Fusion polypeptides in gene cloning: potential problems due to conformational alterations at the junction

Many eukaryotic genes are cloned in bacterial hosts as fusion polypeptides. Prediction of the secondary structures for some common prokaryotic fusion polypeptides shows that many junction sites correspond to important secondary structures. It is suggested that such structures could affect (hinder, etc.) the conformation or drive the folding of the neighboring eukaryotic counterparts. Thus the prokaryotic junction should be better performed in random coil regions, or short fusion prokaryotic polypeptides should be used.     

Intrinsically disordered proteins in crowded milieu: when chaos prevails within the cellular gumbo

Effects of macromolecular crowding on structural and functional properties of ordered proteins, their folding, interactability, and aggregation are well documented. Much less is known about how macromolecular crowding might affect structural and functional behaviour of intrinsically disordered proteins (IDPs) or intrinsically disordered protein regions (IDPRs). To fill this gap, this review represents a systematic analysis of the available literature data on the behaviour of IDPs/IDPRs in crowded environment. Although it was hypothesized that, due to the excluded-volume effects present in crowded environments, IDPs/IDPRs would invariantly fold in the presence of high concentrations of crowding agents or in the crowded cellular environment, accumulated data indicate that, based on their response to the presence of crowders, IDPs/IDPRs can be grouped into three major categories, foldable, non-foldable, and unfoldable. This is because natural cellular environment is not simply characterized by the presence of high concentration of “inert” macromolecules, but represents an active milieu, components of which are engaged in direct physical interactions and soft interactions with target proteins. Some of these interactions with cellular components can cause (local) unfolding of query proteins. In other words, since crowding can cause both folding and unfolding of an IDP or its regions, the outputs of the placing of a query protein to the crowded environment would depend on the balance between these two processes. As a result, and because of the spatio-temporal heterogeneity in structural organization of IDPs, macromolecular crowding can differently affect structures of different IDPs. Recent studies indicate that some IDPs are able to undergo liquid–liquid-phase transitions leading to the formation of various proteinaceous membrane-less organelles (PMLOs). Although interiors of such PMLOs are self-crowded, being characterized by locally increased concentrations of phase-separating IDPs, these IDPs are minimally foldable or even non-foldable at all (at least within the physiologically safe time-frame of normal PMLO existence).     

Faithful chaperones

This review describes the properties of some rare eukaryotic chaperones that each assist in the folding of only one target protein. In particular, we describe (1) the tubulin cofactors, (2) p47, which assists in the folding of collagen, (3) α-hemoglobin stabilizing protein (AHSP), (4) the adenovirus L4-100 K protein, which is a chaperone of the major structural viral protein, hexon, and (5) HYPK, the huntingtin-interacting protein. These various-sized proteins (102–1,190 amino acids long) are all involved in the folding of oligomeric polypeptides but are otherwise functionally unique, as they each assist only one particular client. This raises a question regarding the biosynthetic cost of the high-level production of such chaperones. As the clients of faithful chaperones are all abundant proteins that are essential cellular or viral components, it is conceivable that this necessary metabolic expenditure withstood evolutionary pressure to minimize biosynthetic costs. Nevertheless, the complexity of the folding pathways in which these chaperones are involved results in error-prone processes. Several human disorders associated with these chaperones are discussed.     

Fusion polypeptides in gene cloning: Potential problems due to conformational alterations at the junction

Summary Many eukaryotic genes are cloned in bacterial hosts as fusion polypeptides. Prediction of the secondary structures for some common prokaryotic fusion polypeptides shows that many junction sites correspond to important secondary structures. It is suggested that such structures could affect (hinder, etc.) the conformation or drive the folding of the neighboring eukaryotic counterparts. Thus the prokaryotic junction should be better performed, in random coil regions, or short fusion prokaryotic polypeptides should be sued.1 October 1986     

Hsp70 in mitochondrial biogenesis: From chaperoning nascent polypeptide chains to facilitation of protein degradation

The family of hsp70 (70 kilodalton heat shock protein) molecular chaperones plays an essential and diverse role in cellular physiology, Hsp70 proteins appear to elicit their effects by interacting with polypeptides that present domains which exhibit non-native conformations at distinct stages during their life in the cell. In this paper we review work pertaining to the functions of hsp70 proteins in chaperoning mitochondrial protein biogenesis. Hsp70 proteins function in protein synthesis, protein translocation across mitochondrial membranes, protein folding and finally the delivery of misfolded proteins to proteolytic enzymes in the mitochondrial matrix.     

Molecular guardians for newborn proteins: ribosome-associated chaperones and their role in protein folding

A central dogma in biology is the conversion of genetic information into active proteins. The biosynthesis of proteins by ribosomes and the subsequent folding of newly made proteins represent the last crucial steps in this process. To guarantee the correct folding of newly made proteins, a complex chaperone network is required in all cells. In concert with ongoing protein biosynthesis, ribosome-associated factors can interact directly with emerging nascent polypeptides to protect them from degradation or aggregation, to promote folding into their native structure, or to otherwise contribute to their folding program. Eukaryotic cells possess two major ribosome-associated systems, an Hsp70/Hsp40-based chaperone system and the functionally enigmatic NAC complex, whereas prokaryotes employ the Trigger Factor chaperone. Recent structural insights into Trigger Factor reveal an intricate cradle-like structure that, together with the exit site of the ribosome, forms a protected environment for the folding of newly synthesized proteins. Received 29 June 2005; received after revision 4 August 2005; accepted 18 August 2005     

Distribution of tightened end fragments of globular proteins statistically matches that of topohydrophobic positions: towards an efficient punctuation of protein folding?

Using a set of 372 proteins representative of a variety of 56 distinct globular folds, a statistical correlation was observed between two recently revealed features of protein structures: tightened end fragments or 'closed loops', i. e. sequence fragments that are able in three-dimensional (3D) space to nearly close their ends (a current parameter of polymer physics), and 'topohydrophobic positions', i. e. positions always occupied in 3D space by strong hydrophobic amino acids for all members of a fold family. Indeed, in sequence space, the distribution of preferred lengths for tightened end fragments and that for topohydrophobic separation match. In addition to this statistically significant similarity, the extremities of these 'closed loops' may be preferentially occupied by topohydrophobic positions, as observed on a random sample of various folds. This observation may be of special interest for sequence comparison of distantly related proteins. It is also important for the ab initio prediction of protein folds, considering the remarkable topological properties of topohydrophobic positions and their paramount importance within folding nuclei. Consequently, topohydrophobic positions locking the 'closed loops' belong to the deep cores of protein domains and might have a key role in the folding process. Received 1 February 2001; accepted 7 February 2001     

Structural determinants of protein folding

The last several decades have seen an explosion of knowledge in the field of structural biology. With critical advances in spectroscopic techniques in examining structures of biomacromolecules, in maturation of molecular biology techniques, as well as vast improvements in computation prowess, protein structures are now being elucidated at an unprecedented rate. In spite of all the recent advances, the protein folding puzzle remains as one of the fundamental biochemical challenges. A facet to this empiric problem is the structural determinants of protein folding. What are the driving forces that pivot a polypeptide chain to a specific conformation amongst the vast conformation space? In this review, we shall discuss some of the structural determinants to protein folding that have been identified in the recent decades.     

Modulating RNA structure and catalysis: lessons from small cleaving ribozymes

RNA is a key molecule in life, and comprehending its structure/function relationships is a crucial step towards a more complete understanding of molecular biology. Even though most of the information required for their correct folding is contained in their primary sequences, we are as yet unable to accurately predict both the folding pathways and active tertiary structures of RNA species. Ribozymes are interesting molecules to study when addressing these questions because any modifications in their structures are often reflected in their catalytic properties. The recent progress in the study of the structures, the folding pathways and the modulation of the small ribozymes derived from natural, self-cleaving, RNA motifs have significantly contributed to today’s knowledge in the field.     

The short proline-rich antibacterial peptide family

From the many peptide families that are induced upon bacterial infection and can be isolated from all classes of animals, the short, proline-rich antibacterial peptides enjoy particular interest. These molecules were shown to inactivate an intracellular biopolymer in bacteria without destroying or remaining attached to the bacterial cell membrane, and as such emerged as viable candidates for the treatment of mammalian infections. These peptides were originally isolated from insects, they kill mostly Gram-negative bacteria with high efficiency and they show structural similarities with longer insect- and mammal-derived antimicrobial peptides. However, while the distant relatives appear to carry multiple functional domains, apidaecin, drosocin, formaecin and pyrrhocoricin consist of only minimal determinants needed to penetrate across the cell membrane and bind to the target biopolymer. These peptides appear to inhibit metabolic processes, such as protein synthesis or chaperone-assisted protein folding. Pyrrhocoricin derivatives protect mice from experimental infections in vivo, suggesting the utility of modified analogs in the clinical setting. Sequence variations of the target protein at the peptide-binding site may allow the development of new peptide variants that kill currently unresponsive strains or species. Received 12 December 2001; received after revision 11 February 2002; accepted 19 February 2002     

Biogenesis of bacterial inner-membrane proteins

All cells must traffic proteins into and across their membranes. In bacteria, several pathways have evolved to enable protein transfer across the inner membrane, the periplasm, and the outer membrane. The major route of protein translocation in and across the cytoplasmic membrane is the general secretion pathway (Sec-pathway). The biogenesis of membrane proteins not only requires protein translocation but also coordinated targeting to the membrane beforehand and folding and assembly into their protein complexes afterwards to function properly in the cell. All these processes are responsible for the biogenesis of membrane proteins that mediate essential functions of the cell such as selective transport, energy conversion, cell division, extracellular signal sensing, and motility. This review will highlight the most recent developments on the structure and function of bacterial membrane proteins, focusing on the journey that integral membrane proteins take to find their final destination in the inner membrane.     

Regulation of lactate dehydrogenase activity: reversible and isoenzyme-specific inhibition of the tetramerization process by peptides

The transition of inactive lactic dehydrogenase (LDH) subunits to functional tetramers is controlled by 2 naturally-occurring peptides. One of these peptides inhibits the folding/association/activation process of H-LDH and the other peptide, the reconstitution of M-LDH, NADH and NAD+ are isoenzyme-specific antagonists of the 2 inhibitory peptides.     

嗜热蛋白稳定机理研究进展

嗜热蛋白是一类主要来源于嗜热微生物的热稳定蛋白，能够在高温下长时间保持活性而不变性．通过对嗜热蛋白耐热机理的深入研究，对于人们深入理解蛋白质的折叠、结构与功能、进化以及在蛋白质加工中对蛋白质分子的定向设计和改造有着重要的意义。本文主要介绍了目前对嗜热蛋白的研究概况和主要进展。     

Hsp70 chaperones: Cellular functions and molecular mechanism

Hsp70 proteins are central components of the cellular network of molecular chaperones and folding catalysts. They assist a large variety of protein folding processes in the cell by transient association of their substrate binding domain with short hydrophobic peptide segments within their substrate proteins. The substrate binding and release cycle is driven by the switching of Hsp70 between the low-affinity ATP bound state and the high-affinity ADP bound state. Thus, ATP binding and hydrolysis are essential in vitro and in vivo for the chaperone activity of Hsp70 proteins. This ATPase cycle is controlled by co-chaperones of the family of J-domain proteins, which target Hsp70s to their substrates, and by nucleotide exchange factors, which determine the lifetime of the Hsp70-substrate complex. Additional co-chaperones fine-tune this chaperone cycle. For specific tasks the Hsp70 cycle is coupled to the action of other chaperones, such as Hsp90 and Hsp100.Received 21 October 2004; received after revision 24 November 2004; accepted 6 December 2004     

Emergence of symmetric protein architecture from a simple peptide motif: evolutionary models

Structural symmetry is observed in the majority of fundamental protein folds and gene duplication and fusion evolutionary processes are postulated to be responsible. However, convergent evolution leading to structural symmetry has also been proposed; additionally, there is debate regarding the extent to which exact primary structure symmetry is compatible with efficient protein folding. Issues of symmetry in protein evolution directly impact strategies for de novo protein design as symmetry can substantially simplify the design process. Additionally, when considering gene duplication and fusion in protein evolution, there are two competing models: “emergent architecture” and “conserved architecture”. Recent experimental work has shed light on both the evolutionary process leading to symmetric protein folds as well as the ability of symmetric primary structure to efficiently fold. Such studies largely support a “conserved architecture” evolutionary model, suggesting that complex protein architecture was an early evolutionary achievement involving oligomerization of smaller polypeptides.     

Chemical properties of alcohols and their protein binding sites

Alcohols affect a wide array of biological processes including protein folding, neurotransmission and immune responses. It is becoming clear that many of these effects are mediated by direct binding to proteins such as neurotransmitter receptors and signaling molecules. This review summarizes the unique chemical properties of alcohols which contribute to their biological effects. It is concluded that alcohols act mainly as hydrogen bond donors whose binding to the polypeptide chain is stabilized by hydrophobic interactions. The electronegativity of the O atom may also play a role in stabilizing contacts with the protein. Properties of alcohol binding sites have been derived from X-ray crystal structures of alcohol-protein complexes and from mutagenesis studies of ion channels and enzymes that bind alcohols. Common amino acid sequences and structural features are shared among the protein segments that are involved in alcohol binding. The alcohol binding site is thought to consist of a hydrogen bond acceptor in a turn or loop region that is often situated at the N-terminal end of an alpha-helix. The methylene chain of the alcohol molecule appears to be accommodated by a hydrophobic groove formed by two or more structural elements, frequently a turn and an alpha-helix. Binding at these sites may alter the local protein structure or displace bound solvent molecules and perturb the function of key proteins.     

The perspectives of studying multi-domain protein folding

Most of fundamental studies on protein folding have been performed with small globular proteins consisting of a single domain. In vitro many of these proteins are well characterized by a reversible two-state folding scheme. However, the majority of proteins in the cell belong to the class of larger multi-domain proteins that often unfold irreversibly under in vitro conditions. This makes folding studies difficult or even impossible. In spite of these problems for many multi-domain proteins, folding has been investigated by classical refolding. Co-translational folding of nascent polypeptide chains when synthesized by ribosomes has also been studied. Single molecule techniques represent a promising approach for future studies on the folding of multi-domain proteins, and tremendous advances have been made in these techniques in recent years. In particular, fluorescence-based methods can contribute significantly to an understanding of the fundamental principles of multi-domain protein folding. Received 3 December 2008; accepted 23 December 2008     

Regulated protein degradation in mitochondria

Various adenosine triphosphate (ATP)-dependent proteases were identified within mitochondria which mediate selective mitochondrial protein degradation and fulfill crucial functions in mitochondrial biogenesis. The matrix-localized PIM1 protease, a homologue of theEscherichia coli Lon protease, is required for respiration and maintenance of mitochondrial genome integrity. Degradation of non-native polypeptides by PIM1 protease depends on the chaperone activity of the mitochondrial Hsp70 system, posing intriguing questions about the relation between the proteolytic system and the folding machinery in mitochondria. The mitochondrial inner membrane harbors two ATP-dependent metallopeptidases, them- and thei-AAA protease, which expose their catalytic sites to opposite membrane surfaces and cooperate in the degradation of inner membrane proteins. In addition to its proteolytic activity, them-AAA protease has chaperone-like activity during the assembly of respiratory and ATP-synthase complexes. It constitutes a quality control system in the inner membrane for membrane-embedded protein complexes.