Protein-surfactant interactions: a tale of many states

The scientific study of protein surfactant interactions goes back more than a century, and has been put to practical uses in everything from the estimation of protein molecular weights to efficient washing powder enzymes and products for personal hygiene. After a burst of activity in the late 1960s and early 1970s that established the general principles of how charged surfactants bind to and denature proteins, the field has kept a relatively low profile until the last decade. Within this period there has been a maturation of techniques for more accurate and sophisticated analyses of protein-surfactant complexes such as calorimetry and small angle scattering techniques. In this review I provide an overview of different useful approaches to study these complexes and identify eight different issues which define central concepts in the field. (1) Are proteins denatured by monomeric surfactant molecules, micelles or both? (2) How does unfolding of proteins in surfactant compare with "proper" unfolding in chemical denaturants? Recent work has highlighted the role of shared micelles, rather than monomers, below the critical micelle concentration (cmc) in promoting both protein denaturation and formation of higher order structures. Kinetic studies have extended the experimentally accessible range of surfactant concentrations to far above the cmc, revealing numerous different modes of denaturation by ionic surfactants below and above the cmc which reflect micellar properties as much as protein unfolding pathways. Uncharged surfactants follow a completely different denaturation strategy involving synergy between monomers and micelles. The high affinity of charged surfactants for proteins means that unfolding pathways are generally different in surfactants versus chemical denaturants, although there are common traits. Other issues are as follows: (3) Are there non-denaturing roles for SDS? (4) How reversible is unfolding in SDS? (5) How do solvent conditions affect the way in which surfactants denature proteins? The last three issues compare SDS with "proper" membranes. (6) Do anionic surfactants such as SDS mimic biological membranes? (7) How do mixed micelles interact with globular proteins? (8) How can mixed micelles be used to measure the stability of membrane proteins? The growing efforts to understand the unique features of membrane proteins have encouraged the development of mixed micelles to study the equilibria and kinetics of this class of proteins, and traits which unite globular and membrane proteins have also emerged. These issues emphasise the amazing power of surfactants to both extend the protein conformational landscape and at the same time provide convenient and reversible short-cuts between the native and denatured state for otherwise obdurate membrane proteins.     

Plasmodium-mosquito interactions: a tale of dangerous liaisons

To complete their life cycle, Plasmodium parasites must survive the environment in the insect host, cross multiple barriers including epithelial layers, and avoid destruction by the mosquito immune system. Completion of the Anopheles gambiae and Plasmodium falciparum genomes has opened the opportunity to apply high throughput methods to the analysis of gene function. The burst of information generated by these approaches and the use of molecular markers to investigate the cell biology of these interactions is broadening our understanding of this complex system. This review discusses our current understanding of the critical interactions that take place during the journey of Plasmodium through the mosquito host, with special emphasis on the responses of midgut epithelial cells to parasite invasion.     

Transthyretin Ser 6 gene frequency in individuals without amyloidosis

Transthyretin (TTR) Ser 6 was originally described in a Scottish kindred without amyloidosis. This variant, arising from a GA transition in codon 6 that destroys an MspI site and creates a BsrI site, was present in none of 50 controls, and was therefore throught to be rare. This variant has subsequently been found in a normal human cDNA liver library and in two unrelated patients with familial amyloidosis and other TTR variants, raising the question whether it is actually a common polymorphism. To address this question, we performed PCR and restriction digestion of 574 DNA samples from people without evidence of amyloidosis or a known family history of amyloidosis. The TTR Ser 6 allele frequency was 33/558 (.060) in Caucasians (including 8/192 (.04)) in North American Ashkenazic Jews, 16/218 (.07) in North American non-Jews, and 9/148 (.06) in Portuguese), 3(242 (.01) in African Americans, 0/140 in Africans, and 0/208 in Asians. These data are most suggestive of a single Caucasian founder and the known 25% admixture of Caucasian genes in the African-American population. Alternatively, as this variant arose from a transition at a CG dinucleotide hot spot, it may have arisen on multiple occasions. These data indicate that TTR Ser 6 is a common non-amyloidogenic population polymorphism in Caucasians.     

Dehydrophenylalanine zippers: strong helix-helix clamping through a network of weak interactions

A decapeptide Boc-L-Ala-(Delta Delta Phe)(4)-L-Ala-(Delta Delta Phe)3-Gly-OMe (Peptide I) was synthesized to study the preferred screw sense of consecutive alpha,beta-dehydrophenylalanine (Delta Delta Phe) residues. Crystallographic and CD studies suggest that, despite the presence of two L-Ala residues in the sequence, the decapeptide does not have a preferred screw sense. The peptide crystallizes with two conformers per asymmetric unit, one of them a slightly distorted right-handed 3(10)-helix (X) and the other a left-handed 3(10)-helix (Y) with X and Y being antiparallel to each other. An unanticipated and interesting observation is that in the solid state, the two shape-complement molecules self-assemble and interact with an extensive network of C-H...O hydrogen bonds and pi-pi interactions, directed laterally to the helix axis with amazing regularity. Here, we present an atomic resolution picture of the weak interaction mediated mutual recognition of two secondary structural elements and its possible implication in understanding the specific folding of the hydrophobic core of globular proteins and exploitation in future work on de novo design.     

Protein farnesylation in plants: a greasy tale.

Although farnesylation is required for a number of abscisic acid mediated responses in plants, knowledge of how this lipid modification of proteins regulates specific developmental and physiological processes remains unclear. Recent information from the Arabidopsis genome-sequencing project in combination with mutants deficient in farnesylation should unravel the role(s) of this process in plant signaling.     

Transthyretin Pro55, a variant associated with early-onset,aggressive, diffuse amyloidosis with cardiac and neurologic involvement

Summary Mutations in the protein transthyretin cause amyloidosis involving the heart, peripheral nerves, and other organs. A family from West Virginia developed an unusually aggressive form of widespread transthyretin amyloidosis. Single-strand conformation polymorphism analysis revealed a variant in the transthyretin gene, which was found on sequencing to be a TC transversion at position 2 of codon 55, corresponding to a Leu Pro substitution. The variant sequence was confirmed by restriction analysis and polymerase chain reaction (PCR)-primer introduced restriction analysis.     

Mouse senile amyloidosis. ASSAM amyloidosis in mice presents universally as a systemic age-associated amyloidosis.

Amyloid deposition in 11 inbred strains of mice (A/J, SJL/J, DDD, C57BL/6J, B10.BR, C57BL/10, B10A/SgSn, C3H/HeMs, B10A(5R), DBA/2 and C57BL/6Cr5/c) was studied using the peroxidase antiperoxidase (PAP) method and antisera against ASSAM and murine protein AA. Among the 170 mice examined, in 77 (45.3%) from the nine strains other than C3H/HeMs and DBA/2, there was evidence of spontaneous amyloid deposits in routine histological sections. Immunohistochemical studies using 54 mice with amyloid deposition, demonstrated ASSAM deposition in 45 mice (83.3%) in all nine strains, although the incidence and intensity of the deposition differed somewhat between strains. SJL/J and A/J had ASSAM deposits from the age of 8 months and the incidence increased with advancing age. In the other seven strains, ASSAM was first deposited at an older age than in the SJL/J and A/J strains. In A J, C57BL/6J, C57BL/10, B10.BR, B10A(5R) and C57BL/6Cr5/c, protein AA often coexisted with ASSAM. The distribution pattern of the ASSAM deposits was similar to that observed among the SAM strains. Thus, ASSAM is an ubiquitously distributed senile amyloid protein in the mouse. Determination of the molecular type of apoA-II, a serum precursor of ASSAM, among all 11 strains using the polymerase chain reaction (PCR) revealed the SAM-P/1 type apoA-II variant in SJL/J and A/J strains with a high susceptibility to ASSAM deposition. We concluded from this study that amino acid substitution in precursor apoA-II may be responsible for the early onset and severe amyloid deposition in the mouse.     

Conformational and intermolecular interactions of proteins in amyloidosis

A conception on amyloidosis as a key factor of neuronal death in neurodegenerative diseases is stated. Experimental evidence is presented that amyloidosis is caused by alterations in the activity of a number of enzymes as well as conformational changes in pathogenic proteins. Arguments for amyloidosis as the universal biological mechanism of specific elimination of neurons showing changed metabolic and physiological status of cell differentiation are adduced. The final pattern of cell death seems to differ cardinally from that in both apoptosis and necrosis.     

Transthyretin, a new cryptic protease

Transthyretin (TTR) is a plasma homotetrameric protein that acts physiologically as a transporter of thyroxine (T(4)) and retinol, in the latter case through binding to retinol-binding protein (RBP). A fraction of plasma TTR is carried in high density lipoproteins by binding to apolipoprotein AI (apoA-I). We further investigated the nature of the TTR-apoA-I interaction and found that TTR from different sources (recombinant and plasmatic) is able to process proteolytically apoA-I, cleaving its C terminus after Phe-225. TTR-mediated proteolysis was inhibited by serine protease inhibitors (phenylmethanesulfonyl fluoride, Pefabloc, diisopropyl fluorophosphate, chymostatin, and N(alpha)-p-tosyl-l-phenylala-nine-chloromethyl ketone), suggesting a chymotrypsin-like activity. A fluorogenic substrate corresponding to an apoA-I fragment encompassing amino acid residues 223-228 (Abz-ESFKVS-EDDnp) was used to characterize the catalytic activity of TTR, including optimum reaction conditions (37 degrees C and pH 6.8) and catalytic constant (K(m) = 29 microm); when complexed with RBP, TTR activity was lost, whereas when complexed with T(4), only a slight decrease was observed. Cell lines expressing TTR were able to degrade Abz-ESFKVS-EDDnp 2-fold more efficiently than control cells lacking TTR expression; this effect was reversed by the presence of RBP in cell culture media, therefore proving a TTR-specific proteolytic activity. TTR can act as a novel plasma cryptic protease and might have a new, potentially important role under physiological and/or pathological conditions.     

Solid phase proteomics: dramatic reinforcement of very weak protein-protein interactions

Very weak protein-protein interactions may play a critical role in cell physiology but they are not easily detectable in "in vitro" experiments. To detect these weak interactions, we have developed a strategy that included: (a) design of a rapid and very effective crosslinking of protein-protein complexes with poly-functional reagents; (b) selective adsorption of very large proteins on lowly activated ionic exchangers, based on the need of a multipoint physical adsorption to incorporate the proteins into the matrix; (c) purification by selective adsorption of protein-protein complexes formed by strong protein-protein interactions, via selective adsorption of the complexes on lowly activated ionic exchangers via multi-protein physical adsorption and leaving the non-associated proteins in the solution; (d) reinforcement of very weak protein-protein interactions by selective adsorption of the complex on lowly activated ionic exchange supports via a synergetic cooperation of the weak protein-protein interaction plus the interactions of both proteins with the support enabling the almost full shifting of the equilibrium towards the association position; (e) control of the aggregation state of proteins like BSA, formed by weak protein-protein interactions. In this last case, it seems that the interaction of the protein molecules placed on the borders of the aggregate with the groups on the support partially stabilizes the whole aggregate, although, some molecules of the aggregate cannot interact with the support. The size of the aggregates may be defined by controlling the concentration of ionised groups on the support: the less activated the supports are, the bigger the complexes. In this way, solid-phase proteomics could be a very interesting tool to detect weak protein-protein interactions.     

Emerging trends in molecular recognition: utility of weak aromatic interactions

Aromatic interactions play a vital role in chemistry and biology. As about 20% are aromatic in nature, so the role of aromatic interactions become prominent in drug receptor interactions. Not only in drug receptor interactions but also in crystal engineering, protein folding, stacking interactions in DNA/RNA the role of the interactions is of utmost importance. With the emergence of supramolecular chemistry dendrimers, tweezers, rotaxanes, catenanes, and several supramolecular aggregates are associated with aromatic interactions. The mechanism of such interactions is still unknown by the replacement of a small substituent from the aromatic molecule may lead or destroy the interactions. In the present review several models are being discussed with arene interactions under selected heads.     

Challenges and dreams: physics of weak interactions essential to life

Biological systems display stunning capacities to self-organize. Moreover, their subcellular architectures are dynamic and responsive to changing needs and conditions. Key to these properties are manifold weak “quinary” interactions that have evolved to create specific spatial networks of macromolecules. These specific arrangements of molecules enable signals to be propagated over distances much greater than molecular dimensions, create phase separations that define functional regions in cells, and amplify cellular responses to changes in their environments. A major challenge is to develop biochemical tools and physical models to describe the panoply of weak interactions operating in cells. We also need better approaches to measure the biases in the spatial distributions of cellular macromolecules that result from the integrated action of multiple weak interactions. Partnerships between cell biologists, biochemists, and physicists are required to deploy these methods. Together these approaches will help us realize the dream of understanding the biological “glue” that sustains life at a molecular and cellular level.