| 1. |
- Byström, Anders S, et al.
(författare)
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Differentially expressed trmD ribosomal protein operon of Escherichia coli is transcribed as a single polycistronic mRNA species
- 1989
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Ingår i: Journal of Molecular Biology. - : Academic Press. - 0022-2836 .- 1089-8638. ; 208:4, s. 575-586
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Tidskriftsartikel (refereegranskat)abstract
- The trmD operon is a four-cistron operon in which the first and fourth genes encode ribosomal proteins S16 (rpsP) and L19 (rplS), respectively. The second gene encodes a 21,000 Mr polypeptide of unknown function and the third gene (trmD) encodes the enzyme tRNA(m1G37)methyltransferase, which catalyzes the formation of 1-methylguanosine (m1G) next to the 3' end of the anticodon (position 37) of some tRNAs in Escherichia coli. Here we show under all regulatory conditions studied, transcription initiates at one unique site, and the entire operon is transcribed into one polycistronic mRNA. Between the promoter and the first gene, rpsP, an attenuator-like structure is found (delta G = -18 kcal; 1 cal = 4.184 J), followed by four uridine residues. This structure is functional in vitro, and terminates more than two-thirds of the transcripts. The different parts of the trmD operon mRNA decay at a uniform rate. The stability of the trmD mRNA is not reduced with decreasing growth rate, which is in contrast to what has been found for other ribosomal protein mRNAs. Furthermore, earlier experiments have shown the existence of differential expression as well as non-co-ordinate regulation within the operon. Our results are consistent with the regulation of the trmD operon being due to some mechanism(s) operating at the post-transcriptional level, and do not involve differential degradation of different mRNA segments, internal promoters or internal terminators.
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| 2. |
- Malisauskas, Mantas, et al.
(författare)
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Amyloid protofilaments from the calcium-binding protein equine lysozyme : formation of ring and linear structures depends on pH and metal ion concentration
- 2003
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Ingår i: Journal of Molecular Biology. - 0022-2836 .- 1089-8638. ; 330:4, s. 879-890
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Tidskriftsartikel (refereegranskat)abstract
- The calcium-binding equine lysozyme has been found to undergo conversion into amyloid fibrils during incubation in solution at acidic pH. At pH 4.5 and 57 °C, where equine lysozyme forms a partially unfolded molten globule state, the protein forms protofilaments with a width of ca. 2 nm. In the absence of Ca2+ the protofilaments are present as annular structures with a diameter of 40–50 nm. In the presence of 10 mM CaCl2 the protofilaments of equine lysozyme are straight or curved; they can assemble into thicker threads, but they do not appear to undergo circularisation. At pH 2.0, where the protein is more destabilised compared to pH 4.5, fibril formation occurs at 37 °C and 57 °C. At pH 2.0, both ring-shaped and linear protofilaments are formed, in which periodic repeats of ca 35 nm can be distinguished clearly. The rings constitute about 10% of all fibrillar species under these conditions and they are characterised by a larger diameter of 70–80 nm. All the structures bind Congo red and thioflavine T in a manner similar to fibrils associated with a variety of amyloid diseases. At pH 2.0, fibril formation is accompanied by some acidic hydrolysis, producing specific fragmentation of the protein, leading to the accumulation of two peptides in particular, consisting of residues 1–80 and 54–125. At the initial stages of incubation, however, full-length equine lysozyme represents the dominant species within the fibrils. We propose that the ring-shaped structures observed here, and in the case of disease-associated proteins such as -synuclein, could be a second generic type of amyloid structure in addition to the more common linear fibrils.
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| 3. |
- Wikström, P M, et al.
(författare)
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Non-autogenous control of ribosomal protein synthesis from the trmD operon in Escherichia coli
- 1988
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 203:1, s. 141-152
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Tidskriftsartikel (refereegranskat)abstract
- The trmD operon of Escherichia coli encodes the ribosomal proteins S16 and L19, the tRNA(m1G37)methyltransferase and a 21,000 Mr protein of unknown function. Here we demonstrate that, in contrast to the expression of other ribosomal protein operons, the amount of trmD operon mRNA and the rate of synthesis of the proteins encoded by the operon respond to increased gene dosage. The steady-state level of the mRNA was about 18 times higher, and the relative rate of synthesis of the ribosomal proteins S16 and L19, the tRNA(m1G37)methyltransferase and the 21,000 Mr protein was 15, 9, 25 and 23 times higher, respectively, in plasmid-containing cells than in plasmid-free cells. Overproduced tRNA(m1G37)methyltransferase and 21,000 Mr protein were as stable as E. coli total protein, whereas the two ribosomal proteins were degraded to a large extent. The steady-state amount of S16 and L19 in the plasmid-containing cells exceeded that in plasmid-free cells by threefold and twofold, respectively. No significant effect on the synthesis of the trmD operon proteins from the chromosomally located genes was observed when parts of the operon were expressed on different plasmids. Taken together, these results suggest that the expression of the trmD operon is not subject to transcriptional or translational feedback regulation, and demonstrate that not all ribosomal protein operons are regulated in the same manner. We propose that ribosomal protein operons that do not encode proteins that bind directly to rRNA are not under autogenous control. Metabolic regulation at the transcriptional level and protein degradation are plausible mechanisms for the control of expression of such operons.
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| 4. |
- Aisenbrey, Christopher, et al.
(författare)
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Macromolecular Crowding at Membrane Interfaces : Adsorption and Alignment of Membrane Peptides
- 2008
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Ingår i: Journal of Molecular Biology. - : Elsevier. - 0022-2836 .- 1089-8638. ; 375, s. 376-385
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Tidskriftsartikel (refereegranskat)abstract
- Association of proteins to cellular membranes is involved in various biological processes. Various theoretical models have been developed to describe this adsorption mechanism, commonly implying the concept of an ideal solution. However, due to the two-dimensional character of membrane surfaces intermolecular interactions between the adsorbed molecules become important. Therefore previously adsorbed molecules can influence the adsorption behavior of additional protein molecules and their membrane-associated structure. Using the model peptide LAH4, which upon membrane-adsorption can adopt a transmembrane as well as an in-planar configuration, we carried out a systematic study of the correlation between the peptide concentration in the membrane and the topology of this membrane-associated polypeptide. We could describe the observed binding behavior by establishing a concept, which includes intermolecular interactions in terms of a scaled particle theory.High surface concentration of the peptide shifts the molecules from an in-planar into a transmembrane conformation, a process driven by the reduction of occupied surface area per molecule. In a cellular context, the crowding-dependent alignment might provide a molecular switch for a cell to sense and control its membrane occupancy. Furthermore, crowding might have pronounced effects on biological events, such as the cooperative behavior of antimicrobial peptides and the membrane triggered aggregation of amyloidogenic peptides.
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| 5. |
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| 6. |
- Andersson, Magnus, et al.
(författare)
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A structural basis for sustained bacterial adhesion : Biomechanical properties of CFA/I Pili
- 2012
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Ingår i: Journal of Molecular Biology. - : Elsevier. - 0022-2836 .- 1089-8638. ; 415:5, s. 918-928
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Tidskriftsartikel (refereegranskat)abstract
- Enterotoxigenic Escherichia coli (ETEC) are a major cause of diarrheal disease worldwide. Adhesion pili (or fimbriae), such as the CFA/I (colonization factor antigen I) organelles that enable ETEC to attach efficiently to the host intestinal tract epithelium, are critical virulence factors for initiation of infection. We characterized at single organelle level the intrinsic biomechanical properties and kinetics of individual CFA/I pili, demonstrating that weak external forces (7.5 pN) are sufficient to unwind the intact helical filament of this prototypical ETEC pilus and that it quickly regains its original structure when the force is removed. While the general relationship between exertion of force and an increase in the filament length for CFA/I pili associated with diarrheal disease is analogous to that of P-pili and type 1 pili, associated with urinary tract and other infections, the biomechanical properties of these different pili differ in key quantitative details. Unique features of CFA/I pili, including the significantly lower force required for unwinding, the higher extension speed at which the pili enter a dynamic range of unwinding, and the appearance of sudden force drops during unwinding can be attributed to morphological features of CFA/I pili including weak layer-to-layer interactions between subunits on adjacent turns of the helix, and the approximately horizontal orientation of pilin subunits with respect to the filament axis. Our results indicate that ETEC CFA/I pili are flexible organelles optimized to withstand harsh motion without breaking, resulting in continued attachment to the intestinal epithelium by the pathogenic bacteria that express these pili.
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| 7. |
- Antonyuk, Svetlana V, et al.
(författare)
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The structure of human extracellular copper-zinc superoxide dismutase at 1.7 A resolution : insights into heparin and collagen binding
- 2009
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 388:2, s. 310-326
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Tidskriftsartikel (refereegranskat)abstract
- Extracellular superoxide dismutase (SOD3) is a homotetrameric copper- and zinc-containing glycoprotein with affinity for heparin. The level of SOD3 is particularly high in blood vessel walls and in the lungs. The enzyme has multiple roles including protection of the lungs against hyperoxia and preservation of nitric oxide. The common mutation R213G, which reduces the heparin affinity of SOD3, is associated with increased risk of myocardial infarctions and stroke. We report the first crystal structure of human SOD3 at 1.7 A resolution. The overall subunit fold and the subunit-subunit interface of the SOD3 dimer are similar to the corresponding structures in Cu-Zn SOD (SOD1). The metal-binding sites are similar to those found in SOD1, but with Asn180 replacing Thr137 at the Cu-binding site and a much shorter loop at the zinc-binding site. The dimers form a functional homotetramer that is fashioned through contacts between two extended loops on each subunit. The N- and C-terminal end regions required for tetramerisation and heparin binding, respectively, are highly flexible. Two grooves fashioned by the tetramer interface are suggestive as the probable sites for heparin and collagen binding.
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| 8. |
- Blanch, Ewan W, et al.
(författare)
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Is polyproline II helix the killer conformation? A Raman optical activity study of the amyloidogenic prefibrillar intermediate of human lysozyme.
- 2000
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 301:2, s. 553-563
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Tidskriftsartikel (refereegranskat)abstract
- The amyloidogenic prefibrillar partially denatured intermediate of human lysozyme, prepared by heating the native protein to 57 degrees C at pH 2.0, was studied using Raman optical activity (ROA). A positive band in the room temperature ROA spectrum of the native protein at approximately 1345 cm(-1), assigned to a hydrated form of alpha-helix, is not present in that of the prefibrillar intermediate, where a new strong positive band at approximately 1318 cm(-1) appears instead that is assigned to the poly(l-proline) II (PPII)-helical conformation. A sharp negative band at approximately 1241 cm(-1) in the native protein, assigned to beta-strand, shows little change in the ROA spectrum of the prefibrillar intermediate. The disappearance of a positive ROA band at approximately 1551 cm(-1) assigned to vibrations of tryptophan side-chains indicates that major conformational changes have occurred among the five tryptophan residues present in human lysozyme, four of which are located in the alpha-domain. The various ROA data suggest that a substantial loss of tertiary structure has occurred in the prefibrillar intermediate and that this is located more in the alpha-domain than in the beta-domain. There is no evidence for any increase in beta-structure. The ROA spectrum of hen lysozyme, which does not form amyloid fibrils so readily, remains much more native-like on heating to 57 degrees C at pH 2.0. The thermal behaviour of the alanine-rich alpha-helical peptide AK21 in aqueous solution was found to be similar to that of human lysozyme. Hydrated alpha-helix therefore appears to readily undergo a conformational change to PPII structure on heating, which may be a key step in the conversion of alpha-helix into beta-sheet in the formation of amyloid fibrils in human lysozyme. Since it is extended, flexible, lacks intrachain hydrogen bonds and is fully hydrated in aqueous solution, PPII helix has the appropriate characteristics to be implicated as a critical conformational element in many conformational diseases. Disorder of the PPII type may be a sine qua non for the formation of regular fibrils; whereas the more dynamic disorder of the random coil may lead only to amorphous aggregates.
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| 9. |
- Bokvist, Marcus, et al.
(författare)
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Two Types of Alzheimer’s β-Amyloid (1–40) Peptide Membrane Interactions : Aggregation Preventing Transmembrane Anchoring Versus Accelerated Surface Fibril Formation
- 2004
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 335:4, s. 1039-1049
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Tidskriftsartikel (refereegranskat)abstract
- The 39–42 amino acid long, amphipathic amyloid-β peptide (Aβ) is one of the key components involved in Alzheimer's disease (AD). In the neuropathology of AD, Aβ presumably exerts its neurotoxic action via interactions with neuronal membranes. In our studies a combination of 31P MAS NMR (magic angle spinning nuclear magnetic resonance) and CD (circular dichroism) spectroscopy suggest fundamental differences in the functional organization of supramolecular Aβ1–40 membrane assemblies for two different scenarios with potential implication in AD: Aβ peptide can either be firmly anchored in a membrane upon proteolytic cleavage, thereby being prevented against release and aggregation, or it can have fundamentally adverse effects when bound to membrane surfaces by undergoing accelerated aggregation, causing neuronal apoptotic cell death. Acidic lipids can prevent release of membrane inserted Aβ1–40 by stabilizing its hydrophobic transmembrane C-terminal part (residue 29–40) in an α-helical conformation via an electrostatic anchor between its basic Lys28 residue and the negatively charged membrane interface. However, if Aβ1–40 is released as a soluble monomer, charged membranes act as two-dimensional aggregation-templates where an increasing amount of charged lipids (possible pathological degradation products) causes a dramatic accumulation of surface-associated Aβ1–40 peptide followed by accelerated aggregation into toxic structures. These results suggest that two different molecular mechanisms of peptide–membrane assemblies are involved in Aβ′s pathophysiology with the finely balanced type of Aβ–lipid interactions against release of Aβ from neuronal membranes being overcompensated by an Aβ–membrane assembly which causes toxic β-structured aggregates in AD. Therefore, pathological interactions of Aβ peptide with neuronal membranes might not only depend on the oligomerization state of the peptide, but also the type and nature of the supramolecular Aβ–membrane assemblies inherited from Aβ′s origin.
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| 10. |
- Bosco, Daryl A, et al.
(författare)
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Dissecting the microscopic steps of the cyclophilin a enzymatic cycle on the biological substrate HIV-capsid by NMR
- 2010
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Ingår i: Journal of Molecular Biology. - : Elsevier. - 0022-2836 .- 1089-8638. ; 403:5, s. 723-38
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Tidskriftsartikel (refereegranskat)abstract
- Peptidyl-prolyl isomerases (PPIases) are emerging as key regulators of many diverse biological processes. Elucidating the role of PPIase activity in vivo has been challenging because mutagenesis of active site residues not only reduces the catalytic activity of these enzymes, but also dramatically affects substrate binding. Employing the cyclophilin A (CypA) PPIase together with its biologically relevant and natively folded substrate, the N-terminal domain of the HIV-1 capsid (CA(N)) protein, we demonstrate here how to dissect residue specific contributions to PPIase catalysis versus substrate binding utilizing NMR spectroscopy. Surprisingly, a number of CypA active-site mutants previously assumed to be strongly diminished in activity toward biological substrates based on a peptide assay only, catalyze the HIV capsid with wild-type activity, but with a change in the rate-limiting step of the enzymatic cycle. The results illustrate that a quantitative analysis of catalysis using the biological substrates is critical when interpreting the effects of PPIase mutations in biological assays.
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