Colloidal Liquid Crystals Confined to Synthetic Tactoids.

When a liquid crystal forming particles are confined to a spatial volume with dimensions comparable to that of their own size, they face a complex trade-off between their global tendency to align and the local constraints imposed by the boundary conditions. This interplay may lead to a non-trivial orientational patterns that strongly depend on the geometry of the confining volume. This novel regime of liquid crystalline behavior can be probed with colloidal particles that are macro-aggregates of biomolecules. Here we study director fields of filamentous fd-viruses in quasi-2D lens-shaped chambers that mimic the shape of tactoids, the nematic droplets that form during isotropic-nematic phase separation. By varying the size and aspect ratio of the chambers we force these particles into confinements that vary from circular to extremely spindle-like shapes and observe the director field using fluorescence microscopy. In the resulting phase diagram, next to configurations predicted earlier for 3D tactoids, we find a number of novel configurations. Using Monte Carlo Simulations, we show that these novel states are metastable, yet long-lived. Their multiplicity can be explained by the co-existence of multiple dynamic relaxation pathways leading to the final stable states.

Functional characterization of Rad18 domains for Rad6, ubiquitin, DNA binding and PCNA modification.

Rad18 is a ubiquitin E3 ligase that monoubiquitinates PCNA on stalled replications forks. This allows recruitment of damage-tolerant polymerases for damage bypass and DNA repair. In this activity, the Rad18 protein has to interact with Rad6, the E2 ubiquitin-conjugating enzyme, ubiquitin, PCNA and DNA. Here we analyze the biochemical interactions of specific domains of the Rad18 protein. We found that the Rad6/Rad18 complex forms stable dimers in vitro. Consistent with previous findings, both the Ring domain and a C-terminal region contribute to the Rad6 interaction, while the C-terminus is not required for the interaction with PCNA. Surprisingly we find that the C2HC zinc finger is important for interaction with ubiquitin, apparently analogous to the interactions of classical zinc fingers with ubiquitin such as found in the UBZ and UBM domains in Y-family polymerases. Finally we find that the SAP domain, but not the zinc finger domain, is capable of DNA binding in vitro.

Co-expression of protein complexes in prokaryotic and eukaryotic hosts: experimental procedures, database tracking and case studies.

Structure determination and functional characterization of macromolecular complexes requires the purification of the different subunits in large quantities and their assembly into a functional entity. Although isolation and structure determination of endogenous complexes has been reported, much progress has to be made to make this technology easily accessible. Co-expression of subunits within hosts such as Escherichia coli and insect cells has become more and more amenable, even at the level of high-throughput projects. As part of SPINE (Structural Proteomics In Europe), several laboratories have investigated the use co-expression techniques for their projects, trying to extend from the common binary expression to the more complicated multi-expression systems. A new system for multi-expression in E. coli and a database system dedicated to handle co-expression data are described. Results are also reported from various case studies investigating different methods for performing co-expression in E. coli and insect cells.

Structure and ligand binding of carbohydrate-binding module CsCBM6-3 reveals similarities with fucose-specific lectins and "galactose-binding" domains.

Carbohydrate-binding polypeptides, including carbohydrate-binding modules (CBMs) from polysaccharidases, and lectins, are widespread in nature. Whilst CBMs are classically considered distinct from lectins, in that they are found appended to polysaccharide-degrading enzymes, this distinction is blurring. The crystal structure of CsCBM6-3, a "sequence-family 6" CBM in a xylanase from Clostridium stercorarium, at 2.3 A reveals a similar, all beta-sheet fold to that from MvX56, a module found in a family 33 glycoside hydrolase sialidase from Micromonospora viridifaciens, and the lectin AAA from Anguilla anguilla. Sequence analysis leads to the classification of MvX56 and AAA into a family distinct from that containing CsCBM6-3. Whilst these polypeptides are similar in structure they have quite different carbohydrate-binding specificities. AAA is known to bind fucose; CsCBM6-3 binds cellulose, xylan and other beta-glucans. Here we demonstrate that MvX56 binds galactose, lactose and sialic acid. Crystal structures of CsCBM6-3 in complex with xylotriose, cellobiose, and laminaribiose, 2.0 A, 1.35 A, and 1.0 A resolution, respectively, reveal that the binding site of CsCBM6-3 resides on the same polypeptide face as for MvX56 and AAA. Subtle differences in the ligand-binding surface give rise to the different specificities and biological activities, further blurring the distinction between classical lectins and CBMs.

Identification of cross-linked peptides for protein interaction studies using mass spectrometry and 18O labeling.

A new method is presented to screen proteolytic mass maps of cross-linked protein complexes for the presence of cross-linked peptides and for the verification of proposed structures. On the basis of the incorporation of 18O from isotopically enriched water into the C-termini of proteolytic peptides, cross-linked peptides are readily distinguished in mass spectra by a characteristic 8 amu shift. This is due to the incorporation of two 18O atoms in each C-terminus, so that normal and surface-labeled peptides shift 4 amu and cross-linked peptides containing two C-termini will shift 8 amu compared with their unlabeled counterparts. The method is fast, sensitive, and reliable and can be combined with any available cross-linking reagent and a wide range of proteolytic agents. As proof of principle, we successfully applied the method to a complex of two DNA repair proteins (Rad18-Rad6) and identified the interaction domain.

Differential oligosaccharide recognition by evolutionarily-related beta-1,4 and beta-1,3 glucan-binding modules.

Enzymes active on complex carbohydrate polymers frequently have modular structures in which a catalytic domain is appended to one or more carbohydrate-binding modules (CBMs). Although CBMs have been classified into a number of families based upon sequence, many closely related CBMs are specific for different polysaccharides. In order to provide a structural rationale for the recognition of different polysaccharides by CBMs displaying a conserved fold, we have studied the thermodynamics of binding and three-dimensional structures of the related family 4 CBMs from Cellulomonas fimi Cel9B and Thermotoga maritima Lam16A in complex with their ligands, beta-1,4 and beta-1,3 linked gluco-oligosaccharides, respectively. These two CBMs use a structurally conserved constellation of aromatic and polar amino acid side-chains that interact with sugars in two of the five binding subsites. Differences in the length and conformation of loops in non-conserved regions create binding-site topographies that complement the known solution conformations of their respective ligands. Thermodynamics interpreted in the light of structural information highlights the differential role of water in the interaction of these CBMs with their respective oligosaccharide ligands.

High-resolution crystal structures of the lectin-like xylan binding domain from Streptomyces lividans xylanase 10A with bound substrates reveal a novel mode of xylan binding.

Carbohydrate-binding module (CBM) family 13 includes the "R-type" or "ricin superfamily" beta-trefoil lectins. The C-terminal CBM, CBM13, of xylanase 10A from Streptomyces lividans is a family 13 CBM that is not only structurally similar to the "R-type" lectins but also somewhat functionally similar. The primary function of CBM13 is to bind the polysaccharide xylan, but it retains the ability of the R-type lectins to bind small sugars such as lactose and galactose. The association of CBM13 with xylan appears to involve cooperative and additive participation of three binding pockets in each of the three trefoil domains of CBM13, suggesting a novel mechanism of CBM-xylan interaction. Thus, the interaction of CBM13 with sugars displays considerable plasticity for which we provide a structural rationale. The high-resolution crystal structure of CBM13 was determined by multiple anomalous dispersion from a complex of CBM13 with a brominated ligand. Crystal structures of CBM13 in complex with lactose and xylopentaose revealed two distinct mechanisms of ligand binding. CBM13 has retained its specificity for lactose via Ricin-like binding in all of the three classic trefoil binding pockets. However, CBM13 has the ability to bind either the nonreducing galactosyl moiety or the reducing glucosyl moiety of lactose. The mode of xylopentaose binding suggests adaptive mutations in the trefoil sugar binding scaffold to accommodate internal binding on helical polymers of xylose.