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The Encephaliozoon cuniculi is an obligate intracellular microsporidian parasite with a highly reduced genome, yet it contains several key components of an actin cytoskeleton. In this study, we characterise the α-actinin-like protein from the parasite to gain insight into its role in actin organisation. The protein contains three domains typical of α-actinins: an N-terminal actin-binding domain and a C-terminal calmodulin-like domain, separated by a rod domain. Gel filtration analysis demonstrated that the recombinant protein formed stable dimers, consistent with the canonical antiparallel α-actinin structure. Actin co-sedimentation assays and electron microscopy confirmed that the α-actinin-like protein binds and cross-links actin filaments into tight bundles, whereas the isolated actin-binding domain binds but does not cross-link filaments. AlphaFold modelling predicted an overall structural arrangement compatible with an antiparallel dimer. Our results identify the E. cuniculi α-actinin-like protein as a true α-actinin homologue. The presence of actin-binding proteins in E. cuniculi as well as in other microsporidia with very small genomes implies that an actin-based cytoskeleton is important for their survival.

This class-tested textbook gives an overview of the structure and functions of proteins and explains how amino acids form a defined structural entity with specific properties. The authors also introduce modern methods for purification and separation of proteins as well as different techniques for analyzing their structural and functional properties. A separate part of the book is devoted to enzymes and kinetics of enzymatic reactions. New in the second edition: Since the development of computing techniques has evolved considerably during the last few years, the text benefits from the addition of a chapter on the use of computing, particularly on the use of Alpha Fold in protein research. This AI-based software can determine the protein structures from the amino acid sequence with excellent reliability. Also included a discussion on the use of molecular dynamicsand a real life example of protein purification. Gives a comprehensive overview on the chemistry of proteins and their biological functions.

• Explains the principles of enzymatic regulation and kinetics of biological reactions.

• Written in a student-friendly manner with numerous examples.

Enzymatic catalysis is critically dependent on selectivity, active site architecture, and dynamics. To contribute insights into the interplay of these properties, we established an approach with NMR, crystallography, and MD simulations focused on the ubiquitous phosphotransferase adenylate kinase (AK) isolated from Odinarchaeota (OdinAK). Odinarchaeota belongs to the Asgard archaeal phylum that is believed to be the closest known ancestor to eukaryotes. We show that OdinAK is a hyperthermophilic trimer that, contrary to other AK family members, can use all NTPs for its phosphorylation reaction. Crystallographic structures of OdinAK-NTP complexes revealed a universal NTP-binding motif, while ¹⁹F NMR experiments uncovered a conserved and rate-limiting dynamic signature. As a consequence of trimerization, the active site of OdinAK was found to be lacking a critical catalytic residue and is therefore considered to be "atypical." On the basis of discovered relationships with human monomeric homologs, our findings are discussed in terms of evolution of enzymatic substrate specificity and cold adaptation.

The Australian tree malletwood (Rhodamnia argentea) is unique. The genome of malletwood is the only known plant genome that contains a gene coding for an α-actinin-like protein. Several organisms predating the animal-plant bifurcation express an α-actinin or α-actinin-like protein. Therefore, it appears that plants in general, but not malletwood, have lost the α-actinin or α-actinin-like gene during evolution. In order to characterize its structure and function, we synthesized the gene and expressed the recombinant R. argentea protein. The results clearly show that this protein has all properties of genuine α-actinin. The N-terminal actin-binding domain (ABD), with two calponin homology motifs, is very similar to the ABD of any α-actinin. The C-terminal calmodulin-like domain, as well as the intervening rod domain, are also similar to the corresponding regions in other α-actinins. The R. argentea α-actinin-like protein dimerises in solution and thereby can cross-link actin filaments. Based on these results, we believe the R. argentea protein represents a genuine α-actinin, making R. argentea unique in the plant world.

The biological function of proteins is critically dependent on dynamics inherent to the native structure. Such structural dynamics obey a predefined order and temporal timing to execute the specific reaction. Determination of the cooperativity of key structural rearrangements requires monitoring protein reactions in real time. In this work, we used time-resolved x-ray solution scattering (TR-XSS) to visualize structural changes in the Escherichia coli adenylate kinase (AdK) enzyme upon laser-induced activation of a protected ATP substrate. A 4.3-ms transient intermediate showed partial closing of both the ATP- and AMP-binding domains, which indicates a cooperative closing mechanism. The ATP-binding domain also showed local unfolding and breaking of an Arg131-Asp146 salt bridge. Nuclear magnetic resonance spectroscopy data identified similar unfolding in an Arg131Ala AdK mutant, which refolded in a closed, substrate-binding conformation. The observed structural dynamics agree with a “cracking mechanism” proposed to underlie global structural transformation, such as allostery, in proteins.

The genome of the chlorarchiniophyte Bigelowiella natans codes for a protein annotated as an α-actinin-like protein. Analysis of the primary sequence indicate that this protein has the same domain structure as other α-actinins, a N-terminal actin-binding domain and a C-terminal calmodulin-like domain. These two domains are connected by a short rod domain, albeit long enough to form a single spectrin repeat. To analyse the functional properties of this protein, the full-length protein as well as the separate domains were cloned and isolated. Characerisation showed that the protein is capable of cross-linking actin filaments into dense bundles, probably due to dimer formation. Similar to human α-actinin, calcium-binding occurs to the most N-terminal EF-hand motif in the calmodulin-like C-terminal domain. The results indicate that this Bigelowiella protein is a proper α-actinin, with all common characteristics of a typical α-actinin.

When it comes to crystallization each protein is unique. It can never be predicted beforehand in which condition the particular protein will crystallize or even if it is possible to crystallize. Still, by following some simple checkpoints the chances of obtaining crystals are increased. The primary checkpoints are purity, stability, concentration, and homogeneity. High-quality protein crystals are needed. This protocol will allow an investigator to: clone, express, and crystallize a protein of interest.

The discontinuous polyacrylamide gel electrophoresis system devised by Laemmli (Nature 227:680–685, 1970) has not only been used in numerous laboratories but has also been modified in several ways since its birth. In our laboratories, we use a modified Laemmli SDS-PAGE system for following protein purification as well as for analysis of certain protein-protein interactions, mainly involving filametous actin.

The actin cytoskeleton plays a fundamental role in eukaryotic cells. Its reorganization is regulated by a plethora of actin-modulating proteins, such as a-actinin. In higher organisms, alpha-actinin is characterized by the presence of three distinct structural domains: an N-terminal actin-binding domain and a C-terminal region with EF-hand motif separated by a central rod domain with four spectrin repeats. Sequence analysis has revealed that the central rod domain of alpha-actinin from the fission yeast Schizosaccharomyces pombe consists of only two spectrin repeats. To obtain a firmer understanding of the structure and function of this unconventional alpha-actinin, we have cloned and characterized each structural domain. Our results show that this alpha-actinin isoform is capable of forming dimers and that the rod domain is required for this. However, its actin-binding and cross-linking activity appears less efficient compared to conventional alpha-actinins. The solved crystal structure of the actin-binding domain indicates that the closed state is stabilised by hydrogen bonds and a salt bridge not present in other a-actinins, which may reduce the affinity for actin.