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.     

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.     

Sequence and structural analysis of COVID-19 E and M proteins with MERS virus E and M proteins—A comparative study

The outbreak of SARS in 2003, MERS in 2012, and now COVID-19 in 2019 has demonstrated that Coronaviruses are capable of causing primary lethal infections in humans, and the pandemic is now a global concern. The COVID-19 belongs to the beta coronavirus family encoding 29 proteins, of which four are structural, the Spike, Membrane, Envelope, and Nucleocapsid proteins. Here we have analyzed and compared the Membrane (M) and Envelope (E) proteins of COVID-19 and MERS with SARS and Bat viruses. The sequence analysis of conserved regions of both E and M proteins revealed that many regions of COVID-19 are similar to Bat and SARS viruses while the MERS virus showed variations. The essential binding motifs found in SARS appeared in COVID-19. Besides, the M protein of COVID-19 showed a distinct serine phosphorylation site in the C-terminal domain, which looked like a catalytic triad seen in serine proteases. A Dileucine motif occurred many times in the sequence of the M protein of all the four viruses compared. Concerning the structural part, the COVID-19 E protein showed more similarity to Bat while MERS shared similarity with the SARS virus. The M protein of both COVID-19 and MERS displayed variations in the structure. The interaction between M and E proteins was also studied to know the additional binding regions. Our study highlights the critical motifs and structural regions to be considered for further research to design better inhibitors for the infection caused by these viruses.     

Oligomerization and toxicity of Aβ fusion proteins

This study has found that the Maltose binding protein Aβ42 fusion protein (MBP-Aβ42) forms soluble oligomers while the shorter MBP-Aβ16 fusion and control MBP did not. MBP-Aβ42, but neither MBP-Aβ16 nor control MBP, was toxic in a dose-dependent manner in both yeast and primary cortical neuronal cells. This study demonstrates the potential utility of MBP-Aβ42 as a reagent for drug screening assays in yeast and neuronal cell cultures and as a candidate for further Aβ42 characterization.     

Anchoring proteins and cardiac sudden death: how and why?

Mutations in proteins responsible for ion transport in cardiac tissue can induce a destabilization of electrical function and provoke cardiac sudden death. Identification of a genetic anomaly in a French family that developed the syndrome of cardiac sudden death has revealed a crucial new element in normal cardiac electrical function : Ion channels need to be anchored to specific domains at the plasma membrane by an anchoring protein called ankyrin-B.     

Partial sequence homologies between cytoskeletal proteins,c-myc,Rous sarcoma virus and adenovirus proteins,transducin, and β- and γ-crystallins

Computer based sequence comparisons indicate partial sequence homology between human c-myc, Rous sarcoma virus, adenovirus 7, and simian sarcoma virus proteins and the cytoskeletal proteins desmin, keratin and vimentin. In addition, sections of the oncogene proteins showed partial but significant homology to and subunits of transducin, -II and -BP crystallins showed partial but significant homology to the cytoskeletal proteins keratin, vimentin, desmin, and -tubulin, and to adenovirus 7 and simian sarcoma virus transforming gene proteins. -BP crystallin showed partial but significant homology to Rous sarcoma virus protein, and to and y subunits of transducin. Both crystallins showed partial sequence homology to the GTP-binding protein elongation factor TU fromEscherichia coli . These sequence homologies suggest a link between the mechanisms of normal lens cell differentiation, involving modifications to the cytoskeleton and subsequent changes to the pattern of protein synthesis, and mechanisms of neoplastic transformation. Furthermore the transducin-like region on -crystallin may be important for its interaction with lens membranes and the maintenance of short-range order for lens transparency.     

Comparative analysis of 13C chemical shifts of β-sheet amyloid proteins and outer membrane proteins

Cross-β amyloid fibrils and membrane-bound β-barrels are two important classes of β-sheet proteins. To investigate whether there are systematic differences in the backbone and sidechain conformations of these two families of proteins, here we analyze the ¹³C chemical shifts of 17 amyloid proteins and 7 β-barrel membrane proteins whose high-resolution structures have been determined by NMR. These 24 proteins contain 373 β-sheet residues in amyloid fibrils and 521 β-sheet residues in β-barrel membrane proteins. The ¹³C chemical shifts are shown in 2D ¹³C–¹³C correlation maps, and the amino acid residues are categorized by two criteria: (1) whether they occur in β-strand segments or in loops and turns; (2) whether they are water-exposed or dry, facing other residues or lipids. We also examine the abundance of each amino acid in amyloid proteins and β-barrels and compare the sidechain rotameric populations. The ¹³C chemical shifts indicate that hydrophobic methyl-rich residues and aromatic residues exhibit larger static sidechain conformational disorder in amyloid fibrils than in β-barrels. In comparison, hydroxyl- and amide-containing polar residues have more ordered sidechains and more ordered backbones in amyloid fibrils than in β-barrels. These trends can be explained by steric zipper interactions between β-sheet planes in cross-β fibrils, and by the interactions of β-barrel residues with lipid and water in the membrane. These conformational trends should be useful for structural analysis of amyloid fibrils and β-barrels based principally on NMR chemical shifts.

     

Identification and analysis of extended strands and β-sheets in globular proteins

A method to identify β-sheets in globular proteins from extended strands, using only α-carbon positions, has been developed. The strands that form β-sheets are picked up by means of simple distance criteria. The method has been tested by applying it to three proteins with accurately known secondary structures. It has also been applied to ten other proteins wherein only α-carbon coordinates are available, and the list of β-sheets obtained. The following points are worth noting: (i) The sheets identified by the algorithm are found to agree satisfactorily with the reported ones based on backbone hydrogen bonding, wherever this information is available. (ii) β-Strands that do not form parts of any sheet are a common feature of protein structures. (iii) Such isolated β-strands tend to be short. (iv) The conformation corresponding to the preferred right-handed twist of the sheet is overwhelmingly observed in both the sheet-forming and isolated β-strands.     

Do X and Y spermatozoa differ in proteins?

This article reviews the current knowledge about X- and Y-chromosomal gene expression during spermatogenesis and possible differences between X- and Y-chromosome-bearing spermatozoa (X and Y sperm) in relation to whether an immunological method of separation of X and Y spermatozoa might some day be feasible. Recent studies demonstrated that X- and Y-chromosome-bearing spermatids do express X- and Y-chromosomal genes that might theoretically result in protein differences between X and Y sperm. Most, if not all, of these gene products, however, are expected to be shared among X and Y spermatids via intercellular bridges. Studies on aberrant mouse strains indicate that complete sharing might not occur for all gene products. This keeps open the possibility that X and Y sperm may differ in proteins, but until now, this has not been confirmed by comparative studies between flow-cytometrically sorted X and Y sperm for H-Y antigen or other membrane proteins.     

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     

The angiotensin Ⅱ type 1 receptor and receptor-associated proteins

INTRODUCTIONThe renin-angiotensin system (RAS) is consid-ered to be the major regulator of blood pressure)electrolyte balance and renal, neuronal as well as en-docrine functions related to cardiovascular control.The RAS is the key factor in most cases essential hy-pertension, as indicated by successes in treatment ofhypertensive patients with various angiotensin I con-verting enzyme (ACE) inhibitors and receptor block-ers. Renin was a central subject of intense investigation because of…     

Hydrophilicity and antigenicity of proteins — A case study of myoglobin and hemoglobin

Hydrophilicity index is used to locate antigenic determinants on two related groups of proteins-myoglobin and hemoglobin. The data on 41 species (including 34 mammals) of myoglobin show that average hydrophilicity for the complete myoglobin molecules as well as the average hydrophilicity for all hydrophilic regions put together seem to remain constant; the variation in the size and location of the antigenic determinants in these species is very small indicating that the antigenic sites are not shifted during evolution. In the case of both the proteins there is a good agreement between the antigenic sites picked up by using hydrophilicity index and the experimentally determined antigenic sites. The data on 56 species of hemoglobin α-chains and 44 species of hemoglobinβ-chains showed that although there are few sites on hemoglobin which have remained invariant during evolution, there is a significant variation in other sites in terms of either a splitting of a site, or a drastic change in the hydrophilicity values and/or a length of the site. Comparison of the hydrophilicity data on these two groups of proteins suggests that hemoglobins which perform a variety of functions as compared to myoglobins are evolving faster than myoglobins supporting the contention of earlier workers.     

High-light-inducible proteins HliA and HliB: pigment binding and protein–protein interactions

High-light-inducible proteins (Hlips) are single-helix transmembrane proteins that are essential for the survival of cyanobacteria under stress conditions. The model cyanobacterium Synechocystis sp. PCC 6803 contains four Hlip isoforms (HliA-D) that associate with Photosystem II (PSII) during its assembly. HliC and HliD are known to form pigmented (hetero)dimers that associate with the newly synthesized PSII reaction center protein D1 in a configuration that allows thermal dissipation of excitation energy. Thus, it is expected that they photoprotect the early steps of PSII biogenesis. HliA and HliB, on the other hand, bind the PSII inner antenna protein CP47, but the mode of interaction and pigment binding have not been resolved. Here, we isolated His-tagged HliA and HliB from Synechocystis and show that these two very similar Hlips do not interact with each other as anticipated, rather they form HliAC and HliBC heterodimers. Both dimers bind Chl and β-carotene in a quenching conformation and associate with the CP47 assembly module as well as later PSII assembly intermediates containing CP47. In the absence of HliC, the cellular levels of HliA and HliB were reduced, and both bound atypically to HliD. We postulate a model in which HliAC-, HliBC-, and HliDC-dimers are the functional Hlip units in Synechocystis. The smallest Hlip, HliC, acts as a ‘generalist’ that prevents unspecific dimerization of PSII assembly intermediates, while the N-termini of ‘specialists’ (HliA, B or D) dictate interactions with proteins other than Hlips.

     

Actin-associated proteins and cardiomyopathy—the ‘unknown’ beyond troponin and tropomyosin

It has been known for several decades that mutations in genes that encode for proteins involved in the control of actomyosin interactions such as the troponin complex, tropomyosin and MYBP-C and thus regulate contraction can lead to hereditary hypertrophic cardiomyopathy. In recent years, it has become apparent that actin-binding proteins not directly involved in the regulation of contraction also can exhibit changed expression levels, show altered subcellular localisation or bear mutations that might lead to hereditary cardiomyopathies. The aim of this review is to look beyond the troponin/tropomyosin mechanism and to give an overview of the different types of actin-associated proteins and their potential roles in cardiomyocytes. It will then discuss recent findings relevant to their involvement in heart disease.     

Photochemical reactivity of the homologous proteinsα-lactalbumin and lysozyme

Summary The fluorescent behaviour and the photodynamic effect was studied in native and structurally modified lysozyme and-lactalbumin.The Tyr residues in lysozyme and-lactalbumin show different sensitivities to the photodynamic effect. The effect is zero in the case of Tyr from native lysozyme. In contrast, the Tyr residues in-lactalbumin are susceptible to photooxidation, which indicates a greater degree of exposure to the solvent. The three His residues of-lactalbumin have different degrees of exposure and show two different kinetics of photooxidation whereas the His residue of lysozyme is photooxidized with a single kinetic.Two photooxidation kinetics were obtained for the Trp residues of both native proteins, an indication that in both cases there are Trp residues that are differently exposed to the solvent. The wavelengths of maximum fluorescent emission of the Trp residues were different for the two proteins, an effect which can also be explained in terms of a difference in the environment of these residues. The modified form of these proteins emit at wavelengths longer than those of the native forms. When modified the proteins photooxidize with noticeably greater quantum yields.     

Mannose-binding lectin serine proteases and associated proteins of the lectin pathway of complement: Two genes, five proteins and many functions?

The lectin pathway of the complement system is activated following the binding of carbohydrate-based ligands by recognition molecules such as mannose-binding lectin (MBL) or ficolins. Engagement of the recognition molecules causes activation of associated MBL-associated serine proteases or MASPs, which in turn activate downstream complement molecules to activate the system. Two MASP genes are alternatively spliced during expression to yield 5 proteins, including three proteases (MASP-1, -2 and -3) and two truncated proteins, MAp19 and MAp44. Here we discuss what is currently known about these proteins in terms of their structure and function. MASP-2 is autoactivated following the initial binding events of the pathway and is able to subsequently activate the C4 and C2 substrates required to activate the rest of the pathway. MASP-1 is able to augment MASP-2 activation, but also appears to play other roles, although the physiological significance of these is not yet clear. The roles of the truncated Map19 and Map44 proteins and the MASP-3 protease are currently unknown. The proteases form an interesting sub-family of proteins that clearly should be the focus of future research in order to establish their biological roles.This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.