Biomolecules

873 KiB

Open AccessReview

Protein Stability, Folding and Misfolding in Human PGK1 Deficiency

by Giovanna Valentini, Maristella Maggi and Angel L. Pey

Biomolecules 2013, 3(4), 1030-1052; https://doi.org/10.3390/biom3041030 - 18 Dec 2013

Cited by 16 | Viewed by 9320

Conformational diseases are often caused by mutations, altering protein folding and stability in vivo. We review here our recent work on the effects of mutations on the human phosphoglycerate kinase 1 (hPGK1), with a particular focus on thermodynamics and kinetics of protein [...] Read more.

Conformational diseases are often caused by mutations, altering protein folding and stability in vivo. We review here our recent work on the effects of mutations on the human phosphoglycerate kinase 1 (hPGK1), with a particular focus on thermodynamics and kinetics of protein folding and misfolding. Expression analyses and in vitro biophysical studies indicate that disease-causing mutations enhance protein aggregation propensity. We found a strong correlation among protein aggregation propensity, thermodynamic stability, cooperativity and dynamics. Comparison of folding and unfolding properties with previous reports in PGKs from other species suggests that hPGK1 is very sensitive to mutations leading to enhance protein aggregation through changes in protein folding cooperativity and the structure of the relevant denaturation transition state for aggregation. Overall, we provide a mechanistic framework for protein misfolding of hPGK1, which is insightful to develop new therapeutic strategies aimed to target native state stability and foldability in hPGK1 deficient patients. Full article

(This article belongs to the Special Issue Protein Folding and Misfolding)

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Superimposition of the Cα traces of three PGK structures: Escherichia coli (blue, PDB ID 1ZMR), Saccharomyces cerevisiae (green, PDB ID 1FW8) and Homo sapiens (red, PDB ID 2XE7). The conformation of each individual domain, except for a few source-specific insertions, is similar in all structures. Both inter-domain helix and L hinge are almost completely superposed in all the three PGKs, pointing to a conservative evolution of the structural elements and underlining their essential role in the protein function. The bound ligands are shown as thin stick models. Full article ">Figure 2
Ribbons representation of human PGK1 in open conformation (PDB ID, 2XE7). The bound ligands are shown as stick model. The side chains of amino acids affected by mutations are indicated by black spheres and represented as ball and stick. Full article ">Figure 3
Aggregation of hPGK1 enzymes in vitro and upon expression in E. coli cultures (A) Fraction of soluble hPGK1 existing as a monomer upon expression analyses of hPGK1 enzymes in E. coli. The data are means ± s.d. from three independent expression experiments and were obtained starting from the same amount of total soluble protein and upon purification by ion-exchange and size exclusion chromatography as described [<a href="#B13-biomolecules-03-01030" class="html-bibr">13</a>,<a href="#B45-biomolecules-03-01030" class="html-bibr">45</a>] (B) Normalized thermal denaturation profiles for WT hPGK1 monitored by activity, light scattering (LS) and differential scanning calorimetry (DSC) measurements; Note that the three techniques provide similar denaturation profiles. (C) Arrhenius plots for the thermal denaturation of WT hPGK1 obtained from DSC experiments using a two-state irreversible model [<a href="#B13-biomolecules-03-01030" class="html-bibr">13</a>] and used to determine denaturation rates at 37 °C (indicated by the vertical dotted line). Full article ">Figure 4
Plausible mechanisms linking the reduced kinetic stability (towards aggregation or proteolysis) and the lower unfolding cooperativity in disease-causing hPGK1 mutants. In the case of kinetically stable variants (panel A), the levels of the highly kinetically sensitive intermediate (I) levels are always very low, and aggregation and proteolysis occur mainly through the unfolded state U (left side of the figure). However, in some mutants (panel B), the population of the intermediate may raise (and can be “detected” by a low m-value in urea denaturations), leading to a significant contribution from the I state to the irreversible denaturation kinetics. It must be also noted that in panel B, at low denaturing stress, the I state is much more populated than the U state, and thus, may contribute much more to the kinetics of proteolysis and/or aggregation than the U state. Full article ">Figure 5
A simple scenario for protein folding and misfolding hPGK1 enzymes inside the cell. Folding of hPGK1 may occur in vivo spontaneously after its synthesis, as seen by its spontaneous folding in vitro [<a href="#B13-biomolecules-03-01030" class="html-bibr">13</a>] and the fast and spontaneous folding of yeast PGK in living cells ([<a href="#B26-biomolecules-03-01030" class="html-bibr">26</a>,<a href="#B99-biomolecules-03-01030" class="html-bibr">99</a>]). Alternatively, the native state can be reached via interaction of partially folded states with different classes of molecular chaperones. The red arrows indicate steps that may be affected in vivo upon disease-causing mutations, mostly associated with reduced native stability and folding cooperativity [<a href="#B13-biomolecules-03-01030" class="html-bibr">13</a>,<a href="#B46-biomolecules-03-01030" class="html-bibr">46</a>]. The potential targets for pharmacological correction by proteostasis modulators (PM) and pharmacological chaperones (PC) are also indicated. Full article ">

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Open AccessReview

Toxin Instability and Its Role in Toxin Translocation from the Endoplasmic Reticulum to the Cytosol

by Ken Teter

Biomolecules 2013, 3(4), 997-1029; https://doi.org/10.3390/biom3040997 - 10 Dec 2013

Cited by 18 | Viewed by 7504

Abstract

AB toxins enter a host cell by receptor-mediated endocytosis. The catalytic A chain then crosses the endosome or endoplasmic reticulum (ER) membrane to reach its cytosolic target. Dissociation of the A chain from the cell-binding B chain occurs before or during translocation to [...] Read more.

AB toxins enter a host cell by receptor-mediated endocytosis. The catalytic A chain then crosses the endosome or endoplasmic reticulum (ER) membrane to reach its cytosolic target. Dissociation of the A chain from the cell-binding B chain occurs before or during translocation to the cytosol, and only the A chain enters the cytosol. In some cases, AB subunit dissociation is facilitated by the unique physiology and function of the ER. The A chains of these ER-translocating toxins are stable within the architecture of the AB holotoxin, but toxin disassembly results in spontaneous or assisted unfolding of the isolated A chain. This unfolding event places the A chain in a translocation-competent conformation that promotes its export to the cytosol through the quality control mechanism of ER-associated degradation. A lack of lysine residues for ubiquitin conjugation protects the exported A chain from degradation by the ubiquitin-proteasome system, and an interaction with host factors allows the cytosolic toxin to regain a folded, active state. The intrinsic instability of the toxin A chain thus influences multiple steps of the intoxication process. This review will focus on the host–toxin interactions involved with A chain unfolding in the ER and A chain refolding in the cytosol. Full article

(This article belongs to the Special Issue Protein Folding and Misfolding)

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1336 KiB

Open AccessArticle

Control of Collagen Stability and Heterotrimer Specificity through Repulsive Electrostatic Interactions

by Avanish S. Parmar, Mihir Joshi, Patrick L. Nosker, Nida F. Hasan and Vikas Nanda

Biomolecules 2013, 3(4), 986-996; https://doi.org/10.3390/biom3040986 - 6 Dec 2013

Cited by 8 | Viewed by 6765

Abstract

Charge-pair interactions between acidic and basic residues on the surface of collagen can promote stability as well as control specificity of molecular recognition. Heterotrimeric collagen peptides have been engineered de novo using either rational or computational methods, which in both cases optimize networks [...] Read more.

Charge-pair interactions between acidic and basic residues on the surface of collagen can promote stability as well as control specificity of molecular recognition. Heterotrimeric collagen peptides have been engineered de novo using either rational or computational methods, which in both cases optimize networks of favorable charge-pair interactions in the target structure. Less understood is the role of electrostatic repulsion between groups of like charge in destabilizing structure or directing molecular recognition. To study this, we apply a “charge crowding” approach, where repulsive interactions between multiple aspartate side chains are found to destabilize the homotrimer states in triple helical peptide system and can be utilized to promote the formation of heterotrimers. Neutralizing surface charge by increasing salt concentration or decreasing pH can enhance homotrimer stability, confirming the role of charge crowding on the destabilization of homotrimers via electrostatic repulsion. Charge crowding may be used in conjunction with other approaches to create specific collagen heterotrimers. Full article

(This article belongs to the Special Issue Protein Folding and Misfolding)

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Circular Dichroism (CD) spectra at 4 °C of all 10 mixtures of peptides A, B, C. Full article ">Figure 2
Thermal denaturation (top row) and their respective first derivative plots (bottom row) for peptide mixtures (A and D) 3A, 3B, 3C (B and E) A:B:C, 2A:C, A:2C (C and F) 2A:B, A:2B, 2B:C, B:2C. Full article ">Figure 3
NMR measurement of 1H-13C HSQC spectra. (A) Merged spectra of A and B alone (black) versus A:B mixture (red); (B) Merged spectra of A and C alone (black) versus A:C mixture (red); (C) Merged spectra of B and C alone (black) versus B:C mixture (red); (D) Merged spectra of A, B, C, A:C (all black) versus A:B:C mixture (red). Full article ">Figure 4
Thermal denaturation (top row) and their respective first derivative plots (bottom row) for A, B and C at various salt concentrations. Full article ">Figure 5
Thermal denaturation (top row) and their respective first derivative plots (bottom row) for A, B and C pH7 and pH2.5. Full article ">Figure 6
Sequence of fibrous natural collagens. Glycine is marked in yellow, clusters of like charges in red, clusters of favorable interactions in blue. Full article ">

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Open AccessArticle

Unfolding Thermodynamics of Cysteine-Rich Proteins and Molecular Thermal-Adaptation of Marine Ciliates

by Giorgia Cazzolli, Tatjana Škrbić, Graziano Guella and Pietro Faccioli

Biomolecules 2013, 3(4), 967-985; https://doi.org/10.3390/biom3040967 - 18 Nov 2013

Cited by 5 | Viewed by 6377

Abstract

Euplotes nobilii and Euplotes raikovi are phylogenetically closely allied species of marine ciliates, living in polar and temperate waters, respectively. Their evolutional relation and the sharply different temperatures of their natural environments make them ideal organisms to investigate thermal-adaptation. We perform a comparative [...] Read more.

Euplotes nobilii and Euplotes raikovi are phylogenetically closely allied species of marine ciliates, living in polar and temperate waters, respectively. Their evolutional relation and the sharply different temperatures of their natural environments make them ideal organisms to investigate thermal-adaptation. We perform a comparative study of the thermal unfolding of disulfide-rich protein pheromones produced by these ciliates. Recent circular dichroism (CD) measurements have shown that the two psychrophilic (E. nobilii) and mesophilic (E. raikovi) protein families are characterized by very different melting temperatures, despite their close structural homology. The enhanced thermal stability of the E. raikovi pheromones is realized notwithstanding the fact that these proteins form, as a rule, a smaller number of disulfide bonds. We perform Monte Carlo (MC) simulations in a structure-based coarse-grained (CG) model to show that the higher stability of the E. raikovi pheromones is due to the lower locality of the disulfide bonds, which yields a lower entropy increase in the unfolding process. Our study suggests that the higher stability of the mesophilic E. raikovi phermones is not mainly due to the presence of a strongly hydrophobic core, as it was proposed in the literature. In addition, we argue that the molecular adaptation of these ciliates may have occurred from cold to warm, and not from warm to cold. To provide a testable prediction, we identify a point-mutation of an E. nobilii pheromone that should lead to an unfolding temperature typical of that of E. raikovi pheromones. Full article

(This article belongs to the Special Issue Protein Folding and Misfolding)

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1075 KiB

Open AccessReview

Control of Cell Differentiation by Mitochondria, Typically Evidenced in Dictyostelium Development

by Yasuo Maeda and Junji Chida

Biomolecules 2013, 3(4), 943-966; https://doi.org/10.3390/biom3040943 - 11 Nov 2013

Cited by 19 | Viewed by 11036

Abstract

In eukaryotic cells, mitochondria are self-reproducing organelles with their own DNA and they play a central role in adenosine triphosphate (ATP) synthesis by respiration. Increasing evidence indicates that mitochondria also have critical and multiple functions in the initiation of cell differentiation, cell-type determination, [...] Read more.

In eukaryotic cells, mitochondria are self-reproducing organelles with their own DNA and they play a central role in adenosine triphosphate (ATP) synthesis by respiration. Increasing evidence indicates that mitochondria also have critical and multiple functions in the initiation of cell differentiation, cell-type determination, cell movement, and pattern formation. This has been most strikingly realized in development of the cellular slime mold Dictyostelium. For example, the expression of the mitochondrial ribosomal protein S4 (mt-rps4) gene is required for the initial differentiation. The Dictyostelium homologue (Dd-TRAP1) of TRAP-1 (tumor necrosis receptor-associated protein 1), a mitochondrial molecular chaperone belonging to the Hsp90 family, allows the prompt transition of cells from growth to differentiation through a novel prestarvation factor (PSF-3) in growth medium. Moreover, a cell-type-specific organelle named a prespore-specific vacuole (PSV) is constructed by mitochondrial transformation with the help of the Golgi complex. Mitochondria are also closely involved in a variety of cellular activities including CN-resistant respiration and apoptosis. These mitochondrial functions are reviewed in this article, with special emphasis on the regulation of Dictyostelium development. Full article

(This article belongs to the Special Issue Focus Update in Biomolecules)

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The life cycle of Dictyostelium discoideum axenic strain Ax-2. The vegetative cells are usually grown in liquid medium, by means of pinocytotic incorporation of external nutrients. Under natural conditions, its parental strain D. discoideum NC-4 grows and multiplies by mitosis at the vegetative phase, phagocytosing nearby bacteria such as Escherichia coli and Klebsiella aerogenes. Upon exhaustion of nutrients, however, starving cells initiate differentiation, form multicellular structures (aggregates; mounds), and undergo a series of well-organized morphogenesis to construct fruiting bodies, each of which is consisting of a mass of spores (sorus) and a supporting cellular stalk. (see the text for details). Full article ">Figure 2
A growth/differentiation checkpoint (GDT point) in the cell cycle of a Dictyostelium discoideum Ax-2 cell. The doubling time of axenically growing Ax-2 cells is about 7.2 h and most of their cell cycle is composed of G2-phase with little or no G1-phase and a short period of M- and S-phases. A specific checkpoint (referred to as the GDT point) of GDT is located at the mid–late G2-phase (just after T7 and just before T0). Ax-2 cells progress through their cell cycle to the GDT point, irrespective of the presence or absence of nutrients, and enter the differentiation phase from this point under starvation conditions [<a href="#B2-biomolecules-03-00943" class="html-bibr">2</a>]. T0, T1, and T7 indicates 0, 1, and 7 h, respectively, after a temperature shift from 11.5 °C to 22.0 °C for cell synchrony. The absence of G1 phase in the Dictyostelium cell cycle is not so strange, because there is little or no G1 phase in rapidly dividing cells such as animal cells at the cleavage stage, and also in the true slime mold Physalum and Hydra. (Basically from Maeda [<a href="#B3-biomolecules-03-00943" class="html-bibr">3</a>]). Full article ">Figure 3
Strategy for creating rps4-null cells and their phenotypes. (a) As a starting material, LpCSfo cells in which pCoxIV (MTS)-SfoI is expressed under the tetracycline-minus (−Tet) condition, were prepared. Subsequently, the mutant rps4 gene (Mut-mtDNA for homologous recombination), in which the upstream SfoI-site and a 5'-half of rps4 coding region were deleted, was introduced into LpCSfo cells to obtain heteroplasmic transformants (LpCSfoHR cells) with mitochondria consisting of the Mut-mtDNA and wild-type mtDNA (Wt-mtDNA). Coupled with removal of Tet from growth medium, the fusion protein MTS-SfoI synthesized in the cytoplasm is exclusively transferred into mitochondria of LpCSfoHR cells and selectively digests Wt-mtDNA but not Mut-mtDNA. Since the digested Wt-mtDNA is not duplicated, the Mut-mtDNA becomes dominant during the course of growth under the −Tet condition, thus eventually giving rps4-null cells. (b) These cells were grown in growth medium with (+Tet) or without (−Tet) teteracyclin. Membrane potential of mitochondria was visualized by staining of cells with MitoTracker Orange. As was expected, the staining of mitochondria was almost completely vanished in LpCSfo cells grown without Tet, because their mtDNA with an intact SfoI site would be cleaved by SfoI eventually to become a ρ0 state devoid of mitochondrial DNA. Bars, 200 nm. (c) Development of starved MB35 cells and LpCSfoHR cells on agar. MB35 cells and LpCSfoHR cells grown with (+Tet) or without (−Tet) tetracycline were washed twice in BSS and plated on 1.5% non-nutrient agar at a density of 5 × 106 cells/cm2. This was followed by incubation for the indicated time at 22 °C. Bars, 0.5 mm. (Basically from Chida et al. [<a href="#B22-biomolecules-03-00943" class="html-bibr">22</a>]). Full article ">Figure 4
Schematic drawing showing the behavior of Dd-TRAP1 during the prestarvation response (PSR) and the initiation of differentiation in Dictyostelium cells (See the text for details). Full article ">Figure 5
A working hypothesis for explaining the existence of the GDT-point in the Dictyostelium cell-cycle, by assuming a temporally oscillating PSR activity during synchronized cell growth. This model is based on the fact that there is a good correlation between the expression patterns of the prestarvation (PS) genes and the GDT point-specific genes, and is also constructed from the presumption that the amount of PSFs (i.e., PSF activity) in an ideally synchronized cell population may oscillate in a cell-cycle dependent manner during growth, as shown here. The cell-cycle positions of T0 and T7 offer themselves repeatedly during the course of a completely synchronized growth (see <a href="#biomolecules-03-00943-f002" class="html-fig">Figure 2</a>). The PSR activity reaches the maximum value at the GDT-point (arrow between T7 and T0) in each cell cycle. Although it is presently unknown whether the expressions of Dd-TRAP1 and/or the amount of PSF-3 actually oscillate in a cell-cycle dependent manner, increasing their basal levels during the progression of synchronous cell growth, this issue remains to be examined in future. The horizontal axis of this figure represents incubation time (h) of an ideally synchronized D. discoideum (Ax-2) cell population in growth medium at 22.0 °C. T0 and T7 indicates 0 and 7 h, respectively, after a temperature shift from 11.5 °C to 22.0 °C for cell synchrony, and these are repeatedly represented during a prolonged time of synchronized culture. Full article ">Figure 6
Electron-micrographs of PSV-mitochondrion complexes present in a 47.5%–50% fraction (the intermediate fraction between a lighter pure mitochondria and a heavier pure PSV fractions) of a multilayered sucrose gradient for cell fractionation. (A,B) the outer membrane of a mitochondrion (M) is continuous with the unit membrane of a PSV. (C) a PSV and a mitochondrion are partitioned by a single membrane derived from either the PSV or the mitochondrion, and the electron-dense lining membrane of the PSV is not observed at the contact region. (D) a PSV appears twisted at the contact region (arrow). (E) a completely formed PSV is in close contact with a mitochondrion. (F) a mitochondrial part itself loses its inner structural integrity and seems to be transformed into the PSV. Bar, 500 nm. (From Maeda [<a href="#B49-biomolecules-03-00943" class="html-bibr">49</a>]). Full article ">Figure 7
A diagrammatic representation showing formation of the prespore-specific vacuole (PSV) from a mitochondrion and Golgi vesicles. Prior to PSV formation, mitochondria in differentiating prespore cells undergo drastic transformation to form a sort of vacuole (M vacuole), and some Dd-TRAP1 molecules translocate into the M vacuole. Subsequently, Golgi vesicles containing DIA2, Dd-GRP94 and other materials required for PSV formation fuse with the M vacuole, thus resulting in formation of the lining membrane (red) and the internal fibrous structure in the M vacuole. The mitochondrion-PSV complex is eventually twisted at the junction (arrow) and detached to form the respective organelles. Interestingly, almost all of the DIA2 molecules are selectively translocated to PSVs (possibly M vacuoles) in differentiating prespore cells, and seem to be required for exocytotic secretion of PSVs to form the outer-most membrane of spore cell wall [<a href="#B15-biomolecules-03-00943" class="html-bibr">15</a>]. M-V, M vacuole; GRP94, glucose-regulated protein 94 (endoplasmic reticulum Hsp90). (Slightly modified from Maeda [<a href="#B12-biomolecules-03-00943" class="html-bibr">12</a>]). Full article ">

659 KiB

Open AccessReview

Research Applications of Proteolytic Enzymes in Molecular Biology

by János András Mótyán, Ferenc Tóth and József Tőzsér

Biomolecules 2013, 3(4), 923-942; https://doi.org/10.3390/biom3040923 - 8 Nov 2013

Cited by 144 | Viewed by 27883

Abstract

Proteolytic enzymes (also termed peptidases, proteases and proteinases) are capable of hydrolyzing peptide bonds in proteins. They can be found in all living organisms, from viruses to animals and humans. Proteolytic enzymes have great medical and pharmaceutical importance due to their key role [...] Read more.

Proteolytic enzymes (also termed peptidases, proteases and proteinases) are capable of hydrolyzing peptide bonds in proteins. They can be found in all living organisms, from viruses to animals and humans. Proteolytic enzymes have great medical and pharmaceutical importance due to their key role in biological processes and in the life-cycle of many pathogens. Proteases are extensively applied enzymes in several sectors of industry and biotechnology, furthermore, numerous research applications require their use, including production of Klenow fragments, peptide synthesis, digestion of unwanted proteins during nucleic acid purification, cell culturing and tissue dissociation, preparation of recombinant antibody fragments for research, diagnostics and therapy, exploration of the structure-function relationships by structural studies, removal of affinity tags from fusion proteins in recombinant protein techniques, peptide sequencing and proteolytic digestion of proteins in proteomics. The aim of this paper is to review the molecular biological aspects of proteolytic enzymes and summarize their applications in the life sciences. Full article

(This article belongs to the Special Issue Enzymes and Their Biotechnological Applications)

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778 KiB

Open AccessArticle

Structural Evidence for the Tetrameric Assembly of Chemokine CCL11 and the Glycosaminoglycan Arixtra™

by Andrew B. Dykstra, Matt D. Sweeney and Julie A. Leary

Biomolecules 2013, 3(4), 905-922; https://doi.org/10.3390/biom3040905 - 6 Nov 2013

Cited by 4 | Viewed by 7576

Abstract

Understanding chemokine interactions with glycosaminoglycans (GAG) is critical as these interactions have been linked to a number of inflammatory medical conditions, such as arthritis and asthma. To better characterize in vivo protein function, comprehensive knowledge of multimeric species, formed by chemokines under native [...] Read more.

Understanding chemokine interactions with glycosaminoglycans (GAG) is critical as these interactions have been linked to a number of inflammatory medical conditions, such as arthritis and asthma. To better characterize in vivo protein function, comprehensive knowledge of multimeric species, formed by chemokines under native conditions, is necessary. Herein is the first report of a tetrameric assembly of the human chemokine CCL11, which was shown bound to the GAG Arixtra™. Isothermal titration calorimetry data indicated that CCL11 interacts with Arixtra, and ion mobility mass spectrometry (IM-MS) was used to identify ions corresponding to the CCL11 tetrameric species bound to Arixtra. Collisional cross sections (CCS) of the CCL11 tetramer-Arixtra noncovalent complex were compared to theoretical CCS values calculated using a preliminary structure of the complex deduced using X-ray crystallography. Experimental CCS values were in agreement with theoretical values, strengthening the IM-MS evidence for the formation of the noncovalent complex. Tandem mass spectrometry data of the complex indicated that the tetramer-GAG complex dissociates into a monomer and a trimer-GAG species, suggesting that two CC-like dimers are bridged by Arixtra. As development of chemokine inhibitors is of utmost importance to treatment of medical inflammatory conditions, these results provide vital insights into chemokine-GAG interactions. Full article

(This article belongs to the Special Issue Focus Update in Biomolecules)

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Full article ">Figure 1
Mass spectra of CCL11 under (A) denaturing conditions (50% acetonitrile, 0.1% formic acid) and (B) native conditions (250 mM ammonium acetate). M = monomer; D = dimer; Tr = Trimer; Te = Tetramer. Full article ">Figure 2
(A) Mass spectra of (A) CCL11 and (B) CCL11+Arixtra magnifying the 3,000–4,500 m/z range by a factor of 93 (A) and 145 (B), respectively. Formation of the CCL11 tetramer-Arixtra complex is supported by the presence of peaks corresponding to multiple charge states of the complex at 3,178, 3,584, 3,885, and 4,370 m/z. Higher order oligomers were not observed. D = dimer; Tr = trimer; Te = tetramer; * = species bound to Arixtra. Full article ">Figure 3
Heat map plotting m/z as a function of drift time for the CCL11+Arixtra mixture. Mass range is limited to the 3,000–4,500 m/z range illustrated in <a href="#biomolecules-03-00905-f002" class="html-fig">Figure 2</a>. Ion mobility separation allowed for the characterization of the peak at m/z 3,345 as both [D]5+ and [Te]10+ species. D = dimer; Tr = trimer; Te = tetramer; * = species bound to Arixtra. Full article ">Figure 4
Tandem mass spectrometry of the [Te*]12+ peak at 2,913 m/z showing that as the Te* complex is fragmented, it dissociates into Tr* and M species. M = monomer; D = dimer; Tr = trimer; * = species bound to Arixtra. Full article ">Figure 5
ATDs corresponding to the 8+–11+ charge states of the CCL11 tetramer+Arixtra complex. Full article ">Figure 6
Preliminary X-ray crystallography structure of the CCL11 tetramer-Arixtra complex (shown in color) overlaying the structure of the CCL2 tetramer (1DOL, shown in gray). The Arixtra molecule is shown top center. CCL11 subunits one and two are lower right and upper right, respectively, while subunits three and four are lower left and upper left, respectively. CXC-type dimers are vertically related while CC-type dimers are diagonally related. Full article ">

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Open AccessReview

Biophysical Insights into the Inhibitory Mechanism of Non-Nucleoside HIV-1 Reverse Transcriptase Inhibitors

by Grant Schauer, Sanford Leuba and Nicolas Sluis-Cremer

Biomolecules 2013, 3(4), 889-904; https://doi.org/10.3390/biom3040889 - 1 Nov 2013

Cited by 5 | Viewed by 10029

Abstract

HIV-1 reverse transcriptase (RT) plays a central role in HIV infection. Current United States Federal Drug Administration (USFDA)-approved antiretroviral therapies can include one of five approved non-nucleoside RT inhibitors (NNRTIs), which are potent inhibitors of RT activity. Despite their crucial clinical role in [...] Read more.

HIV-1 reverse transcriptase (RT) plays a central role in HIV infection. Current United States Federal Drug Administration (USFDA)-approved antiretroviral therapies can include one of five approved non-nucleoside RT inhibitors (NNRTIs), which are potent inhibitors of RT activity. Despite their crucial clinical role in treating and preventing HIV-1 infection, their mechanism of action remains elusive. In this review, we introduce RT and highlight major advances from experimental and computational biophysical experiments toward an understanding of RT function and the inhibitory mechanism(s) of NNRTIs. Full article

(This article belongs to the Special Issue Enzymes and Their Biotechnological Applications)

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764 KiB

Open AccessReview

Biotechnological Applications of Transglutaminases

by Natalie M. Rachel and Joelle N. Pelletier

Biomolecules 2013, 3(4), 870-888; https://doi.org/10.3390/biom3040870 - 22 Oct 2013

Cited by 80 | Viewed by 11874

Abstract

In nature, transglutaminases catalyze the formation of amide bonds between proteins to form insoluble protein aggregates. This specific function has long been exploited in the food and textile industries as a protein cross-linking agent to alter the texture of meat, wool, and leather. [...] Read more.

In nature, transglutaminases catalyze the formation of amide bonds between proteins to form insoluble protein aggregates. This specific function has long been exploited in the food and textile industries as a protein cross-linking agent to alter the texture of meat, wool, and leather. In recent years, biotechnological applications of transglutaminases have come to light in areas ranging from material sciences to medicine. There has also been a substantial effort to further investigate the fundamentals of transglutaminases, as many of their characteristics that remain poorly understood. Those studies also work towards the goal of developing transglutaminases as more efficient catalysts. Progress in this area includes structural information and novel chemical and biological assays. Here, we review recent achievements in this area in order to illustrate the versatility of transglutaminases. Full article

(This article belongs to the Special Issue Enzymes and Their Biotechnological Applications)

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Figure 1

Figure 1
Amide bond formation catalyzed by TGase. Peptide- or protein-bound glutamines and lysines serve as substrates, releasing ammonia in the process. Full article ">Figure 2
Crystal structure of MTG (PDB ID: 3IU0). The active site of the zymogen is covered (left) by an α-helix (gold), which is cleaved upon activation, exposing the active site cysteine residue (right, yellow spheres) that is critical for activity. Full article ">Figure 3
Surface representation of MTG (PDB ID: 1UI4), illustrating active site residues investigated by mutagenesis (pink and orange regions) [<a href="#B48-biomolecules-03-00870" class="html-bibr">48</a>]. The active site cleft is indicated by an asterisk. Residues in orange, upon substitution to alanine, resulted in activity of 5% or less than the wild type, revealing their importance. Full article ">Figure 4
Examples of assays used for detection of TGase activity. (A) Colorimetric and fluorescent product release activity assays. The hydroxamate assay (top) remains the standard method to determine and compare TGase activity. TG2 activity can also be quantified by the release of p-nitrophenol (PNP; λmax = 405 nm), umbelliferone (λem = 465 nm), or by the formation of an anilide product (λmax = 278 nm) following conjugation with N,N-dimethyl-1,4-phenylenediamine (DMPDA). (B) Cartoon representation of the TG2 conformational FRET sensor. (C) In vivo activation of MTG allowing for in-cell assaying. Full article ">Figure 5
General scheme for protein labeling using TGase. The protein of interest (P.O.I.) carries an accessible glutamine residue, for TGase-catalysed reaction with an amine-substituted fluorophore; alternatively, the P.O.I. carries a reactive lysine residue for reaction with a glutamine-modified fluorophore. Full article ">Figure 6
Examples of TGases applied for visualization of biomacromolecules. (A) Locations of independently encoded 13-mer peptidyl loop K-tags on bacterial alkaline phosphatase. (B) MTG-aided enzymatic detection of nucleic acids. (C) The paramagnetic agent is cross-linked to a glutamine, generating the CEST effect. Magnetic resonance saturation is transferred to water following saturation of the amide proton. Full article ">

949 KiB

Open AccessArticle

Variation in the Subcellular Localization and Protein Folding Activity among Arabidopsis thaliana Homologs of Protein Disulfide Isomerase

by Christen Y. L. Yuen, Kristie O. Matsumoto and David A. Christopher

Biomolecules 2013, 3(4), 848-869; https://doi.org/10.3390/biom3040848 - 21 Oct 2013

Cited by 25 | Viewed by 8891

Abstract

Protein disulfide isomerases (PDIs) catalyze the formation, breakage, and rearrangement of disulfide bonds to properly fold nascent polypeptides within the endoplasmic reticulum (ER). Classical animal and yeast PDIs possess two catalytic thioredoxin-like domains (a, a′) and two non-catalytic domains ( [...] Read more.

Protein disulfide isomerases (PDIs) catalyze the formation, breakage, and rearrangement of disulfide bonds to properly fold nascent polypeptides within the endoplasmic reticulum (ER). Classical animal and yeast PDIs possess two catalytic thioredoxin-like domains (a, a′) and two non-catalytic domains (b, b′), in the order a-b-b′-a′. The model plant, Arabidopsis thaliana, encodes 12 PDI-like proteins, six of which possess the classical PDI domain arrangement (AtPDI1 through AtPDI6). Three additional AtPDIs (AtPDI9, AtPDI10, AtPDI11) possess two thioredoxin domains, but without intervening b-b′ domains. C-terminal green fluorescent protein (GFP) fusions to each of the nine dual-thioredoxin PDI homologs localized predominantly to the ER lumen when transiently expressed in protoplasts. Additionally, expression of AtPDI9:GFP-KDEL and AtPDI10: GFP-KDDL was associated with the formation of ER bodies. AtPDI9, AtPDI10, and AtPDI11 mediated the oxidative folding of alkaline phosphatase when heterologously expressed in the Escherichia coli protein folding mutant, dsbA⁻. However, only three classical AtPDIs (AtPDI2, AtPDI5, AtPDI6) functionally complemented dsbA⁻. Interestingly, chemical inducers of the ER unfolded protein response were previously shown to upregulate most of the AtPDIs that complemented dsbA⁻. The results indicate that Arabidopsis PDIs differ in their localization and protein folding activities to fulfill distinct molecular functions in the ER. Full article

(This article belongs to the Special Issue Protein Folding and Misfolding)

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Figure 1
Domain arrangements of PDIA1, PDIA6, and PDI-D homologs from Arabidopsis. Red boxes correspond to thioredoxin-like catalytic domains (a, a′, ao), and grey boxes are non-catalytic thioredoxin-fold domains (b, b′). The signal peptides (sp) are represented as white boxes, and acidic regions (c) as yellow boxes. The D domain of PDI-D proteins is depicted as a blue box. The active site sequences of each catalytic domain are shown, with residues deviating from the typical CGHC motif underlined. The last four amino acids are shown at the end of each depicted protein. Full article ">Figure 2
Arabidopsis PDIA1 homologs fused to GFP localize to the ER. (A) PDI5:GFP-KDEL; (B) PDI6:GFP-KDEL; (C) PDI1:GFP-KDEL; (D) PDI2:GFP-KDEL; (E) PDI3:GFP-KDEL; (F) PDI4:GFP-KDEL. Each chimeric fusion was co-expressed in leaf protoplasts with the ER marker, ER:mCherry. GFP signal is shown in Column 1, mCherry signal in Column 2, and a merge of both signal patterns in Column 3. Chlorophyll autofluorescence is shown in Column 4. The white bars in Column 1 represent 10 µm. Full article ">Figure 3
Arabidopsis PDIA6 and PDI-D homologs fused to GFP localize to the ER. (A) PDI9:GFP-KDEL; (B) PDI10:GFP-KDDL; (C) PDI11:GFP; (D) unfused GFP(S65T) control. Each chimeric fusion was co-expressed in leaf protoplasts with the ER marker, ER:mCherry. GFP signal is shown in Column 1, mCherry signal in Column 2, and a merge of both signal patterns in Column 3. Chlorophyll autofluorescence is shown in Column 4. The white bars in Column 1 represent 10 µm. Full article ">Figure 4
SESA1:mCherry partially localizes to punctate bodies induced by PDI9:GFP-KDEL or PDI10:GFP-KDDL expression. (A) PDI9:GFP-KDEL; (B) PDI10:GFP-KDDL; (C) PDI2:GFP-KDEL control (non-ER body-inducing); (D) unfused GFP(S56T) control. Each chimeric fusion was co-expressed in leaf protoplasts with the seed storage protein marker SESA1:mCherry. GFP signal is shown in Column 1, mCherry signal in Column 2, and a merge of both signal patterns in Column 3. Chlorophyll autofluorescence is shown in Column 4. Arrows indicate punctate bodies co-labeled by SESA1:mCherry and either (A) PDI9:GFP-KDEL or (B) PDI10:GFP-KDDL. The white bars in column 1 represent 10 µm. Full article ">Figure 5
Alkaline phosphatase activity of E. coli dsbA− cells expressing Arabidopsis homologs of PDIA1, PDIA6, and PDI-D. Measured alkaline phosphatase activities of wild-type strain RI89 (WT), dsbA− strain RI90 (dsbA−), and RI90 cells transformed with the pFLAG-CTS empty vector (EV) or constructs heterologously expressing the following Arabidopsis PDIs: (A) PDIA1 subfamily members AtPDI1 through AtPDI6; (B) PDIA6 subfamily members AtPDI9 and AtPDI10, and PDI-D subfamily member PDI11; (C) AtPDI1, AtPDI2, and modified variants AtPDI1M (a: CGHC) and AtPDI2M (a: CGAC); (D) AtPDI4 and modified variants AtPDI4M1 (a: CARC), AtPDI4M2 (a: CGHC), and AtPDI4M3 (a: CGHC, a′: CGHC). Error bars represent standard deviations. Full article ">Figure 6
Recombinant PDI expression in the protein folding mutant dsbA− cells. Immunoblot of proteins extracted from mutant E. coli strain RI90 (dsbA−), with or without constructs for the expression of recombinant PDIs. NV, no vector control; Vect, pFLAG-CTS empty vector control; PDI1, PDI2, PDI3, PDI4, PDI5 and PDI6, expression of the indicated PDI1A1 homolog; PDI9, expression of AtPDI9 (PDIA6 homolog); WT, E. coli strain RI89 (dsbA+) control. Lanes denoted with a dash indicate empty lanes. Full article ">Figure 7
Western blot analysis of PDI-GFP fusions expressed in Arabidopsis leaf protoplasts. Crude protein extracts were obtained from Arabidopsis leaf protoplasts transfected with constructs expressing GFP(S65T) alone (control) or fused to members of the Arabidopsis PDI family. The proteins were immobilized on a nitrocellulose membrane and GFP was detected using a rabbit anti-GFP primary antiserum, and an anti-rabbit horseradish peroxidase conjugated secondary antiserum. G, GFP(S65T); 1, PDI1-GFP-KDEL; 2, PDI2-GFP-KDEL; 3, PDI3-GFP-KDEL; 4, PDI4-GFP-KDEL; 5, PDI5-GFP-KDEL; 6, PDI6-GFP-KDEL; PDI9-GFP-KDEL; 10, PDI10-GFP-KDDL; 11, PDI11-GFP. Full article ">

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Open AccessReview

Biocatalysis for Biobased Chemicals

by Rubén De Regil and Georgina Sandoval

Biomolecules 2013, 3(4), 812-847; https://doi.org/10.3390/biom3040812 - 17 Oct 2013

Cited by 42 | Viewed by 14467

Abstract

The design and development of greener processes that are safe and friendly is an irreversible trend that is driven by sustainable and economic issues. The use of Biocatalysis as part of a manufacturing process fits well in this trend as enzymes are themselves [...] Read more.

The design and development of greener processes that are safe and friendly is an irreversible trend that is driven by sustainable and economic issues. The use of Biocatalysis as part of a manufacturing process fits well in this trend as enzymes are themselves biodegradable, require mild conditions to work and are highly specific and well suited to carry out complex reactions in a simple way. The growth of computational capabilities in the last decades has allowed Biocatalysis to develop sophisticated tools to understand better enzymatic phenomena and to have the power to control not only process conditions but also the enzyme’s own nature. Nowadays, Biocatalysis is behind some important products in the pharmaceutical, cosmetic, food and bulk chemicals industry. In this review we want to present some of the most representative examples of industrial chemicals produced in vitro through enzymatic catalysis. Full article

(This article belongs to the Special Issue Enzymes and Their Biotechnological Applications)

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1286 KiB

Open AccessReview

Improvement of Biocatalysts for Industrial and Environmental Purposes by Saturation Mutagenesis

by Francesca Valetti and Gianfranco Gilardi

Biomolecules 2013, 3(4), 778-811; https://doi.org/10.3390/biom3040778 - 8 Oct 2013

Cited by 19 | Viewed by 16891

Abstract

Laboratory evolution techniques are becoming increasingly widespread among protein engineers for the development of novel and designed biocatalysts. The palette of different approaches ranges from complete randomized strategies to rational and structure-guided mutagenesis, with a wide variety of costs, impacts, drawbacks and relevance [...] Read more.

Laboratory evolution techniques are becoming increasingly widespread among protein engineers for the development of novel and designed biocatalysts. The palette of different approaches ranges from complete randomized strategies to rational and structure-guided mutagenesis, with a wide variety of costs, impacts, drawbacks and relevance to biotechnology. A technique that convincingly compromises the extremes of fully randomized vs. rational mutagenesis, with a high benefit/cost ratio, is saturation mutagenesis. Here we will present and discuss this approach in its many facets, also tackling the issue of randomization, statistical evaluation of library completeness and throughput efficiency of screening methods. Successful recent applications covering different classes of enzymes will be presented referring to the literature and to research lines pursued in our group. The focus is put on saturation mutagenesis as a tool for designing novel biocatalysts specifically relevant to production of fine chemicals for improving bulk enzymes for industry and engineering technical enzymes involved in treatment of waste, detoxification and production of clean energy from renewable sources. Full article

(This article belongs to the Special Issue Enzymes and Their Biotechnological Applications)

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3082 KiB

Open AccessReview

Biocatalytic Synthesis of Chiral Alcohols and Amino Acids for Development of Pharmaceuticals

by Ramesh N. Patel

Biomolecules 2013, 3(4), 741-777; https://doi.org/10.3390/biom3040741 - 2 Oct 2013

Cited by 125 | Viewed by 16993

Abstract

Chirality is a key factor in the safety and efficacy of many drug products and thus the production of single enantiomers of drug intermediates and drugs has become increasingly important in the pharmaceutical industry. There has been an increasing awareness of the enormous [...] Read more.

Chirality is a key factor in the safety and efficacy of many drug products and thus the production of single enantiomers of drug intermediates and drugs has become increasingly important in the pharmaceutical industry. There has been an increasing awareness of the enormous potential of microorganisms and enzymes derived there from for the transformation of synthetic chemicals with high chemo-, regio- and enatioselectivities. In this article, biocatalytic processes are described for the synthesis of chiral alcohols and unntural aminoacids for pharmaceuticals. Full article

(This article belongs to the Special Issue Enzymes and Their Biotechnological Applications)

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Open AccessArticle

Isolation, NMR Spectral Analysis and Hydrolysis Studies of a Hepta Pyranosyl Diterpene Glycoside from Stevia rebaudiana Bertoni

by Venkata Sai Prakash Chaturvedula, Steven Chen, Oliver Yu and Guohong Mao

Biomolecules 2013, 3(4), 733-740; https://doi.org/10.3390/biom3040733 - 30 Sep 2013

Cited by 7 | Viewed by 6615

Abstract

From the commercial extract of the leaves of Stevia rebaudiana Bertoni, a minor steviol glycoside, 13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy] ent-kaur-16-en-19-oic acid-[(2-O-(3-O-β-D-glucopyranosyl-α-L-rhamnopyranosyl)-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl) ester] (1); also known as rebaudioside O having seven sugar units has [...] Read more.

From the commercial extract of the leaves of Stevia rebaudiana Bertoni, a minor steviol glycoside, 13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy] ent-kaur-16-en-19-oic acid-[(2-O-(3-O-β-D-glucopyranosyl-α-L-rhamnopyranosyl)-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl) ester] (1); also known as rebaudioside O having seven sugar units has been isolated. Its structural characterization has been achieved by the extensive 1D (¹H and ¹³C), and 2D NMR (COSY, HMQC, HMBC) as well as mass spectral data. Further, hydrolysis studies were performed on rebaudioside O using acid and enzymatic methods to identify aglycone and sugar residues in its structure as well as their configurations. Full article

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Biomolecules, Volume 3, Issue 4 (December 2013) – 14 articles , Pages 733-1052

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