-Vis spectra of dehydrated HRP-PEG dissolved in neat toluene or benzene were very similar to that of HRP in aqueous buffer, and thus the heme environment (heme iron spin, coordination, and redox state) was essentially the same under both conditions. Therefore, the three-dimensional structure of HRP-PEG dissolved in benzene and toluene was similar to that in aqueous solution. The amide I IR spectra of HRP-PEG in aqueous buffer and of dehydrated HRP-PEG dissolved in neat benzene and toluene were also very similar, and the secondary structure compositions (percentages of alpha-helices and beta-sheets) were within the standard error the same. These results are irreconcilable with recent claims that water removal per se could cause substantial amide I IR spectral changes (M. van de Weert, P.I. Haris, W.E. Hennink, and D.J. Crommelin. 2001. Anal. Biochem. 297:160-169). On the contrary, amide I IR spectral changes upon protein dehydration are caused by perturbations in the secondary structure.     

Generation of anisotropic strain dysregulates wild-type cell division at the interface between host and oncogenic tissue

1. Download : Download high-res image (218KB)
2. Download : Download full-size image

     

Packing, specificity, and mutability at the binding interface between the p160 coactivator and CREB-binding protein

Among the most common interaction motifs between nuclear proteins is the recognition of one or more amphipathic helices. In an effort to determine principles behind this recognition, we have investigated the interaction between the p160 coactivator protein ACTR and the ACTR-binding domain of the CREB-binding protein, CBP. The two proteins use relatively small portions of their primary sequences to form a single synergistically folded domain consisting of six intertwined alpha-helices, three from each protein. Neither of the component polypeptides forms a cooperatively folded domain in isolation. However, a considerable amount of residual secondary structure remains in the isolated CBP domain according to CD spectroscopy. Chemical denaturation, differential scanning calorimetry, and ANS binding experiments demonstrate that the isolated CBP domain is not entirely unfolded but forms a helical state with the characteristics of a molten globule. Mutations probing the functional and energetic significance of a buried intermolecular Arg-Asp salt bridge in the interface of the protein complex suggest that these residues are tuned for functional discrimination and not strictly for binding affinity or stability. These results suggest a mechanism for formation of the complex where the unfolded ACTR domain interacts with the partly folded CBP domain in a rapid and specific manner to form the final stable complex.     

Adsorption and desorption studies of phospholipid at the air/water interface

The desorption and adsorption properties of phosphatidylserine (extracted from beef brain) at the air/water interface were studied through surface pressure measurements. The rate of dissolution of phosphatidylserine monolayer into the underlying water of natural pH is extremely slow at room temperature but increases rather suddenly around 40°C. This sudden increase of dissolution rate might be explained as the Kraft point phenomena analogous to the ionic surfactants.     

Macrophages at the fetal-maternal interface express markers of alternative activation and are induced by M-CSF and IL-10

During pregnancy, the maternal immune system is challenged by the presence of the fetus, which must be tolerated despite being semiallogeneic. Uterine mucosal (or decidual) macrophages (M), one of the major leukocyte populations at the fetal-maternal interface, have been implicated in fetal tolerance, but information regarding their regulation is scarce. In this study, we investigated the role of several factors potentially involved in the differentiation and polarization of decidual M with an in vitro M differentiation model. By using flow cytometry, we showed that M-CSF and IL-10 were potent inducers of M2 (immunoregulatory) M markers expressed on human decidual M (CD14, CD163, CD206, CD209). In contrast, proinflammatory stimuli, and unexpectedly also the Th2-associated IL-4 and IL-13, induced different patterns of expression, indicating that a Th2-dominated environment is not required for decidual M polarization. M-CSF/IL-10-stimulated and decidual M also showed similar cytokine secretion patterns, with production of IL-10 as well as IL-6, TNF, and CCL4. Conversely, the proinflammatory, LPS/IFN-γ-stimulated M produced significantly higher levels of TNF and no IL-10. We also used a gene array with 420 M-related genes, of which 100 were previously reported to be regulated in a global gene expression profiling of decidual M, confirming that M-CSF/IL-10-induced M are closely related to decidual M. Taken together, our results consistently point to a central role for M-CSF and in particular IL-10 in the shaping of decidual M with regulatory properties. These cytokines may therefore play an important role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy.     

Conformational transitions of detergent-solubilized Na,K-ATPase are conveniently monitored by the fluorescent probe 6-carboxy-eosin.

6-carboxy-eosin is introduced as a sensitive, non-covalently bound fluorescent probe for monitoring conformational changes in detergent-solubilized Na,K-ATPase. The dissociation constant for 6-carboxy-eosin is about 0.1 microM in 20 mM NaCl at 6 degrees C (pH 7.0) for Na,K-ATPase solubilized in C12E8. It is shown that the slow conformational change from E2 (in K+) to E1 (in Na+) is 4-fold more rapid in the solubilized state than in the membrane-bound state, both for shark rectal gland and pig kidney Na,K-ATPase. The rate of the E1 to E2 transition is rapid and of the same order of magnitude both for the membrane-bound and the solubilized enzyme. All conformational transitions are considerably slower for pig kidney enzyme than for shark enzyme, both in the membrane-bound and in the solubilized state.     

Oxysterol‐binding protein recruitment and activity at the endoplasmic reticulum‐Golgi interface are independent of Sac1

Oxysterol‐binding protein (OSBP) localizes to endoplasmic reticulum (ER)‐Golgi contact sites where it transports cholesterol and phosphatidylinositol 4‐phosphate (PI‐4P), and activates lipid transport and biosynthetic activities. The PI‐4P phosphatase Sac1 cycles between the ER and Golgi apparatus where it potentially regulates OSBP activity. Here we examined whether the ER‐Golgi distribution of endogenous or ectopically expressed Sac1 influences OSBP activity. OSBP and Sac1 co‐localized at apparent ER‐Golgi contact sites in response to 25‐hydroxycholesterol (25OH), cholesterol depletion and p38 MAPK inhibitors. A Sac1 mutant that is unable to exit the ER did not localize with OSBP, suggesting that sterol perturbations cause Sac1 transport to the Golgi apparatus. Ectopic expression of Sac1 in the ER or Golgi apparatus, or Sac1 silencing, did not affect OSBP localization to ER‐Golgi contact sites, OSBP‐dependent activation of sphingomyelin synthesis, or cholesterol esterification in the ER. p38 MAPK inhibition and retention of Sac1 in the Golgi apparatus also caused OSBP phosphorylation and OSBP‐dependent activation of sphingomyelin synthesis at ER‐Golgi contacts. These results demonstrate that Sac1 expression in either the ER or Golgi apparatus has a minimal impact on the PI‐4P that regulates OSBP activity or recruitment to contact sites.      

Investigations into the life cycle of the bacterial predator Bdellovibrio bacteriovorus 109J at an interface by atomic force microscopy

Atomic force microscopy was used to image Bdellovibrio bacteriovorus 109J, a gram-negative bacterial predator that consumes a variety of other gram-negative bacteria. In predator-prey communities grown on filters at hydrated air-solid interfaces, repeated cycles of hunting, invasion, growth, and lysis occurred readily even though the cells were limited to near two-dimensional movement. This system allowed us to image the bacteria directly without extensive preparation or modification, and many of the cells remained alive during imaging. Presented are images of the life cycle in two species of prey organisms, both Escherichia coli (a small prey bacterium that grows two-dimensionally on a surface) and Aquaspirillum serpens (a large prey bacterium that grows three-dimensionally on a surface), including high-resolution images of invaded prey cells called bdelloplasts. We obtained evidence for multiple invasions per prey cell, as well as significant heterogeneity in morphology of bdellovibrios. Mutant host-independent bdellovibrios were observed to have flagella and to excrete a coating that causes the predators to clump together on a surface. Most interestingly, changes in the texture of the cell surface membranes were measured during the course of the invasion cycle. Thus, coupled with our preparation method, atomic force microscopy allowed new observations to be made about Bdellovibrio at an interface. These studies raise important questions about the ways in which bacterial predation at interfaces (air-solid or liquid-solid) may be similar to or different from predation in solution.     

Determination of several antibiotics using voltamperometry at the interface of nitrobenzene and water

A new electroanalytical method of voltamperometry at the interface of two immiscible electrolyte solutions (ITIES) is based on electrochemical polarization of a liquid/liquid interface. The resulting current voltage characteristics completely resemble those obtained with metallic electrodes. The charge transfer processes are either the direct ion transfer across the ITIES or the transfer facilitated by macrocyclic ionophores. Determination of tetracycline antibiotics is based on the direct transfer of the cationic forms of these substances in acid media. Determination of valinomycin, nonactin and monensin acting as ion carriers is connected with the facilitated alkali metal ion transfer. In general, antibiotic concentrations higher than 0.02-0.05 mmol/l can be determined with this method. Monensin can also be determined in the extracts of Streptomyces cinnamonensis.     

Electrostatic repulsion, compensatory mutations, and long-range non-additive effects at the dimerization interface of the HIV capsid protein

In previous studies, thermodynamic dissection of the dimerization interface in CA-C, the C-terminal domain of the capsid protein of human immunodeficiency virus type 1, revealed that individual mutation to alanine of Ser178, Glu180, Glu187 or Gln192 led to significant increases in dimerization affinity. Four related aspects derived from this observation have been now addressed, and the results can be summarized as follows: (i) thermodynamic analyses indicate the presence of an intersubunit electrostatic repulsion between both Glu180 residues. (ii) The mutation Glu180 to Ala was detected in nearly all type 2 human immunodeficiency virus variants, and in several simian immunodeficiency viruses analyzed. However, this mutation was strictly co-variant with mutations Ser178Asp in a neighboring residue, and Glu187Gln. Thermodynamic analysis of multiple mutants showed that Ser178Asp compensated, alone or together with Glu187Gln, the increase in affinity caused by the mutation Glu180Ala, and restored a lower dimerization affinity. (iii) The increase in the affinity constant caused by the multiple mutation to Ala of Ser178, Glu180, Glu187 and Gln192 was more than one order of magnitude lower than predicted if additivity were present, despite the fact that the 178/180 pair and the two other residues were located more than 10A apart. (iv) Mutations in CA-C that caused non-additive increases in dimerization affinity also caused a non-additive increase in the capacity of the isolated CA-C domain to inhibit the assembly of capsid-like HIV-1 particles in kinetic assays. In summary, the study of a protein-protein interface involved in the building of a viral capsid has revealed unusual features, including intersubunit electrostatic repulsions, co-variant, compensatory mutations that may evolutionarily preserve a low association constant, and long-range, large magnitude non-additive effects on association.     

Effect of hydrophobic surfactant protein SP-C on binary phospholipid monolayers. Molecular machinery at the air/water interface

Fluorescent and modified dark-field microscopies were used to investigate the phase behavior of physiologically relevant lipid/protein monomolecular films containing surfactant protein C(SP-C). Synthetic human SP-C(1-34) was labeled at its N-terminus using the fluorescent probe 6-(((4(4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)phenoxy)acetyl)amino)hexanoic acid (BODIPY/TR-X). Using dual fluorescent labeling (lipid and protein) in the monolayers, we have correlated (at physiologically small concentrations of the protein) the lipid phase separation and protein distribution in situ. A comparison of the lipid and protein dye fluorescent micrographs indicates that SP-C(1-34) is preferentially associated with the disordered lipid phase. Three concepts arise from our results. (1) The presence of SP-C alone does not result in the complete dissolution of condensed phase domains in a fashion that we have previously reported for the entire hydrophobic surfactant protein (SP-B/C) fraction (Biophys. J. 77 (1999) 903). Rather, the use of relatively high amounts ( approximately 10 wt.%) of the labeled SP-C protein is needed to reproduce the fluorescence monolayer morphology previously observed for small concentrations ( approximately 1 wt.%) of the natural SP-B/C mixture. (2) Scattered light, dark-field microscopy performed using grazing angle laser illumination reveals the presence of surface-associated, three-dimensional (3D) structures of micrometer-sized dimensions when the labeled BODIPY/TR-X:SP-C(1-34) protein is included in the monolayer, as previously observed with the naturally isolated SP-B/C mixture. The 3D structures are associated exclusively with the presence of the SP-C protein in disordered monolayer phases. (3) To explain these results, we have derived a molecular model accounting for the structure and physico-chemical properties of the SP-C protein in terms of its energetics. The molecular events involved in the SP-C-mediated production of the 3D surface particles are explained using the analogy of a simple molecular machine, namely a loaded spring. This interpretation is supported by an energetic analysis that suggests the major factor contributing to the formation of the 3D particles is the energy liberated by re-expansion of the surrounding phospholipid film into the area vacated by the SP-C protein as it re-orients away from the surface.     

Miscibility of DPPC and DPPA in monolayers at the air/water interface

Monolayers of mixtures of 1,2-dipalmitoylphosphatidylcholine (DPPC) as the substrate and 1,2-dipalmitoylphosphatidic acid (DPPA) as the product of the hydrolysis reaction catalyzed by phospholipase D (PLD) were investigated in the presence of Ca2+. The miscibility behavior and the microstructure of mixed domains have been studied by grazing incidence X-ray diffraction (GIXD), Brewster angle microscopy and film balance measurements. The phase diagram reveals partial miscibility on both sides and a wide miscibility gap, which becomes narrower at high pressure. At low pressure, the segregation of condensed DPPA-rich domains in a fluid-like DPPC matrix was detected already at small DPPA concentrations and their structure was determined. A small amount of DPPC mixed into the segregated DPPA domains induces the transformation from rectangular to an oblique unit cell and increases the tilt angle in the condensed domains. At high pressure, two types of condensed phase domains were found: DPPC-rich and DPPA-rich. A drastic reduction of the tilt angle in the DPPC-rich domains with increasing amount of DPPA was observed. The decrease of the tilt angle must be connected with a change of the head group conformation of DPPC in such mixed domains.