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PiStar-180 References
The last 25 references for the PiStar are listed below. A complete searchable database with all known stopped-flow references can be accessed by logging into the APL members area.
| Authors | Title | Year | Keywords | Journal/Proceedings | |
|---|---|---|---|---|---|
| Soo I. Yang and Takuji Tanaka | Characterization of recombinant prolidase from Lactococcus lactis– changes in substrate specificity by metal cations, and allosteric behavior of the peptidase | 2008 | bitterness, metallopeptidase, overexpression, PepQ, proline | FEBS J, 2008, Vol 275, Issue 2, pp 271-280 | |
| Abstract: The Lactococcus lactis NRRL B-1821 prolidase gene was cloned and overexpressed in Escherichia coli. Under suboptimum growth conditions, recombinant soluble and active prolidase was produced; in contrast, inclusion bodies were formed under conditions preferred for cell growth. Recombinant prolidase retained more than half its full activity between 30 and 60 °C, and was completely inactivated after 30 min at 70 °C. CD analysis confirmed that prolidase was inactivated at 67 °C. The enzyme was active under weak alkali to weak acidic conditions, and showed maximum activity at pH 7.0. Although these characteristics are similar to those for other reported prolidases, this prolidase was distinctive for two kinetic characteristics. Firstly, different substrate specificity was observed for its two preferred metal cations, zinc and manganese: Leu-Pro was preferred with zinc, whereas Arg-Pro was preferred with manganese. Secondly, the enzyme showed an allosteric response to changes in substrate concentrations, with Hill constants of 1.53 for Leu-Pro and 1.57 for Arg-Pro. Molecular modeling of this prolidase suggests that these unique characteristics may be attributed to a loop structure near the active site. | |||||
| Ylva Ivarsson, Carlo Travaglini-Allocatelli, Veronica Morea, Maurizio Brunori, and Stefano Gianni | The folding pathway of an engineered circularly permuted PDZ domain | 2008 | circular permutation, intermediates, kinetics, protein folding, transition state | PROTEIN ENG DES and SEL, 2008, Volume 21, Number 3, pp. 155-160. | |
| Abstract: To understand the role of sequence connectivity in the folding pathway of a multi-state protein, we have analysed the folding kinetics of an engineered circularly permuted PDZ domain. This variant has been designed with the specific aim of posing two of the strands participating in the stabilisation of an early folding nucleus as contiguous elements in the primary structure. Folding of the circularly permuted PDZ2 has been explored by a variety of different experimental approaches including stopped-flow and continuous-flow kinetics, as well as ligand-induced folding experiments. Data reveal that although circular permutation introduces a significant destabilisation of the native state, a folding intermediate is stabilised and accumulated prior folding. Furthermore, quantitative analysis of the observed kinetics indicates an acceleration of the early folding events by more than two orders of magnitude. The results support the importance of sequence connectivity both in the mechanism and the speed of protein folding. | |||||
| Xie, J.-B.; Zhou, J.-M. | Trigger Factor Assisted Folding of Green Fluorescent Protein | 2008 | Protein Folding, FRET | BIOCHEMISTRY-USA, 2008, Vol 47, Issue 1, pp 348-357 | |
| Abstract: Guanidine induced equilibrium and kinetic folding of a variant of green fluorescent protein (F99S/M153T/V163A, GFPuv) was studied. Using manual mixing and stopped-flow techniques, we combined different probes, including tryptophan fluorescence, chromophore fluorescence and reactivity with DTNB, to trace the spontaneous and TF-assisted folding of guanidine denatured GFPuv. We found that both unfolding and refolding of GFPuv occurred in a stepwise manner and a stable intermediate was populated under equilibrium conditions. The thermodynamic parameters obtained show that the intermediate state of GFPuv is quite compact compared to the denatured state and most of the green fluorescence is retained in this state. By studying GFPuv folding assisted by TF and a number of TF mutants, we found that wild-type TF catalyzes proline isomerization and accelerates the folding rate at low TF concentrations, but retards GFPuv folding and decelerates the folding rate at high TF concentrations. This reflects the two activities of TF, as an enzyme and as a chaperone. A general mechanism of TF assisted protein folding is discussed. | |||||
| Christa Jakopitsch, Holger Spalteholz, Paul G. Furtmüller, Jürgen Arnhold and Christian Obinger, | Mechanism of reaction of horseradish peroxidase with chlorite and chlorine dioxide | 2008 | Horseradish peroxidase, Chlorination reaction, Chlorite, Chlorine dioxide, Hypochlorous acid, Compound I, Compound II | J INORG BIOCHEM, 2008, Vol 102, Issue 2, pp 293-302 | |
| Abstract: It is demonstrated that horseradish peroxidase (HRP) mixed with chlorite follows the whole peroxidase cycle. Chlorite mediates the two-electron oxidation of ferric HRP to compound I (k1) thereby releasing hypochlorous acid. Furthermore, chlorite acts as one-electron reductant of both compound I (k2) and compound II (k3) forming chlorine dioxide. The strong pH-dependence of all three reactions clearly suggests that chlorous acid is the reactive species. Typical apparent bimolecular rate constants at pH 5.6 are 1.4 × 105 M-1 s-1 (k1), 2.25 × 105 M-1 s-1 (k2), and 2.4 × 104 M-1 s-1 (k3), respectively. Moreover, the reaction products hypochlorous acid and chlorine dioxide, which are known to induce heme bleaching and amino acid modification upon longer incubation times, also mediate the oxidation of ferric HRP to compound I (2.4 × 107 M-1 s-1 and 2.7 × 104 M-1 s-1, respectively, pH 5.6) but do not react with compounds I and II. A reaction scheme is presented and discussed from both a mechanistic and thermodynamic point of view. It helps to explain the origin of contradictory data so far found in the literature on this topic. | |||||
| Georgios Tsiavaliaris, Setsuko Fujita-Becker, Ulrike Dürrwang, Ralph P. Diensthuber, Michael A. Geeves, and Dietmar J. Manstein | Mechanism, regulation, and functional properties of dictyostelium myosin-1B | 2008 | ENZYME CATALYSIS AND REGULATION | J. Biol. Chem., Vol. 283, Issue 8, 4520-4527, February 22, 2008 | |
| Abstract: Myosin-1B is one of three long tailed class-1 myosins containing an ATP-insensitive actin-binding site in the tail region that are produced in Dictyostelium discoideum. Myosin-1B localizes to actin-rich structures at the leading edge of migrating cells where it has been implicated in the formation and retraction of membrane projections, the recycling of plasma membrane components, and intracellular particle transport. Here, we have used a combination of molecular engineering approaches to describe the kinetic and motile properties of the myosin-1B motor and its regulation by TEDS site phosphorylation. Our results show that myosin-1B is a low duty ratio motor and displays the fastest nucleotide binding kinetics of any of the Dictyostelium class-1 myosins studied so far. Different from Dictyostelium myosin-1D and myosin-1E, dephosphorylated myosin-1B is not inactivated but moves actin filaments efficiently, albeit at an up to 8-fold slower velocity in the in vitro motility assay. A further difference is that myosin-1B lacks the ability to switch between rapid movement and bearing tension upon physiological changes of free Mg2+ ions. In this respect, its motor properties appear to be more closely related to Dictyostelium myosin-2 and rabbit skeletal muscle myosin. | |||||
| Rafael Couñago, Corey J. Wilson, Matthew I. Peña, Pernilla Wittung-Stafshede, and Yousif Shamoo | An adaptive mutation in adenylate kinase that increases organismal fitness is linked to stability–activity trade-offs | 2008 | adaptive evolution, adenylate kinase, molecular evolution ,thermostability, trade-offs | PROTEIN ENG DES SEL, 2008, 21(1):19-27 | |
| Abstract: Protein function is a balance between activity and stability. However, the relevance of stability–activity trade-offs for protein evolution and their impact on organismal fitness have been difficult to determine. Previously, we have linked organismal survival at increasing temperatures to adaptive changes to a single protein sequence through allelic replacement of an essential gene, adenylate kinase (adk), in a thermophile. In vivo continuous evolution of the temperature-sensitive thermophile has shown that the first step toward increased organismal fitness is mutation of glutamine-199 to arginine in the mesophilic enzyme (AKsub Q199R). Here, we show that although substitution of Arg-199 did confer a modest increase in stability (0.6 kcal mol–1 at 20°C; ΔTm = 3.0°C), it is a large change in the activity profile of the enzyme that is responsible for its exceptional robustness during the earlier experimental evolution study. Kinetic studies of AKsub Q199R show that it has a strong loss of enzymatic activity (>50%) at lower temperatures (20–45°C) and a subsequent increase at elevated temperatures. The stability–activity trade-off observed for AKsub Q199R was linked to the rigidification of the overall structure through stabilization of a polypeptide loop containing Arg-199 that is part of the ATP-binding site of the enzyme. Structural analysis revealed the formation of new ionic interactions facilitated by Arg-199. Our results suggest that stability–activity trade-offs are employed readily as an evolutionary strategy during natural selection to increase organismal fitness. | |||||
| Adele Di Matteo, Stefano Gianni, M. Eugenia Schininà, Alessandra Giorgi, Fabio Altieri, Nicoletta Calosci, Maurizio Brunori, and Carlo Travaglini-Allocatelli | A Strategic Protein in Cytochrome c Maturation: THREE-DIMENSIONAL STRUCTURE OF CcmH AND BINDING TO APOCYTOCHROME c | 2007 | Cytochrome C Maturation,Cyt-C, protein structure, protein folding. | J BIOL CHEM, 2007, Vol 282, Issue 37, pp 27012-27019 | |
| Abstract: CcmH (cytochromes c maturation protein H) is an essential component of the assembly line necessary for the maturation of c-type cytochromes in the periplasm of Gram-negative bacteria. The protein is a membrane-anchored thiol-oxidoreductase that has been hypothesized to be involved in the recognition and reduction of apocytochrome c, a prerequisite for covalent heme attachment. Here, we present the 1.7Å crystal structure of the soluble periplasmic domain of CcmH from the opportunistic pathogen Pseudomonas aeruginosa (Pa-CcmH*). The protein contains a three-helix bundle, i.e. a fold that is different from that of all other thiol-oxidoreductases reported so far. The catalytic Cys residues of the conserved LRCXXC motif (Cys25 and Cys28), located in a long loop connecting the first two helices, form a disulfide bond in the oxidized enzyme. We have determined the pKa values of these 2 Cys residues of Pa-CcmH* (both >8) and propose a possible mechanistic role for a conserved Ser36 and a water molecule in the active site. The interaction between Pa-CcmH* and Pa-apocyt c551 (where cyt c551 represents cytochrome c551) was characterized in vitro following the binding kinetics by stopped-flow using a Trp-containing fluorescent variant of Pa-CcmH* and a dansylated peptide, mimicking the apocytochrome c551 heme binding motif. The kinetic results show that the protein has a moderate affinity to its apocyt substrate, consistent with the role of Pa-CcmH as an intermediate component of the assembly line for c-type cytochrome biogenesis. | |||||
| Changyuan Lu, Masahiro Mukai, Yu Lin, Guanghui Wu, Robert K. Poole, and Syun-Ru Yeh | Structural and Functional Properties of a Single Domain Hemoglobin from the Food-borne Pathogen Campylobactor jejuni | 2007 | Hemoglobin, haemoglobin, Hgb | J BIOL CHEM, 2007, Vol 282, Iss 35, pp 25917-25928 | |
| Abstract: Campylobacter jejuni contains two globins, a truncated hemoglobin, Ctb, and a single domain hemoglobin, Cgb. The physiological function of Ctb remains unclear, whereas Cgb has been linked to NO detoxification. With resonance Raman scattering, the iron-histidine stretching mode of Cgb was identified at 251 cm-1. This frequency is unusually high, suggesting an imidazolate character of the proximal histidine as a result of the H-bonding network linking the catalytic triad involving the F8His, H23Glu, and G5Tyr residues. In the CO-complex, two conformers were identified with the {nu}C-O/{nu}Fe-CO at 529/1914 cm-1 and 492/1963 cm-1. The former is assigned to a `closed` conformation, in which the heme-bound CO is stabilized by the H-bond(s) donated from the B10Tyr-E7Gln residues, whereas the latter is assigned to an `open` conformer, in which the H-bonding interaction is absent. The presence of the two alternative conformations demonstrates the plasticity of the protein matrix. In the O2-complex, the iron-O2 stretching frequency was identified at 554 cm-1, which is unusually low, indicating that the heme-bound O2 is stabilized by strong H-bond(s) donated by the B10Tyr-E7Gln residues. This scenario is consistent with its low O2 off-rate (0.87 s-1). Taken together the data suggest that the NO-detoxifying activity of Cgb is facilitated by the imidazolate character of the proximal F8His and the distal positive polar environment provided by the B10Tyr-E7Gln. They may offer electronic `push` and `pull,` respectively, for the O-O bond cleavage reaction required for the isomerization of the presumed peroxynitrite intermediate to the product, nitrate. | |||||
| Jody M. Mason, Urs B. Hagemann, and Katja M. Arndt | Improved Stability of the Jun-Fos Activator Protein-1 Coiled Coil Motif: A STOPPED-FLOW CIRCULAR DICHROISM KINETIC ANALYSIS | 2007 | stopped-flow circular dichroism kinetic analysis, protein structure, protein folding | J BIOL CHEM, 2007, Vol 282, Iss 32, pp 23015-23024 | |
| Abstract: Two c-Jun leucine zipper variants that bind with high affinity to c-Fos have been selected using semirational design combined with protein-fragment complementation assays (JunW) or phage display selection (JunWPh1). Enriched winners differ from each other in only two of ten semi-randomized positions, with {Delta}Tm values of 28 °C and 37 °C over wild-type. cFos-JunW, cFos-JunWPh1, and two intermediate mutants (cFos-JunWQ21R and cFos-JunWE23K) display biphasic kinetics in the folding direction, indicating the existence of a folding intermediate. The first reaction phase is fast and concentration-dependent, showing that the intermediate is readily populated and dimeric. The second phase is independent of concentration and is exponential. In contrast, in the unfolding direction, all molecules display two-state kinetics. Collectively this implies a transition state between unfolded helices and dimeric intermediate that is readily traversed in both directions. We demonstrate that the added stability of cFos-JunWPh1 relative to cFos-JunW is achieved via a combination of kinetic rate changes; cFos-JunWE23K has an increased initial dimerization rate, prior to the major transition state barrier while cFos-JunWQ21R displays a decreased unfolding rate. The former implies that improved hydrophobic burial and helix-stabilizing mutations exert their effect on the initial, rapid, monomer-collision event. In contrast, electrostatic interactions exert their effect late in the folding pathway. Although our focus is the leucine zipper region of the oncogenic transcription factor Activator Protein-1, coiled coils are ubiquitous and highly specific in their recognition of partners. Consequently, generating kinetic-based rules to predict and engineer their stability is of major significance in peptide-based drug design and nano-biotechnology. | |||||
| Hui-Yong Lian, Hong Zhang, Zai-Rong Zhang, Harriët M. Loovers, Gary W. Jones, Pamela J. E. Rowling, Laura S. Itzhaki, Jun-Mei Zhou, and Sarah Perrett | Hsp40 Interacts Directly with the Native State of the Yeast Prion Protein Ure2 and Inhibits Formation of Amyloid-like Fibrils | 2007 | Hsp40, Amyloid-like gibrils, prion protein, PrP, oligomerization | J BIOL CHEM, 2007, Vol 282, Issue 16, pp 11931-11940 | |
| Abstract: Ure2 is the protein determinant of the [URE3] prion phenotype in Saccharomyces cerevisiae and consists of a flexible N-terminal prion-determining domain and a globular C-terminal glutathione transferase-like domain. Overexpression of the type I Hsp40 member Ydj1 in yeast cells has been found to result in the loss of [URE3]. However, the mechanism of prion curing by Ydj1 remains unclear. Here we tested the effect of overexpression of Hsp40 members Ydj1, Sis1, and Apj1 and also Hsp70 co-chaperones Cpr7, Cns1, Sti1, and Fes1 in vivo and found that only Ydj1 showed a strong curing effect on [URE3]. We also investigated the interaction of Ydj1 with Ure2 in vitro. We found that Ydj1 was able to suppress formation of amyloid-like fibrils of Ure2 by delaying the process of fibril formation, as monitored by thioflavin T binding and atomic force microscopy imaging. Controls using bovine serum albumin, Sis1, or the human Hsp40 homologues Hdj1 or Hdj2 showed no significant inhibitory effect. Ydj1 was only effective when added during the lag phase of fibril formation, suggesting that it interacts with Ure2 at an early stage in fibril formation and delays the nucleation process. Using surface plasmon resonance and size exclusion chromatography, we demonstrated a direct interaction between Ydj1 and both wild type and N-terminally truncated Ure2. In contrast, Hdj2, which did not suppress fibril formation, did not show this interaction. The results suggest that Ydj1 inhibits Ure2 fibril formation by binding to the native state of Ure2, thus delaying the onset of oligomerization. | |||||
| Ylva Ivarsson, Carlo Travaglini-Allocatelli, Per Jemth, Francesco Malatesta, Maurizio Brunori, and Stefano Gianni | An On-pathway Intermediate in the Folding of a PDZ Domain | 2007 | J BIOL CHEM, 2007, Vol 282, Issue 12, pp 8568-8572. | ||
| Abstract: The folding pathways of some proteins include the population of partially structured species en route to the native state. Identification and characterization of these folding intermediates are particularly difficult as they are often only transiently populated and play different mechanistic roles, being either on-pathway productive species or off-pathway kinetic traps. To define the role of folding intermediates, a quantitative analysis of the folding and unfolding rate constants over a wide range of denaturant concentration is often required. Such a task is further complicated by the reversible nature of the folding reaction, which implies the observed kinetics to be governed by a complex combination of different microscopic rate constants. Here, we tackled this problem by measuring directly the folding rate constant under highly denaturing conditions, namely by inducing the folding of a PDZ domain through a quasi-irreversible binding reaction with a specific peptide. In analogy with previous works based on hydrogen exchange experiments, we present evidence that the folding pathway of the PDZ domain involves the formation of an obligatory on-pathway intermediate. The results presented exemplify a novel type of kinetic test to detect an on-pathway folding intermediate. | |||||
| Michel O. Steinmetz, Ilian Jelesarov, William M. Matousek, Srinivas Honnappa, Wolfgang Jahnke, John H. Missimer, Sabine Frank, Andrei T. Alexandrescu, and Richard A. Kammerer | Molecular basis of coiled-coil formation | 2007 | autonomous folding unit | protein folding | trigger sequence | leucine zipper | {alpha}-helix | PROC NAT ACAD SCI USA, 2007, Vol 104, Issue 17, pp 7062-7067 | |
| Abstract: Coiled coils have attracted considerable interest as design templates in a wide range of applications. Successful coiled-coil design strategies therefore require a detailed understanding of coiled-coil folding. One common feature shared by coiled coils is the presence of a short autonomous helical folding unit, termed `trigger sequence,` that is indispensable for folding. Detailed knowledge of trigger sequences at the molecular level is thus key to a general understanding of coiled-coil formation. Using a multidisciplinary approach, we identify and characterize here the molecular determinants that specify the helical conformation of the monomeric early folding intermediate of the GCN4 coiled coil. We demonstrate that a network of hydrogen-bonding and electrostatic interactions stabilize the trigger-sequence helix. This network is rearranged in the final dimeric coiled-coil structure, and its destabilization significantly slows down GCN4 leucine zipper folding. Our findings provide a general explanation for the molecular mechanism of coiled-coil formation. | |||||
| Graham D. Balkwill, Huw E. L. Williams and Mark S. Searle | Structure and folding dynamics of a DNA hairpin with a stabilising d(GNA) trinucleotide loop: influence of base pair mis-matches and point mutations on conformational equilibria | 2007 | DNA, hairpin, trinucleotide, circular dichroism, CD, spectroscopy | ORG BIOMOL CHEM, 2007, Vol 5, Iss 5, pp 832-839 | |
| Abstract: Hairpins are known to play specific roles in DNA– and RNA–protein recognition. Various disease states are thought to originate from the ill-timed formation of a hairpin loop during transcription, particularly in the context of triplet repeats which are associated with myotonic dystrophy, fragile X syndrome and other genetic disorders. An understanding of nucleic acid folding mechanisms requires a detailed appreciation of the timescales of these local folding events, a characterisation of the conformational equilibria that exist in solution and the influence of point mutations on the relative stabilities of the different species. We investigate using NMR and CD spectroscopy the structure and dynamics of a DNA hairpin containing a highly stabilising cGNAg loop. The single-stranded 13-mer 5-d(GCTACGNAGTCGC) with N = T folds to form a hairpin structure which accommodates a C–T mis-matched base pair within the double-stranded stem region. The hairpin is in equilibrium with a double-stranded duplex form with the mixture of two interconverting conformations in slow exchange on the NMR timescale (1–2 s–1 at 308 K). We are able to characterise the dynamics of the interconversion process by NMR magnetisation transfer and by CD stopped-flow kinetic experiments. The latter shows that the hairpin folds too rapidly to detect by this method (>500 s–1) and forms in a kinetic overshoot followed by a much slower equilibration to a mixture of conformations (0.13 s–1 at 298 K). A point mutation that converts the GTA to a GAA loop sequence destabilises the intermolecular duplex structure and enables us to unambiguously assign the various dynamic processes that are taking place. | |||||
| Edward FRANKLIN, Seamus BROWNE, Jerrard HAYES, Coilin BOLAND, Aisling DUNNE, Gordon ELLIOT and Timothy J. MANTLE | Activation of biliverdin-IXa reductase by inorganic phosphate and related anions | 2007 | anion activation, biliverdin-IXa reductase (BVR-A), NAD, pH optima, Xenopus tropicalis. | BIOCHEM J, 2007, Vol 405, pp 61-67 | |
| Abstract: The effect of pH on the initial-rate kinetic behaviour of BVR-A (biliverdin-IXa reductase) exhibits an alkaline optimum with NADPH as cofactor, but a neutral optimum with NADH as cofactor. This has been described as dual cofactor and dual pH dependent behaviour; however, no mechanism has been described to explain this phenomenon. We present evidence that the apparent peak of activity observed at neutral pH with phosphate buffer and NADH as cofactor is an anion-dependent activation, where inorganic phosphate apparently mimics the role played by the 2´-phosphate of NADPH in stabilizing the interaction between NADH and the enzyme. The enzymes from mouse, rat and human all exhibit this behaviour. This behaviour is not seen with BVR-A from Xenopus tropicalis or the ancient cyanobacterial enzyme from Synechocystis PCC 6803, which, in addition to being refractory to activation by inorganic phosphate, are also differentiated by an acid pH optimum with both nicotinamide nucleotides. | |||||
| Sungmun Lee, Erik J. Fernandez, and Theresa A. Good | Role of aggregation conditions in structure, stability, and toxicity of intermediates in the Abeta fibril formation pathway | 2007 | Alzheimer's disease; amyloid; aggregation; guanidine hydrochloride; unfolding | PROTEIN SCI, 2007, Vol 16, pp 723-732 | |
| Abstract: beta-amyloid peptide (Abeta) is one of the main protein components of senile plaques associated with Alzheimer's disease (AD). Abeta readily aggregates to forms fibrils and other aggregated species that have been shown to be toxic in a number of studies. In particular, soluble oligomeric forms are closely related to neurotoxicity. However, the relationship between neurotoxicity and the size of Abeta aggregates or oligomers is still under investigation. In this article, we show that different Abeta incubation conditions in vitro can affect the rate of Abeta fibril formation, the conformation and stability of intermediates in the aggregation pathway, and toxicity of aggregated species formed. When gently agitated, Abeta aggregates faster than Abeta prepared under quiescent conditions, forming fibrils. The morphology of fibrils formed at the end of aggregation with or without agitation, as observed in electron micrographs, is somewhat different. Interestingly, intermediates or oligomers formed during Abeta aggregation differ greatly under agitated and quiescent conditions. Unfolding studies in guanidine hydrochloride indicate that fibrils formed under quiescent conditions are more stable to unfolding in detergent than aggregation associated oligomers or Abeta fibrils formed with agitation. In addition, Abeta fibrils formed under quiescent conditions were less toxic to differentiated SH-SY5Y cells than the Abeta aggregation associated oligomers or fibrils formed with agitation. These results highlight differences between Abeta aggregation intermediates formed under different conditions and provide insight into the structure and stability of toxic Abeta oligomers. | |||||
| I. Papapanagiotou, S. D. Streeter, P. D. Cary, and G. G. Kneale | DNA structural deformations in the interaction of the controller protein C.AhdI with its operator sequence | 2007 | nucleic acid, structural deformation, protein structure | NUCL ACID RES, 2007, Vol 35, Iss 8, pp 2643-2650 | |
| Abstract: Controller proteins such as C.AhdI regulate the expression of bacterial restriction–modification genes, and ensure that methylation of the host DNA precedes restriction by delaying transcription of the endonuclease. The operator DNA sequence to which C.AhdI binds consists of two adjacent binding sites, OL and OR. Binding of C.AhdI to OL and to OL + OR has been investigated by circular permutation DNA-bending assays and by circular dichroism (CD) spectroscopy. CD indicates considerable distortion to the DNA when bound by C.AhdI. Binding to one or two sites to form dimeric and tetrameric complexes increases the CD signal at 278 nm by 40 and 80% respectively, showing identical local distortion at both sites. In contrast, DNA-bending assays gave similar bend angles for both dimeric and tetrameric complexes (47 and 38°, respectively). The relative orientation of C.AhdI dimers in the tetrameric complex and the structural role of the conserved Py-A-T sequences found at the centre of C-protein-binding sites are discussed. | |||||
| Wildemann, D.; Schiene-Fischer, C.; Aumuller, T.; Bachmann, A.; Kiefhaber, T.; Lucke, C.; Fischer, G. | A Nearly Isosteric Photosensitive Amide-Backbone Substitution Allows Enzyme Activity Switching in Ribonuclease S | 2007 | protein, RNase, thioxylation, peptide bond, thermodynamic stability, enzymatic activity, photoisomerization, polypeptide | J AM CHEM SOC, 2007, Vol 129, Iss 16, pp 4910-4918 | |
| Abstract: [CS-NH]4-RNase S, a site specific modified version of RNase S obtained by thioxylation (O/S exchange) at the Ala4-Ala5- peptide bond, was used to evaluate the impact of protein backbone photoswitching on bioactivity. [CS-NH]4-RNase S was yielded by recombination of the S-protein and the respective chemically synthesized thioxylated S-peptide derivative. Comparison with RNase S revealed similar thermodynamic stability of the complex and an unperturbed enzymatic activity toward cytidine 2',3'-cyclic monophosphate (cCMP). Reversible photoisomerization with a highly increased cis/trans isomer ratio of the thioxopeptide bond of [CS-NH]4-RNase S in the photostationary state occurred under UV irradiation conditions (254 nm). The slow thermal reisomerization (t1/2 = 180 s) permitted us to determine the enzymatic activity of cis [CS-NH]4-RNase S by measurement of inital rates of cCMP hydrolysis. Despite thermodynamic stability of cis [CS-NH]4-RNase S, its enzymatic activity is completely abolished but recovers after reisomerization. We conclude that the thioxopeptide bond modified polypeptide backbone represents a versatile probe for site-directed photoswitching of proteins. | |||||
| Hsi-Chen Hsiao, Svetlana Boycheva, Nicholas J. Watmough and Thomas Brittain | Activation of the cytochrome c peroxidase of Pseudomonas aeruginosa. The role of a heme-linked protein loop: A mutagenesis studies | 2007 | Cytochrome c peroxidase; Activity; Mutagenesis | J INORG BIOCHEM, 2007, Vol 101, Iss 8, pp 1133-1139 | |
| Abstract: Mutagenesis studies have been used to investigate the role of a heme ligand containing protein loop (67–79) in the activation of di-heme peroxidases. Two mutant forms of the cytochrome c peroxidase of Pseudomonas aeruginosa have been produced. One mutant (loop mutant) is devoid of the protein loop and the other (H71G) contains a non-ligating Gly at the normal histidine ligand site. Spectroscopic data show that in both mutants the distal histidine ligand of the peroxidatic heme in the un-activated enzyme is lost or is exchangeable. The un-activated H71G and loop mutants show, respectively, 75% and 10% of turnover activity of the wild-type enzyme in the activated form, in the presence of hydrogen peroxide and the physiological electron donor cytochrome c551. Both mutant proteins show the presence of constitutive reactivity with peroxide in the normally inactive, fully oxidised, form of the enzyme and produce a radical intermediate. The radical product of the constitutive peroxide reaction appears to be located at different sites in the two mutant proteins. These results show that the loss of the histidine ligand from the peroxidatic heme is, in itself, sufficient to produce peroxidatic activity by providing a peroxide binding site and that the formation of radical intermediates is very sensitive to changes in protein structure. Overall, these data are consistent with a major role for the protein loop 67–79 in the activation of di-heme peroxidases and suggest a “charge hopping” mechanism may be operative in the process of intra-molecular electron transfer. | |||||
| Michael C. Lane, Paul W. O'Toole, and Stanley A. Moore | Molecular Basis of the Interaction between the Flagellar Export Proteins FliI and FliH from Helicobacter pylori | 2006 | Flagellar Export Proteins, Bacterial flagellar protein, Helicobacter pylori | J BIOL CHEM, 2006, Vol 281, Iss 1, pp 508-517 | |
| Abstract: Bacterial flagellar protein export requires an ATPase, FliI, and presumptive inhibitor, FliH. We have explored the molecular basis for FliI/FliH interaction in the human gastric pathogen Helicobacter pylori. By using bioinformatic and biochemical analyses, we showed that residues 1–18 of FliI very likely form an amphipathic {alpha}-helix upon interaction with FliH, and that residues 21–91 of FliI resemble the N-terminal oligomerization domain of the F1-ATPase catalytic subunits. A truncated FliI-(2–91) protein was shown to be folded, although the N-terminal 18 residues were likely unstructured. Deletion and scanning mutagenesis showed that residues 1–18 of FliI were essential for the FliI/FliH interaction. Scanning mutation of amino acids in the N-terminal 10 residues of FliI indicated that a cluster of hydrophobic residues in this segment was critical for the interaction with FliH. The interaction between FliI and FliH has similarities to the interaction between the N-terminal {alpha}-helix of the F1-ATPase {alpha}-subunit and the globular domain of the F1-ATPase δ-subunit, respectively. This similarity suggests that FliH may function as a molecular stator. | |||||
| Eitan Gross, Dao-Quan Peng, Stanley L. Hazen, and Jonathan D. Smith | A Novel Folding Intermediate State for Apolipoprotein A-I: Role of the Amino and Carboxy Termini | 2006 | Stopped-flow, circular dichroism, fluorescence spectroscopy, apolipoprotein. | BIOPHYS J, 2006, Vol 90, pp 1362-1370 | |
| Abstract: Intramolecular interactions between the amino and carboxy termini of apolipoprotein A-I (apoAI) are believed to stabilize the helix bundle conformation of the protein. During lipid assembly the protein undergoes conformational changes that result in an exposure of the carboxy terminus and its insertion into the lipid phase. To determine the role of the two termini in the energetics of unfolding, we studied the guanidine-hydrochloride-induced unfolding and refolding of apoAI as well as its N-terminal deletion (del[1–43]), C-terminal deletion (del[186–243]), and the double deletion containing only the central residues 44–185. Thermodynamic analysis of the equilibrium unfolding measured by fluorescence spectroscopy revealed the presence of an intermediate unfolded state (Iequil) in addition to the native (N) and unfolded states. Refolding kinetics of apoAI, measured by stopped-flow circular dichroism, revealed two kinetic intermediates, Iburst and Irecovery. Computer modeling suggested that the first resembles the partially unfolded protein, whereas the second overlaps with the native state of the protein. The free energy changes for the N -> Iequil transition of the N-terminal and double deletions were lower then that of the full-length form, whereas that for the C-terminal deletion was higher. Our findings suggest that the N-terminus of apoAI stabilizes the native state of the protein by increasing the Eyring energy barrier for the N -> Iequil unfolding transition; whereas the carboxyl terminus destabilizes that state. | |||||
| Xavier I. Ambroggio and Brian Kuhlman | Computational Design of a Single Amino Acid Sequence that Can Switch between Two Distinct Protein Folds | 2006 | protein function, protein folding, conformational change,amino acid sequence, monte carlo simulation | J AM CHEM SOC, 2006, Vol 128, Iss 4, pp 1154-1161 | |
| Abstract: The functions of many proteins are mediated by specific conformational changes, and therefore the ability to design primary sequences capable of secondary and tertiary changes is an important step toward the creation of novel functional proteins. To this end, we have developed an algorithm that can optimize a single amino acid sequence for multiple target structures. The algorithm consists of an outer loop, in which sequence space is sampled by a Monte Carlo search with simulated annealing, and an inner loop, in which the effect of a given mutation is evaluated on the various target structures by using the rotamer packing routine and composite energy function of the protein design software, RosettaDesign. We have experimentally tested the method by designing a peptide, Sw2, which can be switched from a 2Cys-2His zinc finger-like fold to a trimeric coiled-coil fold, depending upon the pH or the presence of transition metals. Physical characterization of Sw2 confirms that it is able to reversibly adopt each intended target fold. | |||||
| Xiaofeng Liu, Kijeong Kim, Terrance Leighton, and Elizabeth C. Theil | Paired Bacillus anthracis Dps (Mini-ferritin) Have Different Reactivities with Peroxide | 2006 | Bacillus anthracis, mini-ferritins, protein nanocages, ferroxidase activity | J BIOL CHEM, 2006, Vol 281, Iss 38, pp 27827-27835 | |
| Abstract: Dps (DNA protection during starvation) proteins, mini-ferritins in the ferritin superfamily, catalyze Fe2+/H2O2/O2 reactions and make minerals inside protein nanocages to minimize radical oxygen-chemistry (metal/osmotic/temperature/nutrient/oxidant) and sometimes to confer virulence. Paired Dps proteins in Bacillus, rare in other bacteria, have 60% sequence identity. To explore functional differences in paired Bacilli Dps protein, we measured ferroxidase activity and DNA protection (hydroxyl radical) for Dps protein dodecamers from Bacillus anthracis (Ba) since crystal structures and iron mineralization (iron-stain) were known. The self-assembled (200 kDa) Ba Dps1 (Dlp-1) and Ba Dps2 (Dlp-2) proteins had similar Fe2+/O2 kinetics, with space for minerals of 500 iron atoms/protein, and protected DNA. The reactions with Fe2+ were novel in several ways: 1) Ba Dps2 reactions (Fe2+/H2O2) proceeded via an A650 nm intermediate, with similar rates to maxi-ferritins (Fe2+/O2), indicating a new Dps protein reaction pathway, 2) Ba Dps2 reactions (Fe2+/O2 versus Fe2+/O2 + H2O2) differed 3-fold contrasting with Escherichia coli Dps reactions, with 100-fold differences, and 3) Ba Dps1, inert in Fe2+/H2O2 catalysis, inhibited protein-independent Fe2+/H2O2 reactions. Sequence similarities between Ba Dps1 and Bacillus subtilis DpsA (Dps1), which is regulated by general stress factor (SigmaB) and Fur, and between Ba Dps2 and B. subtilis MrgA, which is regulated by H2O2 (PerR), suggest the function of Ba Dps1 is iron sequestration and the function of Ba Dps2 is H2O2 destruction, important in host/pathogen interactions. Destruction of H2O2 by Ba Dps2 proceeds via an unknown mechanism with an intermediate similar spectrally (A650 nm) and kinetically to the maxi-ferritin diferric peroxo complex. | |||||
| Szabolcs Osváth, Levente Herényi, Péter Závodszky, Judit Fidy, and Gottfried Köhler | Hierarchic Finite Level Energy Landscape Model: TO DESCRIBE THE REFOLDING KINETICS OF PHOSPHOGLYCERATE KINASE | 2006 | protein folding kinetics, stopped-flow, guanidine, tryptophan fluorescence | J BIOL CHEM, 2006, Vol 281, Iss 34, pp 24375-24380 | |
| Abstract: One of the most intriguing predictions of energy landscape models is the existence of non-exponential protein folding kinetics caused by hierarchical structures in the landscapes. Here we provide the strongest evidence so far of such hierarchy and determine the time constants and weights of the kinetic components of the suggested hierarchic energy landscape. To our knowledge, the idea of hierarchical folding energy barriers has never been tested over such a broad timescale. Refolding of yeast phosphoglycerate kinase was initiated from the guanidine-unfolded state by stopped-flow or manual mixing and monitored by tryptophan fluorescence from 1 ms to 15 min. The strategy to build a model that describes folding of yeast phosphoglycerate kinase was to start from the simplest paradigm and modify it stepwise to the necessary minimal extent after repeated comparisons with the experiments. We made no a priori assumptions about the folding landscape. The result was a hierarchic finite level landscape model that quantitatively describes the refolding of yeast phosphoglycerate kinase from 1 ms to 15 min. The early steps of the folding process happen in the upper region of the landscape, where the surface has a hierarchic structure. This leads to stretched kinetics in the early phase of the folding. The lower region of the energy landscape is dominated by a trap that reflects the accumulation of molten globule intermediate state. From this intermediate, the protein can reach the global energy minimum corresponding to the native state through a cross-barrier folding step. | |||||
| Alessandro Borgia, Daniele Bonivento, Carlo Travaglini-Allocatelli, Adele Di Matteo, and Maurizio Brunori | Unveiling a Hidden Folding Intermediate in c-Type Cytochromes by Protein Engineering | 2006 | state-of-the-art kinetics, folding intermediate, mutagenesis, protein folding | J BIOL CHEM, 2006, Vol 281, Iss 14, pp 9331-9336 | |
| Abstract: Several investigators have highlighted a correlation between the basic features of the folding process of a protein and its topology, which dictates the folding pathway. Within this conceptual framework we proposed that different members of the cytochrome c (cyt c) family share the same folding mechanism, involving a consensus partially structured state. Pseudomonas aeruginosa cyt c551 (Pa cyt c551) folds via an apparent two-state mechanism through a high energy intermediate. Here we present kinetic evidence demonstrating that it is possible to switch its folding mechanism from two to three state, stabilizing the high energy intermediate by rational mutagenesis. Characterization of the folding kinetics of one single-site mutant of the Pa cyt c551 (Phe7 to Ala) indeed reveals an additional refolding phase and a fast unfolding process which are explained by the accumulation of a partially folded species. Further kinetic analysis highlights the presence of two parallel processes both leading to the native state, suggesting that the above mentioned species is a non obligatory on-pathway intermediate. Determination of the crystallographic structure of F7A shows the presence of an extended internal cavity, which hosts three `bound` water molecules and a H-bond in the N-terminal helix, which is shorter than in the wild type protein. These two features allow us to propose a detailed structural interpretation for the stabilization of the native and especially the intermediate states induced by a single crucial mutation. These results show how protein engineering, x-ray crystallography and state-of-the-art kinetics concur to unveil a folding intermediate and the structural determinants of its stability. | |||||
| Yan Jiang, Yong-Bin Yan, and Hai-Meng Zhou | Polyvinylpyrrolidone 40 Assists the Refolding of Bovine Carbonic Anhydrase B by Accelerating the Refolding of the First Molten Globule Intermediate | 2006 | protein, protein aggregation, protein science, protein refolding, guanidine hydrochloride | J BIOL CHEM, 2006, Vol 281, Iss 14, pp 9058-9065 | |
| Abstract: Protecting proteins from aggregation is one of the most important issues in both protein science and protein engineering. In this research, the mechanism of enhancing the refolding of guanidine hydrochloride-denatured carbonic anhydrase B by polyvinylpyrrolidone 40 (PVP40) was studied by both kinetic and equilibrium refolding experiments. The reactivation and refolding kinetics indicated that the rate constant of refolding the first refolding intermediate (I1) to the second one (I2) is promoted by the addition of PVP. Fluorescence quenching studies further indicated that PVP could bind to the aggregation-prone species I1, resulting in the protection of the exposed hydrophobic surface, a minimization of the protein surface, and more importantly, an increase of the refolding rate of I1. These properties were quite different from those of poly(ethylene glycol) (PEG), which has been shown to have a strong and stoichiometric binding to I1 and does not interfere with the refolding pathway. Unlike PEG, the binding of PVP to I1 does not block the aggregation pathway directly but decreases the energy barrier for I1 to refold to I2 and thus reduces the accumulation of I1. These results suggested that PVP works by a quite different mechanism from those well established ones in chaperones and chemical promoters. PVP is more like a folding catalyst rather than a chemical chaperone. The distinct mechanism of enhancing protein aggregation by PVP is expected to facilitate the attempt to develop new chemical compounds as well as new strategies to protect proteins from aggregation. | |||||