Rosetta Stone proteins: "chance and necessity"?

A response to Functional associations of proteins in entire genomes by means of exhaustive detection of gene fusions by AJ Enright, CA Ouzounis. Genome Biology 2000, 2:research0034.1-0034.7     

Rosetta Stone proteins: "chance and necessity"?

A response to Functional associations of proteins in entire genomes by means of exhaustive detection of gene fusions by AJ Enright, CA Ouzounis. Genome Biology 2000, 2:research0034.1-0034.7     

Why are "natively unfolded" proteins unstructured under physiologic conditions?

"Natively unfolded" proteins occupy a unique niche within the protein kingdom in that they lack ordered structure under conditions of neutral pH in vitro. Analysis of amino acid sequences, based on the normalized net charge and mean hydrophobicity, has been applied to two sets of proteins: small globular folded proteins and "natively unfolded" ones. The results show that "natively unfolded" proteins are specifically localized within a unique region of charge-hydrophobicity phase space and indicate that a combination of low overall hydrophobicity and large net charge represent a unique structural feature of "natively unfolded" proteins.     

Do telomeres ask checkpoint proteins: "gimme shelter-in"?

Telomeres are complicated structures designed to allow one thing and avoid another. They allow replication of chromosome ends, an issue mostly about telomerase, which we seem to understand (though details of its regulation are works in progress). Telomeres must also avoid being detected as DNA breaks. This is important for two reasons: DNA breaks activate checkpoints that cause arrest of cell division, and DNA breaks engage repair machinery. Clearly, normal telomeres neither activate cell cycle arrest nor allow themselves to be repaired; arrest blocks cell division, and repair fuses chromosomes.     

A novel fractionation method of the rough ER integral membrane proteins; Resident proteins versus exported proteins?

We treated the high salt‐washed canine pancreatic rough ER (KRM) with 0.18% Triton X‐100, separated the extract from the residual membrane (0.18%Tx KRM), and processed the extract with SM‐2 beads to recover membrane proteins in proteoliposomes. To focus on integral membrane proteins, KRM, 0.18%Tx KRM and proteoliposomes were subjected to sodium carbonate treatment, and analyzed by 2‐D gel electrophoresis. Consequently we found that a distinct group of integral membrane protein of KRM preferentially extracted from the membrane and recovered in proteoliposomes did exist, while majority of KRM integral membrane proteins were fractionated in 0.18%Tx KRM, which retained the basic structure and functions of KRM. Protein identification showed that the former group was enriched with proteins exported from the ER and the latter group comprised mostly of ER resident proteins. This result will potentially affect the prevailing view of the ER membrane structure as well as protein sorting from the ER.     

The enigma of the CLIC proteins: Ion channels, redox proteins, enzymes, scaffolding proteins?

Chloride intracellular channel proteins (CLICs) are distinct from most ion channels in that they have both soluble and integral membrane forms. CLICs are highly conserved in chordates, with six vertebrate paralogues. CLIC-like proteins are found in other metazoans. CLICs form channels in artificial bilayers in a process favoured by oxidising conditions and low pH. They are structurally plastic, with CLIC1 adopting two distinct soluble conformations. Phylogenetic and structural data indicate that CLICs are likely to have enzymatic function. The physiological role of CLICs appears to be maintenance of intracellular membranes, which is associated with tubulogenesis but may involve other substructures.     

Fluorescent proteins in microbial biotechnology—new proteins and new applications

The recent advances over the past 5 years in the utilisation of fluorescent proteins in microbial biotechnology applications, including recombinant protein production, food processing, and environmental biotechnology, are reviewed. We highlight possible areas where fluorescent proteins currently used in other bioscience disciplines could be adapted for use in biotechnological applications and also outline novel uses for recently developed fluorescent proteins.     

Latent TGF-β-binding proteins

The LTBPs (or latent transforming growth factor β binding proteins) are important components of the extracellular matrix (ECM) that interact with fibrillin microfibrils and have a number of different roles in microfibril biology. There are four LTBPs isoforms in the human genome (LTBP-1, − 2, − 3, and − 4), all of which appear to associate with fibrillin and the biology of each isoform is reviewed here.The LTBPs were first identified as forming latent complexes with TGFβ by covalently binding the TGFβ propeptide (LAP) via disulfide bonds in the endoplasmic reticulum. LAP in turn is cleaved from the mature TGFβ precursor in the trans-golgi network but LAP and TGFβ remain strongly bound through non-covalent interactions. LAP, TGFβ, and LTBP together form the large latent complex (LLC). LTBPs were originally thought to primarily play a role in maintaining TGFβ latency and targeting the latent growth factor to the extracellular matrix (ECM), but it has also been shown that LTBP-1 participates in TGFβ activation by integrins and may also regulate activation by proteases and other factors. LTBP-3 appears to have a role in skeletal formation including tooth development. As well as having important functions in TGFβ regulation, TGFβ-independent activities have recently been identified for LTBP-2 and LTBP-4 in stabilizing microfibril bundles and regulating elastic fiber assembly.     

Hydrophilic framework in proteins?

The spatial neighborhood composition of residues was determined in a 511-structure set by taking only side-chain atoms into account to generate a hydrophobicity scale. This scale is symmetrical and has been divided into seven functional groups. Hydrophobic (LIVFMCAWYG) and hydrophilic (PTHSQRNKED) residues obey an equipartition rule: not only are they found in equal proportions, but they play equivalent roles in many of their properties. The nearest neighbors of all residues are always hydrophilic. However, hydrophobic residues are mostly surrounded by other hydrophobic residues located at a peak at 3.9 Å, while hydrophilic residues show three peaks at 5.0, 6.5, and 8.0 Å, suggesting a hydrophilic structural framework. This leads us to question the importance of hydrophobic cores believed to be at the origin of protein folding.     

Why are proteins O-glycosylated?

The O-linked oligosaccharides of glycoproteins are usually clustered within heavily glycosylated regions of the peptide chain. Steric interactions between carbohydrate and peptide within these clusters induce the peptide core to adopt a stiff and extended conformation and this conformational effect appears to represent a major function of O-glycosylation.     

How do proteins avoid becoming too stable? Biophysical studies into metastable proteins

The vast majority of theoretical and experimental folding studies have shown that as a protein folds, it attempts to adopt a conformation that occurs at its lowest free energy minimum. However, studies on a small number of proteins have now shown that this is a generality. In this review we discuss recent data on how two proteins, -lytic protease and ₁-antitrypsin, successfully fold to their metastable native states, whilst avoiding more stable but inactive conformations.     

Small heat shock proteins and α-crystallins: dynamic proteins with flexible functions

The small heat shock proteins (sHSPs) and the related α-crystallins (αCs) are virtually ubiquitous proteins that are strongly induced by a variety of stresses, but that also function constitutively in multiple cell types in many organisms. Extensive research has demonstrated that a majority of sHSPs and αCs can act as ATP-independent molecular chaperones by binding denaturing proteins and thereby protecting cells from damage due to irreversible protein aggregation. As a result of their diverse evolutionary history, their connection to inherited human diseases, and their novel protein dynamics, sHSPs and αCs are of significant interest to many areas of biology and biochemistry. However, it is increasingly clear that no single model is sufficient to describe the structure, function or mechanism of action of sHSPs and αCs. In this review, we discuss recent data that provide insight into the variety of structures of these proteins, their dynamic behavior, how they recognize substrates, and their many possible cellular roles.     

How do proteins fold?

This article emphasizes the importance of getting students to understand the ways in which polypeptides fold to form protein molecules with complex higher-ordered structures. Modern views on how this folding occurs in vitro and in the cell are summarized and set within an appropriate biological context.     

Are coiled-coil proteins evolutionarily related?

A modification to the standard Needleman-Wunsch sequence comparison scheme is presented. In cases where high levels of sequence similarity may arise from a common structural motif, this method discriminates between common ancestry and similarity due to structural constraints alone. Use of this algorithm is illustrated with the coiled-coil motif in the cases of idealized coiled-coil sequences, intermediate filaments and reovirus hemagluttinin.     

How do uncoupling proteins uncouple?

According to the proton buffering model, introduced by Klingenberg, UCP1 conducts protons through a hydrophilic pathway lined with fatty acid head groups that buffer the protons as they move across the membrane. According to the fatty acid protonophore model, introduced by Garlid, UCPs do not conduct protons at all. Rather, like all members of this gene family, they are anion carriers. A variety of anions are transported, but the physiological substrates are fatty acid (FA) anions. Because the carboxylate head group is translocated by UCP, and because the protonated FA rapidly diffuses across the membrane, this mechanism permits FA to behave as regulated cycling protonophores. Favoring the latter mechanism is the fact that the head group of long-chain alkylsulfonates, strong acid analogues of FA, is also translocated by UCP.     

How are tonoplast proteins degraded?

Protein turnover is fundamental both for development and cellular homeostasis. The mechanisms responsible for the turnover of integral membrane proteins in plant cells are however still largely unknown. Recently, considerable attention has been devoted to the degradation of plasma membrane proteins. We have now studied the turnover of a tonoplast protein, the potassium channel TPK1, in fully differentiated Arabidopsis leaf cells and showed that its degradation occurs upon internalization into the vacuole. Here, we discuss the possible mechanisms and triggering events involved.     

Rapid prototyping of proteins: Mail order gene fragments to assayable proteins within 24 hours

In this study, we present a minimal template design and accompanying methods to produce assayable quantities of custom sequence proteins within 24 hr from receipt of inexpensive gene fragments from a DNA synthesis vendor. This is done without the conventional steps of plasmid cloning or cell-based amplification and expression. Instead the linear template is PCR amplified, circularized, and isothermally amplified using a rolling circle polymerase. The resulting template can be used directly with cost-optimized, scalably-manufactured Escherichia coli extract and minimal supplement reagents to perform cell-free protein synthesis (CFPS) of the template protein. We demonstrate the utility of this template design and 24 hr process with seven fluorescent proteins (sfGFP, mVenus, mCherry, and four GFP variants), three enzymes (chloramphenicol acetyltransferase, a chitinase catalytic domain, and native subtilisin), a capture protein (anti-GFP nanobody), and 2 antimicrobial peptides (BP100 and CA(1–7)M(2–9)). We detected each of these directly from the CFPS reaction using colorimetric, fluorogenic, and growth assays. Of especial note, the GFP variant sequences were found from genomic screening data and had not been expressed or characterized before, thus demonstrating the utility of this approach for phenotype characterization of sequenced libraries. We also demonstrate that the rolling circle amplified version of the linear template exhibits expression similar to that of a complete plasmid when expressing sfGFP in the CFPS reaction. We evaluate the cost of this approach to be $61/mg sfGFP for a 4 hr reaction. We also detail limitations of this approach and strategies to overcome these, namely proteins with posttranslational modifications.     

A search method for homologs of small proteins. Ubiquitin-like proteins in prokaryotic cells?

The question of protein homology versus analogy arises when proteins share a common function or a common structural fold without any statistically significant amino acid sequence similarity. Even though two or more proteins do not have similar sequences but share a common fold and the same or closely related function, they are assumed to be homologs, descendant from a common ancestor. The problem of homolog identification is compounded in the case of proteins of 100 or less amino acids. This is due to a limited number of basic single domain folds and to a likelihood of identifying by chance sequence similarity. The latter arises from two conditions: first, any search of the currently very large protein database is likely to identify short regions of chance match; secondly, a direct sequence comparison among a small set of short proteins sharing a similar fold can detect many similar patterns of hydrophobicity even if proteins do not descend from a common ancestor. In an effort to identify distant homologs of the many ubiquitin proteins, we have developed a combined structure and sequence similarity approach that attempts to overcome the above limitations of homolog identification. This approach results in the identification of 90 probable ubiquitin-related proteins, including examples from the two prokaryotic domains of life, Archaea and Bacteria.     

Structure determination of α-helical membrane proteins by solution-state NMR: Emphasis on retinal proteins

The biochemical processes of living cells involve a numerous series of reactions that work with exceptional specificity and efficiency. The tight control of this intricate reaction network stems from the architecture of the proteins that drive the chemical reactions and mediate protein–protein interactions. Indeed, the structure of these proteins will determine both their function and interaction partners. A detailed understanding of the proximity and orientation of pivotal functional groups can reveal the molecular mechanistic basis for the activity of a protein. Together with X-ray crystallography and electron microscopy, NMR spectroscopy plays an important role in solving three-dimensional structures of proteins at atomic resolution. In the challenging field of membrane proteins, retinal-binding proteins are often employed as model systems and prototypes to develop biophysical techniques for the study of structural and functional mechanistic aspects. The recent determination of two 3D structures of seven-helical trans-membrane retinal proteins by solution-state NMR spectroscopy highlights the potential of solution NMR techniques in contributing to our understanding of membrane proteins. This review summarizes the multiple strategies available for expression of isotopically labeled membrane proteins. Different environments for mimicking lipid bilayers will be presented, along with the most important NMR methods and labeling schemes used to generate high-quality NMR spectra. The article concludes with an overview of types of conformational restraints used for generation of high-resolution structures of membrane proteins. This article is part of a Special Issue entitled: Retinal Proteins — You can teach an old dog new tricks.     

Classification of β-hairpin repeat proteins

In recent years, a number of new protein structures that possess tandem repeats have emerged. Many of these proteins are comprised of tandem arrays of β-hairpins. Today, the amount and variety of the data on these β-hairpin repeat (BHR) structures have reached a level that requires detailed analysis and further classification. In this paper, we classified the BHR proteins, compared structures, sequences of repeat motifs, functions and distribution across the major taxonomic kingdoms of life and within organisms. As a result, we identified six different BHR folds in tandem repeat proteins of Class III (elongated structures) and one BHR fold (up-and-down β-barrel) in Class IV (“closed” structures). Our survey reveals the high incidence of the BHR proteins among bacteria and viruses and their possible relationship to the structures of amyloid fibrils. It indicates that BHR folds will be an attractive target for future structural studies, especially in the context of age-related amyloidosis and emerging infectious diseases. This work allowed us to update the RepeatsDB database, which contains annotated tandem repeat protein structures and to construct sequence profiles based on BHR structural alignments.