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The kinetic adsorption-desorption behaviour of porcine gastric mucin in the presence of physiologically relevant concentrations of the polyphenol epigallocatechin gallate (EGCG) was investigated using high-resolution kinetic optical waveguide lightmode spectroscopy (OWLS) and atomic force microscopy (AFM). Comparison with dynamic light scattering results from EGCG-mucin mixtures indicates that discrete particles are formed whose size increases with increasing EGCG:mucin ratio. These particles are deduced to be the adsorbing entities, which fuse on the surface to form complex surface layers. At low molar EGCG:mucin ratios (<∼1000), aggregates fuse on the surface to form a monolayer similar to one of pure mucin. With increasing EGCG concentration, the surface assembly of aggregates becomes consistent with their rearrangement and spreading in the shape of a spherical segment. At the highest molar ratios investigated (>12,000) the particles begin to destabilize. The presence of EGCG leads to birefringence hysteresis during adsorption-desorption, indicating structural rearrangement, even at molar ratios ∼1000. The intensification of the phenomenon with increasing EGCG:mucin ratio mimics what was previously observed with the increase of mucin concentration in an EGCG-free system.

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Biodesulfurization (BDS) of dibenzothiophene (DBT) was carried out by Rhodococcus erythropolis IGST8 decorated with magnetic Fe3O4 nanoparticles, synthesized in-house by a chemical method, with an average size of 45-50 nm, in order to facilitate the post-reaction separation of the bacteria from the reaction mixture. Scanning electron microscopy (SEM) showed that the magnetic nanoparticles substantially coated the surfaces of the bacteria. It was found that the decorated cells had a 56% higher DBT desulfurization activity in basic salt medium (BSM) compared to the nondecorated cells. We propose that this is due to permeabilization of the bacterial membrane, facilitating the entry and exit of reactant and product, respectively. Model experiments with black lipid membranes (BLM) demonstrated that the nanoparticles indeed enhance membrane permeability.

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A phenomenological theory of salt-induced Hofmeister phenomena is presented, based on a relation between protein solubility in salt solutions and protein-water interfacial tension. As a generalization of previous treatments, it implies that both kosmotropic salting out and chaotropic salting in are manifested via salt-induced changes of the hydrophobic/hydrophilic properties of protein-water interfaces. The theory is applied to describe the salt-dependent free energy profiles of proteins as a function of their water-exposed surface area. On this basis, three classes of protein conformations have been distinguished, and their existence experimentally demonstrated using the examples of bacteriorhodopsin and myoglobin. The experimental results support the ability of the new formalism to account for the diverse manifestations of salt effects on protein conformation, dynamics, and stability, and to resolve the puzzle of chaotropes stabilizing certain proteins (and other anomalies). It is also shown that the relation between interfacial tension and protein structural stability is straightforwardly linked to protein conformational fluctuations, providing a keystone for the microscopic interpretation of Hofmeister effects. Implications of the results concerning the use of Hofmeister effects in the experimental study of protein function are discussed.

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PC12 pheochromocytoma cells expressing a dominant inhibitory mutant of Ha-Ras (M-M17-26) and PC12 cells transfected with normal c-RasH (M-CR3B) have been used to investigate the role of nitrosylation and farnesylation of Ras on the production of homocysteine and the activities of the redox-sensitive transcription factors NF-kappaB and c-Fos. We found that under serum and nerve growth factor withdrawal conditions undifferentiated apoptotic M-CR3B cells accumulated more homocysteine than M-M17-26 cells, and the production of homocysteine decreased in the presence of manumycin and increased in the presence of l-NAME. Furthermore, we have shown that manumycin increased the activity of c-Fos in the M-CR3B cells and decreased the activity of NF-kappaB, while l-NAME decreased the activities of both transcription factors, and accelerated apoptosis of M-CR3B cells. In contrast, in M-M17-26 cells manumycin did not change the activity of c-Fos, nor the activity of NF-kappaB. We conclude that trophic factor withdrawal stimulates Ras, which apparently through the Rac/NADPH oxidase system induces permanent oxidative stress, modulates the activities of NF-kappaB and c-Fos, induces production of homocysteine and accelerates apoptosis. Nitrosylation of Ras is necessary for maintaining the survival of PC12 cells, while farnesylation of Ras stimulates apoptosis under withdrawal conditions.

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Integrated optics technology has a great potential for medical device applications. This article explains the technology and how it can be used with particular reference to in vitro diagnostics and endoscopy.

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The association of myelin basic protein charge isomers with the lipid part of the myelin membrane was investigated at the microscopic (molecular) level in a model membrane system, using optical waveguide lightmode spectrometry to determine with high precision the kinetics of association and dissociation to planar phospholipid membranes under controlled hydrodynamic conditions and over a range of protein concentrations. Detailed analysis of the data revealed a rich and intricate behaviour and clearly showed that the membrane protein affinity is characterized by at least four independent parameters: (i) the association rate coefficient characterizing the protein-membrane interaction energy as the protein approaches the fluid-membrane interface; (ii) the protein-membrane adhesion, i.e., the probability that it will remain at the membrane after arrival; (iii) the protein conformation at the membrane; and (iv) the protein's tendency to cluster at the membrane. Some of these parameters varied in characteristic ways as the bulk solution concentration of the protein was varied, giving further clues to the detailed molecular comportment of the protein. The parameters and their characteristic variations with bulk concentration were markedly different for the different isomers. Implications of these results for neurological disorders involving demyelination, such as multiple sclerosis, are discussed.

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By incorporating a grating in a planar optical waveguide one creates a device with which the spectrum of guided lightmodes can he measured. When the surface of the waveguide is exposed to different solutions, the peaks in the spectrum shift due to molecular interactions with the surface. Optical waveguide lightmode spectroscopy (OWLS) is a highly sensitive technique that is capable of real-time monitoring of these interactions. Since this integrated optical method is based on the measurement of the polarizability density (i.e., refractive index) in the vicinity of the waveguide surface, radioactive, fluorescent or other kinds of labeling are not required. In addition, measurement of at least two guided modes enables the absolute mass of adsorbed molecules to be determined. In this article, the technique will be described in some detail, and applications from different areas will be discussed. Selected examples will be presented to demonstrate how monitoring the modification of different metal oxides with polymers and the response of the coated oxides to biofluids help in the design of novel biomaterials; how OWLS is useful for accurate bioaffinity sensing, which is a key issue in the development of new drugs; and how the quantitative study of protein-DNA/RNA and cell surface interactions can enhance the understanding of processes in molecular and cellular biology.

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TipAL is a Streptomyces transcriptional activator assigned to the MerR/SoxR family based both on homology within its putative DNA recognition domain and the fact that its operator binding sites lie within a region of its promoter normally occupied by RNA polymerase. The tipA gene is also independently translated as the C-terminal ligand-binding domain of TipAL (TipAS; residues 111-254). Both TipAS and TipAL share broad recognition specificity for cyclic thiopeptide antibiotics. The molecular mechanism by which TipAL catalyzes prokaryotic transcriptional activation at the tipA promoter (ptipA) in response to thiostrepton was studied using a combination of analytical ultracentrifugation (AU), circular dichroism (CD), optical waveguide lightmode spectroscopy (OWLS; a sensitive in situ binding assay), and mutational analyses. AU showed that TipAL, but not TipAS, was a dimer in solution in the presence or absence of thiostrepton. This indicated that activation of TipAL by thiostrepton was not mediated by changes in multimerization and mapped the dimerization domain to its N-terminal 110 amino acids, presumably within amino acids predicted to form a coil-coil domain (residues 77-109). CD spectra showed that TipAL had more alpha-helical content than TipAS, probably because of the presence of the additional N-terminal region. The helicity of TipAL and TipAS both increased slightly after binding thiostrepton demonstrating conformation changes upon thiostrepton binding. OWLS experiments determined the overall binding constants via measurements of association and dissociation rates for both TipA proteins and RNA polymerase with ptipA. Thiostrepton slightly enhanced the rate of specific association of TipAL with ptipA, but drastically lowered the rate of dissociation from the binding site. TipAL-thiostrepton increased the affinity of RNA polymerase for ptipA more than 10-fold. In conjunction with genetic experiments, we propose that, while there are some similarities, the mechanism by which TipAL activates transcription is distinctly different from the established MerR/SoxR paradigm.

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The distributions of synthesis rates of expressed proteins in a liquid batch culture of the prokaryote S. coelicolor during 3 days' growth have been analyzed by using a law governing the relation between the synthesis rates and the corresponding ranks in a list of rates (the so-called simplified canonical law, scl), which we have found previously to characterize the distribution of prokaryotic protein expression. The scl remains valid throughout development and the two parameters of the distribution, q and r, evolve in a highly characteristic and revealing way. q is a measure of the degree to which available genomic resources are used, in the sense of exploiting their potential diversity. The passage from one developmental phase to another is marked by a sharp peak in q, as these resources are fully mobilized to deal with a crisis (i.e., exhaustion of the habitual food supply). This is followed by an even more pronounced trough, as the organism briefly focuses its resources on synthesizing just those proteins most essential for survival, especially those hitherto unavailable and needed for metabolizing the new nutrient source. The parameter r indicates redundancy among the most abundantly expressed proteins: higher r corresponds to more diversity; i.e., less duplication of function, hence less robustness. This parameter is relatively steady throughout the development of the culture, except for a pronounced peak during the developmental phase transition. This corresponds to the "emergency mode" characterized by extremely low q, during which a minimum repertoire of proteins is expressed.

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The effector domain (ED) of MARCKS proteins can associate with calmodulin (CaM) as well as with phospholipids. It is not clear, however, whether a complex between MARCKS proteins and CaM can form at the surface of phospholipid membranes or whether CaM and membranes compete for ED binding. Using two-mode waveguide spectroscopy, we have investigated how CaM regulates the association of MARCKS-related protein (MRP) with planar supported phospholipid bilayer membranes. Bringing a solution containing CaM into contact with membranes on which MRP had previously been deposited results in low-affinity binding of CaM to MRP. A preformed, high-affinity CaM MRP complex in the aqueous phase binds much more slowly than pure MRP to membranes. Similar observations were made when a peptide corresponding to the ED of MRP was used instead of MRP. Hence CaM cannot form a stable complex with MRP once the latter is bound at the membrane surface. CaM can, however, strongly retard the association of MRP with lipid membranes. The most likely interpretation of these results is that CaM and the phospholipid membrane share the same binding region at the ED and that the ED is forced by membrane binding to adopt a conformation unfavorable for CaM binding.

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MARCKS (myristoylated alanine-rich C kinase substrate, 32 kDa) and its 20 kDa brother MARCKS-related protein (MRP) are abundant, widely distributed proteins unusually rich in alanine and glutamic acid, and with lysines, serines and phenylalanines concentrated in a compact "effector domain" (ED) near the middle of the sequence. Its conformation in solution appears to be labile, with little evidence for definite secondary structure. MARCKS (and MRP) interact inter alia with lipid bilayer membranes (via the myristoyl group and the ED), with protein kinases (which phosphorylate the serines in the ED), and with calmodulin (via the ED); synergies between these diverse interactions present an unusually rich array of possibilities for a variety of regulatory rôles. The proteins appear to be essential for controlling cell shape changes, possibly via involvement in cytoskeleton-membrane linkage. MRP deficiency leads to neural tube defects in brain development; MARCKS overexpression strongly depresses the proliferation of cancer cells.

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The association of various protein constructs of MARCKS-related protein (MRP) lacking the myristoyl moiety or the basic effector domain (ED) or both to neutral and acidic supported planar phospholipid bilayer membranes has been monitored using two-mode optical waveguide spectroscopy. The importance of the myristoyl moiety for interaction with both neutral and acidic membranes is demonstrated but unmyristoylated MRP still binds appreciably to neutral membranes, albeit less than to acidic membranes. Only when both the myristoyl moiety and the ED are excised does the interaction fall to zero in the case of the acidic membranes, with very small residual binding still detectable in the presence of neutral membranes. These results point to the importance of hydrophobic interactions apart from those associated with the myristoyl moiety in the association of MRP with membranes. The ED is well endowed with hydrophobic as well as with basic residues, and the former are chiefly responsible for binding unmyristoylated MRP to neutral membranes: The very small residual attraction between MRP lacking both the myristoyl moiety and the ED is completely outweighed by electrostatic repulsion between the net acidic MRP and the acidic lipid head groups.

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Thick layers of the protein lysozyme have been deposited on mica, and their force-distance hysteresis measured using atomic force microscopy in the presence of different salts. Sodium thiocyanate, which is known to lower the melting temperature of proteins and increase their solubility, increases lysozyme deformability and lowers the viscosity of the protein layer, compared with sodium chloride. Sodium phosphate, known to raise the melting temperature and lower the solubility, decreases deformability and increases the viscosity.

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We have recently shown that unmyristoylated MARCKS-related protein (MRP) does not bind to neutral phospholipid vesicles, unless negatively charged phospholipids are present. Similar behaviour has also been reported for MARCKS itself. Here we have compared the binding of MRP to neutral and negatively charged supported planar lipid bilayer membranes (SPLM) using two-mode waveguide spectroscopy. We find appreciable binding of unmyristoylated MRP to neutral SPLM. We propose that hydrophobic residues in the effector domain constitute an additional factor capable of mediating MRP-membrane interaction.

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Multilayer assemblies were prepared by alternating adsorption of monolayers of monoclonal antibody against horse radish peroxidase (anti-HRP) and dextran sulfate (DS) on solid supports at acid pH. After crosslinking with glutaraldehyde, DS was washed out of the film with buffered physiological saline, while the antibody remained immobilised on the support. Assembly was monitored in situ on germanium supports by infrared multi-internal reflection spectroscopy. The binding capacity of the immobilised antibodies for HRP was measured by ELISA and by optical waveguide light mode spectroscopy. The activity of an immobilised anti-HRP bilayer was approximately twice that of a monolayer prepared by simple physiosorption. An addition of further anti-HRP layers could increase the activity only up to 2.5 of the monolayer activity independently of a number of layers in the assembly. The non-specific adsorption of proteins from human blood plasma was three times lower on the immobilised anti-HRP multilayer film than on the surface covered only with a physiosorbed anti-HRP monolayer.

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The myristoylated alanine-rich C kinase substrate (MARCKS) protein family has two known members, MARCKS itself and MARCKS-related protein (MRP, also called MacMARCKS or F52). They are essential for brain development and are believed to regulate the structure of the actin cytoskeleton at the plasma membrane. Hence membrane binding is central to their function. MARCKS has been quite extensively characterized; MRP much less so. Despite the fact that MRP is only two thirds the size of MARCKS, it has hitherto been assumed that the two proteins have similar properties. Here we make a detailed study, including the effects of myristoylation, lipid composition, calmodulin and phosphorylation of the binding of MRP to phospholipid vesicles. We show that both the N-terminal myristoyl moiety and the central effector domain mediate binding. MRP behaves like MARCKS in the presence of neutral phospholipids. In contrast to MARCKS, however, the incorporation of 20% of negatively-charged phospholipids only marginally increases the affinity of myristoylated MRP. Co-operativity between the myristoyl moiety and the effector domain of MRP is weak and the protein has a significantly lower affinity for these vesicles compared with MARCKS. Furthermore, calmodulin or phosphorylation of the effector domain by the catalytic subunit of protein kinase C do not significantly decrease the binding of myristoylated MRP to negatively-charged phospholipid vesicles. Our results show that the mechanisms regulating the interactions of MARCKS and MRP with phospholipid vesicles are, at least quantitatively, different. In agreement with cellular studies, we therefore propose that MARCKS and MRP have different subcellular localization and, consequently, different functions.

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The interaction of the nonionic detergent Triton X-100 with supported phosphatidylcholine planar lipid bilayers has been investigated by optically monitoring changes in the bilayer, using the technique of optical waveguide lightmode spectroscopy (OWLS). This technique has several advantages over the methods applied to the problem hitherto, including: high sensitivity; measurement in situ with good time resolution; the fact that the free detergent concentration is well-defined, and the lipid concentration in solution is zero; ease of studying the reversibility of the interaction; and the readiness with which absolute rather than effective amounts of detergent incorporated into the lipid can be determined. The main finding is that as the free Triton concentration increases, the detergent is first incorporated reversibly into the bilayer, then partly but never completely removes lipid, and finally (at or above the cmc) completely solubilizes the bilayer. The behaviour of the lanar supported lipid bilayers is thus similar to that previously reported for lipid vesicles.

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The interaction of phospholipid vesicles with planar metal oxide supports has been previously reported as a means of preparing supported lipid bilayers, which are useful models of biological membranes. Nevertheless, extant evidence that bilayers are actually formed is rather circumstantial, and the necessary and sufficient conditions for their formation have never been delineated. Here, we tackle this problem by using smooth planar optical waveguides as the support. Analysis of the lightmode spectra of the waveguides, measured in situ during the deposition process, yields the mass of lipid deposited at the solid/liquid interface. By comparing the optogeometric parameters of the structures assembled from the vesicles with those of a lipid bilayer of known structure assembled using the Langmuir-Blodgett technique, we show that in many cases the vesicles remain intact and form a supported layer of vesicles rather than a bilayer, and often mixed structures (intact vesicles embedded in a bilayer partially covering the surface) occur. Careful analysis of the lipid deposition kinetics corroborates this result. We have also found that divalent cations dramatically promote attachment of mixed phosphatidylcholine/phosphatidylglycerol vesicles to form supported vesicle layers, and bilayer formation from pure phosphatidylcholine vesicles.

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Advances in experimental and computational methodologies have led to a recent renewed interest in the Hofmeister series and its molecular origins. New results are surveyed and assessed. Insights into the underlying mechanisms have been gained, although deeper molecular understanding still seems to be elusive. The principal reason appears to be that the Hofmeister series emerges from a combination of a general effect of cosolutes (salts, etc.) on solvent structure, and of specific interactions between the cosolutes and the solute (protein or other biopolymer). Hence every system needs to be studied individually in detail, a state of affairs which is likely to continue for some time. A deeper understanding of the Hofmeister series can be an extraordinarily valuable guide to designing experiments, including not only those probing the series per se, but also those designed to elucidate the adsorption, aggregation and stabilization phenomena which underlie so many biological events. The aim of this review is to provide an up-to-date framework to guide such understanding, consolidating recent advances in the many fields on which the Hofmeister series impinges.

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