Shira Albeck

Control of Aragonite or Calcite Polymorphism by Mollusk Shell Macromolecules

Many mineralizing organisms selectively form either calcite or aragonite, two polymorphs of calcium carbonate with very similar crystalline structures. Understanding how these organisms achieve this control has represented a major challenge in the field of biomineralization. Macromolecules extracted from the aragonitic shell layers of some mollusks induced aragonite formation in vitro when preadsorbed on a substrate of β-chitin and silk fibroin. Macromolecules from calcitic shell layers induced mainly calcite formation under the same conditions. The results suggest that these macromolecules are responsible for the precipitation of either aragonite or calcite in vivo.

Kemp elimination catalysts by computational enzyme design

The design of new enzymes for reactions not catalysed by naturally occurring biocatalysts is a challenge for protein engineering and is a critical test of our understanding of enzyme catalysis. Here we describe the computational design of eight enzymes that use two different catalytic motifs to catalyse the Kemp elimination—a model reaction for proton transfer from carbon—with measured rate enhancements of up to 105 and multiple turnovers. Mutational analysis confirms that catalysis depends on the computationally designed active sites, and a high-resolution crystal structure suggests that the designs have close to atomic accuracy. Application of in vitro evolution to enhance the computational designs produced a >200-fold increase in kcat/Km (kcat/Km of 2,600 M-1s-1 and kcat/kuncat of >106). These results demonstrate the power of combining computational protein design with directed evolution for creating new enzymes, and we anticipate the creation of a wide range of useful new catalysts in the future.

Rational design of faster associating and tighter binding protein complexes

A protein design strategy was developed to specifically enhance the rate of association (kon) between a pair of proteins without affecting the rate of dissociation (koff). The method is based on increasing the electrostatic attraction between the proteins by incorporating charged residues in the vicinity of the binding interface. The contribution of mutations towards the rate of association was calculated using a newly developed computer algorithm, which predicted accurately the rate of association of mutant protein complexes relative to the wild type. Using this design strategy, the rate of association and the affinity between TEM1 β-lactamase and its protein inhibitor BLIP was enhanced 250-fold, while the dissociation rate constant was unchanged. The results emphasize that long range electrostatic forces specifically alter kon, but do not effect koff. The design strategy presented here is applicable for increasing rates of association and affinities of protein complexes in general.

Control Over Aragonite Crystal Nucleation and Growth: An In Vitro Study of Biomineralization

Control over calcium carbonate deposition in an in vitro system containing the major macromolecular components present in mollusk shell is shown to depend on the presence of a specific protein fraction in the appropriate microenvironment, in this case, silk fibroin adsorbed on β-chitin. Experiments with synthetic polypeptides elucidate aspects of the sequence dependency of nucleation of the aragonite polymorph, shown here growing in the nacreous layer of the bivalve Atrina serrata (note the orientation of the small, newly formed crystals and their merging together in more mature areas).

A De Novo Protein Binding Pair By Computational Design and Directed Evolution

The de novo design of protein-protein interfaces is a stringent test of our understanding of the principles underlying protein-protein interactions and would enable unique approaches to biological and medical challenges. Here we describe a motif-based method to computationally design protein-protein complexes with native-like interface composition and interaction density. Using this method we designed a pair of proteins, Prb and Pdar, that heterodimerize with a Kd of 130 nM, 1000-fold tighter than any previously designed de novo protein-protein complex. Directed evolution identified two point mutations that improve affinity to 180 pM. Crystal structures of an affinity-matured complex reveal binding is entirely through the designed interface residues. Surprisingly, in the in vitro evolved complex one of the partners is rotated 180° relative to the original design model, yet still maintains the central computationally designed hotspot interaction and preserves the character of many peripheral interactions. This work demonstrates that high-affinity protein interfaces can be created by designing complementary interaction surfaces on two noninteracting partners and underscores remaining challenges.

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.

Crystal-protein interactions studied by overgrowth of calcite on biogenic skeletal elements

Abstract A key parameter in the biological control of crystal formation is the interaction of a group of acidic macromolecules with the mineral phase. Here we study protein-calcite interactions using epitaxial overgrowth of synthetic calcite crystals under conditions in which local release of occluded macromolecules from the biogenic substrate occurs. The macromolecules subsequently interact with the newly formed overgrown crystals, resulting in modified calcite morphology. This novel method provides a means of mapping crystal-protein interactions under conditions that minimally affect the conformational states of the acidic macromolecules. We show that proteins released from calcitic sponge spicules and mollusc prisms specifically interact with {001} and {01 l } faces of calcite, whereas proteins released from echinoderm skeletal elements only interact with {01 l } faces. The extent to which the overgrown crystals are affected by the proteins varies even in the same organism and within the same element, depending on the site and crystallographic orientation of the skeletal elements.

Regulation of calcite crystal morphology by intracrystalline acidic proteins and glycoproteins.

Many biologically formed calcite crystals contain intracrystalline macromolecules. The ways in which they interact with growing calcite crystals were evaluated by monitoring changes in the morphology of calcite crystals grown in vitro in their presence. Macromolecules were extracted from within isolated prisms from the prismatic layer of the shell of the mollusk Atrina rigida and from spines of the sea urchin Paracentrotus lividus. Two modes of interaction were identified; the interaction of highly acidic proteins with calcite planes perpendicular to the c crystallographic axis and the interaction of glycoproteins with planes roughly parallel to the c axis. By different preparative procedures we demonstrated that the polysaccharide moieties of the sea urchin spine glycoproteins are directly involved in the latter mode of interactions. We suggest that organisms utilize the abilities of these macromolecules to interact in different ways with calcite crystals, and in so doing fine-tune aspects of the control of crystal growth in vivo.

Communication between viruses guides lysis–lysogeny decisions

Temperate viruses can become dormant in their host cells, a process called lysogeny. In every infection, such viruses decide between the lytic and the lysogenic cycles, that is, whether to replicate and lyse their host or to lysogenize and keep the host viable. Here we show that viruses (phages) of the SPbeta group use a small-molecule communication system to coordinate lysis–lysogeny decisions. During infection of its Bacillus host cell, the phage produces a six amino-acids-long communication peptide that is released into the medium. In subsequent infections, progeny phages measure the concentration of this peptide and lysogenize if the concentration is sufficiently high. We found that different phages encode different versions of the communication peptide, demonstrating a phage-specific peptide communication code for lysogeny decisions. We term this communication system the ‘arbitrium’ system, and further show that it is encoded by three phage genes: aimP, which produces the peptide; aimR, the intracellular peptide receptor; and aimX, a negative regulator of lysogeny. The arbitrium system enables a descendant phage to ‘communicate’ with its predecessors, that is, to estimate the amount of recent previous infections and hence decide whether to employ the lytic or lysogenic cycle.

Core Promoter Binding by Histone-Like TAF Complexes

ABSTRACT A major function of TFIID is core promoter recognition. TFIID consists of TATA-binding protein (TBP) and 14 TBP-associated factors (TAFs). Most of them contain a histone fold domain (HFD) that lacks the DNA-contacting residues of histones. Whether and how TAF HFDs contribute to core promoter DNA binding are yet unresolved. Here we examined the DNA binding activity of TAF9, TAF6, TAF4b, and TAF12, which are related to histones H3, H4, H2A, and H2B, respectively. Each of these TAFs has intrinsic DNA binding activity adjacent to or within the HFD. The DNA binding domains were mapped to evolutionarily conserved and essential regions. Remarkably, HFD-mediated interaction enhanced the DNA binding activity of each of the TAF6-TAF9 and TAF4b-TAF12 pairs and of a histone-like octamer complex composed of the four TAFs. Furthermore, HFD-mediated interaction stimulated sequence-specific binding by TAF6 and TAF9. These results suggest that TAF HFDs merge with other conserved domains for efficient and specific core promoter binding.