Current Protein and Peptide Science - Volume 8, Issue 5, 2007

The structure of Escherichia coli chaperonin GroEL was studied using various experimental tools. Such studies produced information about its structure with increasing details. Moreover, remarkable advances in experimental methods provided a step forward in understanding the reaction cycle involved in GroEL-mediated protein folding. In the current review we summarize recent progress, focus on the structure of GroEL and understand the mechanism involved in GroEL-mediated protein folding. This review is divided into the following sections: (i) Section 1 provides basic understanding on protein folding, (ii) Section 2 not only describes various tools used to elucidate the structural aspects of GroEL but also provides details about its structure with particular emphasis, (iii) Section 3 describes allosteric transitions and the reaction cycle involved in GroEL-mediated protein folding, (iv) Section 4 explains iterative annealing and smoothing of the energy landscape model and finally (v) Section 5 discusses applications and recent progress.

Mitochondria and chloroplasts are both of endosymbiotic origin. Upon symbiosis the ancestral bacteria had to be incorporated into the regulatory cellular network. A long known phenomenon is thereby the alteration of the positioning of the organelles in response to extra- and intracellular stimuli. For chloroplasts, the repositioning is a response to light intensity changes in order to optimize the photosynthetic process. Mitochondria movement ensures a positioning of the organelle close to the place where its function is required, e.g. in metabolic pathways, apoptosis, regulation of cytosolic calcium levels and ATP production. Even though the morphological description of the movement was circumstantiated decades ago, only recent research gave some insights into the molecular concepts behind the movement of organelles and its regulation. Mitochondrial movement is influenced for instance by calcium but also by small molecules like lysophosphatidic acid or metals like zinc. In turn, chloroplasts move in response to light. The light quality giving the impulse for movement depends on the plants investigated. As for mitochondria, calcium is an important second messenger for that process. The organelle movement is achieved by actin or tubulin. The recent discovery of proteins involved in the modulation of the movement like milton and miro in mitochondria or phot1 and phot2 and the organelle localized protein chup1, possibly facilitating the cytoskeleton contact, marked a new area of understanding of the process. This review will focus on a comprehensive overview on recent discoveries of the regulatory components.

Structure-based drug design (SBDD) has played an integral role in the development of highly specific, potent protease inhibitors resulting in a number of drugs in clinical trials and on the market. Possessing biochemical assays and structural information on both the target protease and homologous family members helps ensure compound selectivity. We have redesigned the path from clone to protein eliminating many of the traditional bottlenecks associated with protein production to ensure a constant supply to feed many diverse protease drug discovery programs. The process was initiated with the design of a multi-system vector, capable of expression in both eukaryotic and prokaryotic hosts; this vector also facilitated high-throughput cloning, expression and purification. When combined into an expression screen, supplemented with salvage screens for detergent extraction and refolding, a route for protein production was established rapidly. Using this process-orientated approach we have successfully expressed and purified all mechanistic classes of active human and viral proteases for enzymatic assays and crystallization studies. While exploiting recent developments in high-throughput biochemistry, we still employ classical biophysical techniques such as light-scattering and analytical ultracentrifugation, to ensure the highest quality protein enters crystallization trials. We have drawn on examples from our own research programs to illustrate how these strategies have been successfully used in the production of proteases for SBDD.

Defensins are a family of peptides with potent antimicrobial activity. They are found in various organisms. The intent of this article is to review the structures and mechanisms of antimicrobial actions of defensins produced by different organisms including humans, other mammals, birds, reptiles, fish, mollusks, arthropods, plants and fungi. The evolution and possible applications of these defensins are discussed.

Biological systems are organized in intricate and highly structured networks with hierarchies and multiple scales. Cells can be considered as “meso-scale level” systems placed between the “macro-scale level” (systems of cellular networks) and the “micro-scale level” (systems of molecular networks). In fact, cells represent complex biochemical machineries made by networks of molecules connected by biochemical reactions. Thus, the brain should be studied as a system of “networks of networks”. Recently, the existence of a Global Molecular Network (GMN) enmeshing the entire CNS was proposed. This proposal is based on the evidence that the extra-cellular matrix is a dynamic molecular structure capable of storing and releasing signals and of interacting with receptors and proteins on the cell membranes. Proteins have a special role in molecular networks since they can be assembled into high-order molecular complexes, which have been defined as Protein Mosaics (PM). Protein monomers in a PM (the “tesserae” of the mosaic) can interact via classical and non-classical cooperativity behaviour involving allosteric interactions. In the present paper, new features of allostery and cooperativity for protein folding, assemblage and topological features of PM will be discussed. Against this background, alterations in PM via allosteric modulations and non-classical cooperativity mechanisms may lead to protein aggregates like beta amyloid fibrils. Such aggregates cause pathological changes in the GMN structure and function leading to neurodegenerative diseases such as Alzheimer's disease. Thus, a novel view of the so called Protein Conformational Diseases (PCD) is proposed.

The 9-aminoacridines play an important role in medicine. They were applied first in a treatment of protozoal infections in the beginning of the last century. Recently, it has been shown that the 9-aminoacridines are successful candidates for treatment of cancer, viral and prion diseases. Their conjugation with biomolecules such as peptides and proteins may modulate their activity, bioavailability and applicability. This review deals with the synthesis of 9-aminoacridine, its conjugation with variety of molecules and utilization of such conjugates in several fields of science.

Seminal studies by Richardson [1] and Thornton [2] defined the constraints imposed by protein structure on disulfide formation and flagged forbidden regions of primary or secondary structure seemingly incapable of forming disulfide bonds between resident cysteine pairs. With respect to secondary structure, disulfide bonds were not found between cysteine pairs: A. on adjacent β-stands [1]; B. in a single helix or strand [2]; C. on non-adjacent strands of the same β-sheet [2]. In primary structure, disulfide bonds were not found between cysteine pairs: D. adjacent in the sequence [2]. In the intervening years it has become apparent that all these forbidden regions are indeed occupied by disulfide-bonded cysteines, albeit rather strained ones. It has been observed that sources of strain in a protein structure, such as residues in forbidden regions of the Ramachandran plot and cis-peptide bonds, are found in functionally important regions of the protein and warrant further investigation [3-5]. Like the Ramachandran plot, the earlier studies by Richardson [1] and Thornton [2] have identified a fundamental truth in protein stereochemistry: “forbidden” disulfides adopt strained conformations, but there is likely a functional reason for this. Emerging evidence supports a role for forbidden disulfides in redoxregulation of proteins.

Knowledge regarding the regulation of hepatic cytochrome P450 (P450) is crucial to the fields of drug therapy and drug development, as well as to our understanding of the mechanisms underlying the metabolic activation of toxic and carcinogenic compounds. P450 is a membrane-anchored protein that shows a variety of interaction with membrane phospholipids, which affect the membrane topology and catalytic activities of the protein. In particular, anionic phospholipids, nonbilayer forming lipids, and the degree of saturation of the lipid fatty acyl chain play important roles in the functional regulation of P450, as well as in the bilayer structure of the membrane. However, despite the importance of phospholipids in the regulation of P450s, the interaction of the protein with membrane phospholipids, and the membrane properties induced by phospholipids which regulate P450, are unclear. In this review, we describe the effect of the physicochemical properties of the phospholipid constituents of biological membranes on hepatic P450 catalytic activity, membrane insertion (and/or penetration), and structural changes.

Burn-induced immunosuppression not only increases susceptibility to infection, but also predisposes burn patients to related adverse sequelae, including systemic inflammatory response syndrome and sepsis. Although burn-related immunosuppression is not fully understood, it is characterized by decreased T- and B-lymphocyte function and by impaired functions of circulating leukocytes and complement. Alterations in defensins, a family of cationic, naturally occurring antimicrobial peptides, may underlie these immune deficiency patterns. Defensins are considered important components of the innate immune system, as they inhibit bacterial, fungal, and viral colonization. They also chemoattract immature dendritic cells and T lymphocytes, recruit neutrophils, macrophages, and monocytes, modulate complement and adjuvant activity, and promote inflammation and wound healing. Infectious states are associated with upregulation of circulating defensins, which suggests an underlying antimicrobial role. In addition, data from our laboratory demonstrated diminished levels of certain defensins in burned tissue. The inference is that decreased defensin levels in burn injury may facilitate infection and subsequent sepsis. It may also alter functions of T- and B-lymphocytes, neutrophils, macrophages, and complement, thereby contributing to the pathophysiology of burn-related systemic inflammatory responses. This article is a comprehensive review on the role of antimicrobial peptides in burns and wounds.