Mapping sonic hedgehog-receptor interactions by steric interference

We have defined regions in the Sonic hedgehog (Shh) molecule that are important for Patched (Ptc) receptor binding by targeting selected surface amino acid residues with probes of diverse sizes and shapes and assessing the effects of these modifications on function. Eleven amino acid residues that surround the surface of the protein were chosen for these studies and mutated to cysteine residues. These cysteines were then selectively modified with thiol-specific probes, and the modified proteins were tested for hedgehog receptor binding activity and their ability to induce differentiation of C3H10T1/2 cells into osteoblasts. Based on these analyses, approximately one-third of the Shh surface can be modified without effect on function regardless of the size of the attachment. These sites are located near to where the C terminus protrudes from the surface of the protein. All other sites were sensitive to modification, indicating that the interaction of Shh with its primary receptor Ptc is mediated over a large surface of the Shh protein. For sites Asn-50 and Ser-156, function was lost with the smallest of the probes tested, indicating that these residues are in close proximity to the Ptc-binding site. The epitope for the neutralizing mAb 5E1 mapped to a close but distinct region of the structure. The structure-activity data provide a unique view of the interactions between Shh and Ptc that is not readily attainable by conventional mapping strategies.     

Probing Rad51-DNA interactions by changing DNA twist

In eukaryotes, Rad51 protein is responsible for the recombinational repair of double-strand DNA breaks. Rad51 monomers cooperatively assemble on exonuclease-processed broken ends forming helical nucleo-protein filaments that can pair with homologous regions of sister chromatids. Homologous pairing allows the broken ends to be reunited in a complex but error-free repair process. Rad51 protein has ATPase activity but its role is poorly understood, as homologous pairing is independent of adenosine triphosphate (ATP) hydrolysis. Here we use magnetic tweezers and electron microscopy to investigate how changes of DNA twist affect the structure of Rad51-DNA complexes and how ATP hydrolysis participates in this process. We show that Rad51 protein can bind to double-stranded DNA in two different modes depending on the enforced DNA twist. The stretching mode is observed when DNA is unwound towards a helical repeat of 18.6 bp/turn, whereas a non-stretching mode is observed when DNA molecules are not permitted to change their native helical repeat. We also show that the two forms of complexes are interconvertible and that by enforcing changes of DNA twist one can induce transitions between the two forms. Our observations permit a better understanding of the role of ATP hydrolysis in Rad51-mediated homologous pairing and strand exchange.     

Affinity-based isolation of a bacterial lipase through steric chaperone interactions

Lipases are important as additives in detergent formulations but their biocatalytic potential is increasingly exploited in the synthesis of high-added value chemicals, in fine-chemical production and in the pharmaceutical industry. Traditionally, conventional purification schemes comprise several chromatographic steps. Here we report a new purification procedure of the lipase (LipA) that is endogenously secreted by the Gram-negative bacterium Burkholderia glumae. This affinity purification combines the specific binding scaffold of a lipase-specific foldase (Lif) and the intrinsic resistance to chemical denaturation of LipA. The newly devised method is less labor-intensive, is fast, leads to a homogeneous preparation and can be easily scaled up. The novel and the conventional purification strategies were evaluated in parallel and characteristics of the B. glumae lipase were analyzed via CD spectroscopy. Lipopolysaccharide (LPS) was still present in the samples purified via the conventional purification scheme and was shown to increase the thermostability of the lipase.     

Electrostatic and steric interactions determine bacteriorhodopsin single-molecule biomechanics

Bacteriorhodopsin (bR) is a haloarchaeal membrane protein that converts the energy of single photons into large structural changes to directionally pump protons across purple membrane. This is achieved by a complex combination of local dynamic interactions controlling bR biomechanics at the submolecular level, producing efficient amplification of the retinal photoisomerization. Using single molecule force spectroscopy at different salt concentrations, we show that tryptophan (Trp) residues use steric specific interactions to create a rigid scaffold in bR extracellular region and are responsible for the main unfolding barriers. This scaffold, which encloses the retinal, controls bR local mechanical properties and anchors the protein into the membrane. Furthermore, the stable Trp-based network allows ion binding to two specific sites on the extracellular loops (BC and FG), which are involved in proton release and lateral transport. In contrast, the cytoplasmic side of bR is mainly governed by relatively weak nonspecific electrostatic interactions that provide the flexibility necessary for large cytoplasmic structural rearrangements during the photocycle. The presence of an extracellular Trp-based network tightly enclosing the retinal seems common to most haloarchaeal rhodopsins, and could be relevant to their exceptional efficiency.     

Engineering receptor-mediated cytotoxicity into human ribonucleases by steric blockade of inhibitor interaction

Several nonmammalian members of the RNase A superfamily exhibit anticancer activity that appears to correlate with resistance to the cytosolic ribonuclease inhibitor (RI). We mutated two human ribonucleases-pancreatic RNase (hRNAse) and eosinophil-derived neurotoxin (EDN)-to incorporate cysteine residues at putative sites of close contact to RI, but distant from the catalytic sites. Coupling of Cys89 of RNase and Cys87 of EDN to proteins at these sites via a thioether bond produced enzymatically active conjugates that were resistant to RI. To elicit cellular targeting as well as to block RI binding, transferrin was conjugated to a mutant human RNase, rhRNase(Gly89)-->Cys) and a mutant EDN (Thr87-->Cys). The transferrin-rhRNase(Gly89-->Cys) thioether conjugate was 5000-fold more toxic to U251 cells than recombinant wild-type hRNase. In addition, transferrin-targeted EDN exhibited tumor cell toxicities similar to those of hRNase. Thus, we endowed two human RI-sensitive RNases with greater cytotoxicity by increasing their resistance to RI. This strategy has the potential to generate a novel set of recombinant human proteins useful for targeted therapy of cancer.     

Formation of 9,10-epoxypalmitate and 9,10-dihydroxypalmitate from palmitoleic acid by a soluble system from Bacillus megaterium.

A soluble, cytochrome P-450-containing system from $\text{Bacillus}\text{megaterium}$ which catalyzes the monohydroxylation of long-chain saturated fatty acids has now been found to convert palmitoleic acid to 9,10-epoxypalmitate and 9,10-dihydroxypalmitate in addition to the expected isomeric mixture of monohydroxypalmitoleic acids.     

Phosphorescence quenching in evaluation of the steric interactions of heme-containing proteins

The rate constants of exchange phosphorescence quenching of eosin by ferricyanide, hemin and hem-proteins (cytochrome c, myoglobin , hemoglobin, leghemoglobin) are measured. The steric factor of hem of cytochrome c is evaluated from the constant of eosin phosphorescence quenching by cytochrome c.     

Engineering a "steric doorstop" in rhodopsin: converting an inverse agonist to an agonist

The crystal structures of rhodopsin depict the inactive conformation of rhodopsin in the dark. The 11-cis retinoid chromophore, the inverse agonist holding rhodopsin inactive, is well-resolved. Thr118 in helix 3 is the closest amino acid residue next to the 9-methyl group of the chromophore. The 9-methyl group of retinal facilitates the transition from an inactive metarhodopsin I to the active metarhodopsin II intermediate. In this study, a site-specific mutation of Thr118 to the bulkier Trp was made with the idea to induce an active conformation of the protein. The data indicate that such a mutation does indeed result in an active protein that depends on the presence of the ligand, specifically the 9-methyl group. As a result of this mutation, 11-cis retinal has been converted to an agonist. The apoprotein form of this mutant is no more active than the wild-type apoprotein. However, unlike wild-type rhodopsin, the covalent linkage of the ligand can be attacked by hydroxylamine in the dark. The combination of the Thr118Trp mutation and the 9-methyl group of the chromophore behaves as a "steric doorstop" holding the protein in an open and active conformation.     

Writhe of DNA induced by a terminal twist

This paper considers the three-dimensional structure of B-form DNA. The molecule may be open or covalently closed. For the former, its two ends are not allowed to move or rotate freely in space unless the molecule is under the influence of rigid body motions of the ambient space. Implied by the elastic rod model for DNA, the molecule writhes immediately when subject to a terminal twist as long as its axis is none of the following curves: lines, circular arcs, circular helices. This result is remarkably different from well-known results about DNA of other conformations. For example, if a DNA is regarded as an elastic rod whose axis is a circle, then it has no induced writhe when subject to a terminal twist until the latter meets a critical extent. To my mother for her 70th birthday An erratum to this article is available at .     

Fluorescence properties of a benzo(a)pyrene 7,8 dihydrodiol 9,10-oxide-DNA adduct. Conformation and effects of intermolecular DNA interactions

The pyrene-like fluorescence of the covalent benzo(a)pyrene diol-epoxide-DNA complex prepared by reacting 7,8,-dihydrodiol 9,10-epoxy benzo(a)pyrene (BPDE) with DNA in aqueous solution $\text{in vitro}$, has been investigated. It is shown that this fluorescence is $\text{sensitive}$ to molecular oxygen, to the concentration of $\text{native}$ DNA and to the ionic strength (KCl concentration), but is $\text{insensitive}$ to the concentration of $\text{denatured}$ DNA. These effects are related to the conformation of the pyrene-like chromophore of BPDE. Most of the fluorescence of a $\text{dilute}$ solution of the DNA-bound benzo(a)pyrene derivative originates from binding sites in which the pyrene moiety is not intercalated between the DNA base pairs, but is located on the outside of the DNA double helix.     

Regulation by phosphorylation. Yet another twist in the WASP story

Cell migration and other complex cellular processes involve a variety of signaling molecules and require the integration of multiple signals into a coherent cytoskeletal response. Two papers in the May issue of Molecular Cell now demonstrate that phosphorylation plays a critical role in WASP function as a regulator of Arp2/3-mediated actin polymerization.     

Role of interchain interactions in the stabilization of the right-handed twist of beta-sheets

Conformational energy computations have been carried out for parallel and antiparallel beta-sheets composed of poly-L-Val and poly-L-Ile peptide chains, each consisting of four and of six residues, respectively, with CH3CO- and-NHCH3 end groups. The beta-sheets considered contained three and five equivalent chains, respectively. All computed minimum-energy beta-sheets were found to have a large right-handed twist of a magnitude that corresponds to the mean twist of beta-sheets observed in globular proteins. The twist has the same sign but is much larger than in beta-sheets of poly-L-Ala, because of intra- and interchain interactions between the bulky beta-branched side-chains. While the right-handed twist is a result of intrachain interactions between side-chains in the case of poly-L-Val, these interactions would favor a left-handed twist in poly-L-Ile, and the right-handed twist in the latter is a result of interchain interactions. Parallel beta-sheets are more stable than antiparallel sheets for both poly-L-Val and poly-L-Ile, in contrast to poly-L-Ala. This result agrees with observations on the preferred orientation of the chains in oligopeptides that form beta-structures. It also explains the observed high relative frequencies of occurrence of Val and Ile residues in parallel beta-sheets, as compared with antiparallel sheets, in globular proteins.     

Effect of electron and steric interactions on the radioprotective properties of indolylalkylamines

Molecular mechanisms of radioprotective action of some substituted indolylalkylamines are discussed in terms of the statistical correlation analysis. It was established that electron and steric properties of the substituents are the factors influencing the radioprotective efficiency of these compounds. It was shown that the correlation obtained, relating the structure of the compounds to the radioprotective effect, may be applied in studies of the mechanism of action of the preparations and for the purposeful synthesis of new compounds.     

Characterization of yeast cultivations by steric sedimentation field-flow fractionation.

The characterization and quantification of biomass is often time consuming and dependent on the cultivation media and gives no detailed information between cell size and shape and their productivity. By monitoring the bioprocess with steric sedimentation field-flow fractionation (Sd/StFFF) in combination with laser light scattering, not only cell growth, but also the variation of cell size and shape during the cultivation, can be observed. In this work, the feasibility of separating and characterizing cell populations by steric sedimentation field-flow fractionation is demonstrated by its application to three different yeast cultivation broths. For this purpose samples which were collected at different cultivation times were injected into an FFF system. Fractograms were obtained in less than 4 min. Due to the relatively high resolution of the method, a cell sample could be fractionated in several subpopulations differing in their size as well as in their number of buds.