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PubMed2005

Ab initio construction of all-atom loop conformations.

In this study, a new ab initio method named CLOOP has been developed to build all-atom loop conformations. In this method, a loop main-chain conformation is generated by sampling main-chain dihedral angles from a restrained varphi/psi set, and the side-chain conformations are built randomly. The CHARMM all-atom force field was used to evaluate the loop conformations. Soft core potentials were used to treat the non-bond interactions, and a designed energy-minimization technique was used to close and optimize the loop conformations. It is shown that the two strategies improve the computational efficiency and the loop-closure rate substantially compared to normal minimization methods. CLOOP was used to construct the conformations of 4-, 8-, and 12-residue loops in Fiser's test set. The average main-chain root-mean-square deviations obtained in 1,000 trials for the 10 different loops of each size are 0.33, 1.27, and 2.77 A, respectively. CLOOP can build all-atom loop conformations with a sampling accuracy comparable with previous loop main-chain construction algorithms. [Figure: see text].

Algorithms↗
PubMed1985

Loop arrays in mouse brain demonstrated with antisera to cytokeratins and monoclonal antibodies to several classes of intermediate filaments: strain differences and developmental expression.

Some monoclonal antibodies raised against mouse brain antigens display a novel loop array apparently localized within the cytoplasm of neurons in fresh frozen sections of adult mouse brain. By indirect immunofluorescence, these loops are detectable in the cerebral cortex, thalamus, brainstem, and are particularly striking in association with pyramidal neurons of the hippocampus. The loops are also seen with polyclonal antibodies to the cytokeratin class of intermediate filaments. The antibodies which react with these loops also react with ependymal cells. Western blot analysis of crude insoluble cytoskeletal components of mouse brain with antibodies of cytokeratins confirm the presence of reactive bands in the range of 40-60 kdalton, appropriate in molecular weight for this class of cytoskeletal filaments. This evidence suggests that the loops share antigenic determinants with non-neural cytokeratins. During development, immunoreactive structures are first seen as small punctate or curvilinear profiles, which change into a loop array at approximately 14 days postnatal age in several mouse strains. However, in 8 of 15 different mouse strains, these immature punctate profiles remain without morphological alteration to loops throughout adult age. The F1 crosses between strains with and without the loops develop loops, but on average they are of smaller size than in the positive parent.

Animals↗
PubMed1989

The effect of postplacental insertion of the spiked and the standard Lippes loop on uterine involution.

The spiked Lippes loop was inserted in 53 women and the standard loop in 55 women immediately following the delivery of the placenta after normal vaginal delivery. A group of 50 recently delivered women with no IUD was studied as a control. The cases were followed up for 1 year. Ultrasonic scanning was performed during the 6th week after insertion for 47 cases with the spiked loop and 43 cases with the standard loop. All loops were correctly positioned within the uterine cavity except in two cases; one using the spiked and the other using the standard loop. The loops were found displaced downwards and as there was excessive bleeding, the devices were removed. Regarding the effect of the IUD on uterine involution, it was found that the fundus to internal os length for the cases with IUDs was shorter than that among the controls, also this length among the standard loop users was significantly shorter than that of the controls (P less than 0.01). Postplacental IUD insertion did not delay uterine involution, on the contrary, it helped involution. Regarding the effect of lactation, the fundus to internal os length for the fully lactating women either using a loop or not, was found to be significantly shorter than that of the non-lactating cases (P less than 0.002).

Adult↗
PubMed1992

Taxonomy and conformational analysis of loops in proteins.

We propose a general classification scheme for loops, aperiodic segments of protein structure. In an effort to avoid the geometric complexity created by non-repeating phi psi angles, a morphologic definition that focuses upon the linearity and planarity of loops is utilized. Out of 432 loops (4 to 20 residues in length) extracted from 67 proteins, 205 are classified as linear (straps), 133 as non-linear and planar (omegas), and 86 as non-linear and non-planar (zetas). The remaining 8 are classified as compound loops because they contain a combination of strap, omega, and zeta morphologies. We introduce a structural alphabet as a shorthand notation for describing local conformation. The symbols of this alphabet are based on the virtual dihedral angle joining four consecutive alpha carbons. The notation is used to provide a compact description of loop motifs in phosphate binding and calcium binding proteins. Since similar loop conformations form similar "words", the structural sequence facilitates the search for common structural motifs in a family of loops. Contrary to the view of loops as "random coils", we find loops to have positional preferences for amino acid residues analogous to those previously described for beta-turns.

Amino Acid Sequence↗
PubMed2005

Is ambulatory monitoring for "community-acquired" syncope economically attractive? A cost-effectiveness analysis of a randomized trial of external loop recorders versus Holter monitoring.

BACKGROUND: Out patient ambulatory monitoring is often performed in patients with syncope that present in the primary care setting to include or exclude an arrhythmia. The cost-effectiveness of 2 monitoring strategies was assessed in a prospective randomized trial. METHODS: One hundred patients referred for ambulatory monitoring with syncope or presyncope were randomized to a 1-month external loop recorder (n = 49) or 48-hour Holter monitor (n = 51). Patients were offered crossover if there was failed activation or no symptom recurrence. The primary end point was symptom-rhythm correlation during monitoring. Direct costs were calculated based on the 2003 Ontario Health Insurance Plan fee schedule, combined with calculation of labor, materials, service, and overhead for diagnostic testing and related equipment. RESULTS: Before enrollment, the cost of all previous health care resource use was USD 472 +/- USD 397 (range USD 21-USD 1965). In the loop recorder group, 63% of patients had symptom recurrence and successful activation, compared with 24% in the Holter group (P < .0001). The cost per Holter was USD 177.64, and per loop recorder, USD 533.56, with a similar cost per diagnosis with the 2 techniques. The incremental cost-effectiveness ratio of the loop recorder was USD 901.74 per extra successful diagnosis. A strategy of Holter followed by offered loop recorder trended toward lower cost than initial loop recorder followed by Holter (USD 481 +/- USD 267 vs USD 551 +/- USD 83, P = .08), but was associated with a lower overall diagnostic yield (49% vs 63%) and a resultant higher cost per diagnosis (USD 982 vs USD 871, P = .08). Bootstrapping suggested that 90% of incremental cost-effectiveness ratios were less than USD 1250. CONCLUSION: Despite the increased upfront cost of external loop recorders, the marked improvement in diagnostic yield offsets the cost. External loop recorders are an economically attractive alternative. First-line use of external loop recorders in patients with "community-acquired" syncope and presyncope should be considered to optimize diagnostic yield given its value.

Cost-Benefit Analysis↗
PubMed2006

Quantitative characterization of morphological polymorphism of handwritten characters loops.

A methodology based on Fourier descriptors that was previously validated has been applied to 13 writers in order to quantify the polymorphism degree of the shape of the loops of the handwritten characters a, d, o and q. In a first step, the discriminating power of the parameters extracted from these letters was investigated. The loop of the letter d appeared to be the most discriminant with a correct classification rate of 82.4%, whereas the least discriminant one was the loop of the letter o (69.7%). The second aim of the study was to extract grouping characteristics which make it possible to discriminate between writer sets, whatever the letter. Trends in the writing of loops could effectively be shown: the 13 writers of the study were separated into five main groups according to the shape and surface of their loops. The most discriminating features between the writer groups were the importance of the loops elongation and the surface of the loops. Finally, the differences between writers belonging to distinct groups could be characterized more precisely, and differences between writers belonging to the same group were revealed; the individual writings were distinguished by the variability of the parameters of shape and surface of their loops and the morphological distances between its different letters. The correct classification rates reached in this study suggest that carrying out an expertise of fragmentary samples of handwriting comprising only some loops is completely possible.

Journal Article↗
PubMed2004

Dynamics of DNA loop capture by the SfiI restriction endonuclease on supercoiled and relaxed DNA.

The SfiI endonuclease is a prototype for DNA looping. It binds two copies of its recognition sequence and, if Mg(2+) is present, cuts both concertedly. Looping was examined here on supercoiled and relaxed forms of a 5.5 kb plasmid with three SfiI sites: sites 1 and 2 were separated by 0.4 kb, and sites 2 and 3 by 2.0 kb. SfiI converted this plasmid directly to the products cut at all three sites, though DNA species cleaved at one or two sites were formed transiently during a burst phase. The burst revealed three sets of doubly cut products, corresponding to the three possible pairings of sites. The equilibrium distribution between the different loops was evaluated from the burst phases of reactions initiated by adding MgCl(2) to SfiI bound to the plasmid. The short loop was favored over the longer loops, particularly on supercoiled DNA. The relative rates for loop capture were assessed after adding SfiI to solutions containing the plasmid and MgCl(2). On both supercoiled and relaxed DNA, the rate of loop capture across 0.4 kb was only marginally faster than over 2.0 kb or 2.4 kb. The relative strengths and rates of looping were compared to computer simulations of conformational fluctuations in DNA. The simulations concurred broadly with the experimental data, though they predicted that increasing site separations should cause a shallower decline in the equilibrium constants than was observed but a slightly steeper decline in the rates for loop capture. Possible reasons for these discrepancies are discussed.

Computer Simulation↗
PubMed2024

Cell-type-specific loops linked to RNA polymerase II elongation in human neural differentiation.

DNA is folded into higher-order structures that shape and are shaped by genome function. The role of long-range loops in the establishment of new gene expression patterns during cell fate transitions remains poorly understood. Here, we investigate the link between cell-specific loops and RNA polymerase II (RNA Pol II) during neural lineage commitment. We find thousands of loops decommissioned or gained de novo upon differentiation of human induced pluripotent stem cells (hiPSCs) to neural progenitor cells (NPCs) and post-mitotic neurons. During hiPSC-to-NPC and NPC-to-neuron transitions, genes changing from RNA Pol II initiation to elongation are >4-fold more likely to anchor cell-specific loops than repressed genes. Elongated genes exhibit significant mRNA upregulation when connected in cell-specific promoter-enhancer loops but not invariant promoter-enhancer loops or promoter-promoter loops or when unlooped. Genes transitioning from repression to RNA Pol II initiation exhibit a slight mRNA increase independent of loop status. Our data link cell-specific loops and robust RNA Pol II-mediated elongation during neural cell fate transitions.

Humans↗
PubMed2003

Conformational dynamics of the active site loop of S-adenosylmethionine synthetase illuminated by site-directed spin labeling.

S-adenosylmethionine synthetase (ATP: L-methionine S-adenosyltransferase, methionine adenosyltransferase, a.k.a. MAT) is one of numerous enzymes that have a flexible polypeptide loop that moves to gate access to the active site in a motion that is closely coupled to catalysis. Crystallographic studies of this tetrameric enzyme have shown that the loop is closed in the absence of bound substrates. However, the loop must open to allow substrate binding and a variety of data indicate that the loop is closed during the catalytic steps. Previous kinetic studies indicate that during turnover loop motion occurs on a time scale of 10(-2)s, ca. 10-fold faster than chemical transformations and turnover. Site-directed spin labeling has been used to introduce nitroxide groups at two positions in the loop to illuminate how the motion of the loop is affected by substrate binding. The two loop mutants constructed, G105C and D107C, retain wild type levels of MAT activity; attachment of a methanethiosulfonate spin label to convert the cysteine to the "R1" residue reduced the k(cat) only for the labeled D107R1 form (7-fold). The K(m) value for methionine increased 2- to 4-fold for the cysteine mutants and 2- to 7-fold for the labeled proteins, whereas the K(m) for ATP was changed by at most 2-fold. EPR spectra for both labeled proteins are nearly identical and show the presence of two major spin label environments with rotational diffusion rates differing by approximately 10-fold; the slower rate is ca. 4-fold faster than the estimated protein rotational rate. The spectra are not altered by addition of substrates or products. At both positions the less mobile conformation constitutes ca. 65% of the total species, indicating an equilibrium that only slightly favors one form, that in which the label is more immobilized. The equilibrium constant that relates the two forms is comparable to the equilibrium constant of 1.5 for a conformational change that was previously deduced from the viscosity dependence of the rate of AdoMet formation. The results suggest that the motion of the loop may be an intrinsic property of the protein and not be strictly ligand modulated.

Cysteine↗
PubMed2003

Fluorescence resonance energy transfer over approximately 130 basepairs in hyperstable lac repressor-DNA loops.

Lac repressor (LacI) binds two operator DNA sites, looping the intervening DNA. DNA molecules containing two lac operators bracketing a sequence-directed bend were previously shown to form hyperstable LacI-looped complexes. Biochemical studies suggested that orienting the operators outward relative to the bend direction (in construct 9C14) stabilizes a positively supercoiled closed form, with a V-shaped LacI, but that the most stable loop construct (11C12) is a more open form. Here, fluorescence resonance energy transfer (FRET) is measured on DNA loops, between fluorescein and TAMRA attached near the two operators, approximately 130 basepairs apart. For 9C14, efficient LacI-induced energy transfer ( approximately 74% based on donor quenching) confirms that the designed DNA shape can force the looped complex into a closed form. From enhanced acceptor emission, correcting for observed donor-dependent quenching of acceptor fluorescence, approximately 52% transfer was observed. Time-resolved FRET suggests that this complex exists in both closed- and open form populations. Less efficient transfer, approximately 10%, was detected for DNA-LacI sandwiches and 11C12-LacI, consistent with an open form loop. This demonstration of long-range FRET in large DNA loops confirms that appropriate DNA design can control loop geometry. LacI flexibility may allow it to maintain looping with other proteins bound or under different intracellular conditions.

Bacterial Proteins↗
PubMed1993

Gating of the active site of triose phosphate isomerase: Brownian dynamics simulations of flexible peptide loops in the enzyme.

The enzyme triose phosphate isomerase has flexible peptide loops at its active sites. The loops close over these sites upon substrate binding, suggesting that the dynamics of the loops could be of mechanistic and kinetic importance. To investigate these issues, the loop motions in the dimeric enzyme were simulated by Brownian dynamics. The two loops, one on each monomer, were represented by linear chains of appropriately parameterized spheres, each sphere corresponding to an amino acid residue. The loops moved in the electrostatic field of the rest of the enzyme, which was held rigid in its crystallographically observed conformation. In the absence of substrate, the loops exhibited gating of the active site with a period of about 1 ns and occupied "closed" conformations for about half of the time. As the period of gating is much shorter than the enzyme-substrate relaxation time, the motion of the loops does not reduce the rate constant for the approach of substrate from its simple diffusion-controlled value. This suggests that the flexible loops may have evolved to create the appropriate environment for catalysis while, at the same time, minimizing the kinetic penalty for gating the active site.

Amino Acid Sequence↗
PubMed2003

Elucidation of the nature of the conformational changes of the EF-interhelical loop in bacteriorhodopsin and of the helix VIII on the cytoplasmic surface of bovine rhodopsin: a time-resolved fluorescence depolarization study.

The conformation of the AB-loop and EF-loop of bacteriorhodopsin and of the fourth cytoplasmic loop (helix VIII) of bovine rhodopsin were assessed by a combination of time-resolved fluorescence depolarization and site-directed fluorescence labeling. The fluorescence anisotropy decays were measured employing a tunable Ti:sapphire laser/microchannel plate based single-photon counting apparatus with picosecond time resolution. This method allows measurement of the diffusional dynamics of the loops directly on a nanosecond time-scale. We implemented the method to study model peptides and two-helix systems representing sequences of bacteriorhodopsin. Thus, we systematically analyzed the anisotropic behavior of four different fluorescent dyes covalently bound to a single cysteine residue on the protein surface and assigned the anisotropy decay components to the modes of motion of the protein and its segments. We have identified two mechanisms of loop conformational changes in the functionally intact proteins bacteriorhodopsin and bovine rhodopsin. First, we found a surface potential-dependent transition between two conformational states of the EF-loop of bacteriorhodopsin, detected with the fluorescent dye bound to position 160. A transition between the two conformational states at 150mM KCl and 20 degrees C requires a surface potential change that corresponds to Deltasigma approximately -1.0e(-)/bacteriorhodopsin molecule. We suggest, that the surface potential-based switch of the EF-loop is the missing link between the movement of helix F and the transient surface potential change detected during the photocycle of bacteriorhodopsin. Second, in the visual pigment rhodopsin, with the fluorescent dye bound to position 316, a particularly striking pH-dependent conformational change of the fourth loop on the cytoplasmic surface was analyzed. The loop mobility increased from pH 5 to 8. The midpoint of this transition is at pH 6.2 and correlates with the midpoint of the pH-dependent equilibrium between the active metarhodopsin II and the inactive metarhodopsin I state.

Animals↗
PubMed2003

Engineering the exo-loop of Trichoderma reesei cellobiohydrolase, Cel7A. A comparison with Phanerochaete chrysosporium Cel7D.

The exo-loop of Trichoderma reesei cellobiohydrolase Cel7A forms the roof of the active site tunnel at the catalytic centre. Mutants were designed to study the role of this loop in crystalline cellulose degradation. A hydrogen bond to substrate made by a tyrosine at the tip of the loop was removed by the Y247F mutation. The mobility of the loop was reduced by introducing a new disulphide bridge in the mutant D241C/D249C. The tip of the loop was deleted in mutant Delta(G245-Y252). No major structural disturbances were observed in the mutant enzymes, nor was the thermostability of the enzyme affected by the mutations. The Y247F mutation caused a slight k(cat) reduction on 4-nitrophenyl lactoside, but only a small effect on cellulose hydrolysis. Deletion of the tip of the loop increased both k(cat) and K(M) and gave reduced product inhibition. Increased activity was observed on amorphous cellulose, while only half the original activity remained on crystalline cellulose. Stabilisation of the exo-loop by the disulphide bridge enhanced the activity on both amorphous and crystalline cellulose. The ratio Glc(2)/(Glc(3)+Glc(1)) released from cellulose, which is indicative of processive action, was highest with Tr Cel7A wild-type enzyme and smallest with the deletion mutant on both substrates. Based on these data it seems that the exo-loop of Tr Cel7A has evolved to facilitate processive crystalline cellulose degradation, which does not require significant conformational changes of this loop.

Amino Acid Sequence↗
PubMed1999

Forearm loop, upper arm straight, and brachial-internal jugular vein dialysis grafts: a comparison study of graft survival utilizing a combined percutaneous endovascular and surgical maintenance approach.

PURPOSE: To determine rates and duration of patency achievable in forearm loop, upper arm straight, and brachial-internal jugular (IJ) vein hemodialysis grafts utilizing a combined percutaneous endovascular and surgical maintenance approach. MATERIALS AND METHODS: A retrospective analysis of 74 hemodialysis grafts (forearm loop, n = 22; upper arm straight, n = 34; and brachial-IJ vein, n = 18) in 50 patients with end-stage renal disease was conducted. Operative notes, interventional procedural reports, and hospital records were used to construct a history for each of these grafts from the time of surgical placement until the time the graft was abandoned for an alternative method of dialysis. All procedures performed to maintain and/or restore patency during the usable lifetime of the grafts were documented. RESULTS: Survival analysis using the Kaplan-Meier method demonstrated the following probabilities of primary patency at 6, 12, and 16 months, respectively: forearm loop graft = .46, .26, and .26; upper arm straight graft = .39, .22, and .16; and brachial-IJ vein graft = .19, .06, and .06 (forearm loop vs upper arm straight grafts, P > .05; forearm loop and upper arm straight vs brachial-IJ grafts, P < .001, P < .001, respectively). The probabilities of secondary patency at 12, 24, and 48 months, respectively, were: forearm loop graft = .89, .30, and NA; upper arm straight graft = .52, .35, and .17; and brachial-IJ vein graft = .54, .42, and .21 (P < .05 for all three comparisons: forearm loop > brachial-IJ > upper arm straight). Six percutaneous and two surgical procedures were compared and no significant differences in utilization were determined among the three graft types (ANOVA, P range, .38-.88). CONCLUSION: Kaplan-Meier analysis determined the probability of primary patency for forearm loop grafts to be similar to that for upper arm straight grafts, and both were significantly greater than for brachial-IJ vein grafts. The secondary patency rates for forearm loop grafts are greater than for upper arm and brachial-IJ vein grafts, while that for the brachial-IJ vein graft is greater than the upper arm straight graft. Utilization of interventional and surgical resources required to maintain patency do not significantly differ among the three types of upper extremity hemodialysis grafts.

Arm↗
PubMed1993

Theoretical predictions of DNA hairpin loop conformations: correlations with thermodynamic and spectroscopic data.

A computational procedure for generating conformations of DNA hairpin loop structures from a broad range of low-energy starting states is described. The starting point of the modeling is the distribution of oligonucleotide chain conformations obtained from Monte Carlo simulations of feasible dinucleotide steps. Structures which meet the spatial criteria for hairpin loop formation are selected from the distributions and subsequently minimized using all-atom molecular mechanics. Both d(CTnG) and d(CAnG) oligomers, where n = 3, 4, or 5, are modeled. These sequences are chosen because of the large number of published NMR and thermodynamic studies on DNA hairpins containing thymine or adenine residues. The minimized three-dimensional hairpin loop structures are compared with one another as well as analyzed in terms of available experimental data. The computational approach provides the first detailed analysis of DNA hairpin loop structure in terms of a multistate conformational model. Investigation of the minimized conformations reveals several interesting structural features. First, hairpin loops of the same sequence adopt several distinctly different conformations, as opposed to minor variants of the same equilibrium structure, that could potentially interconvert in solution. Second, in contrast to double-helical nucleic acids, the hairpin loop models exhibit hydrophobic and hydrophilic surfaces. The different disposition of hydrophobic groups in loops versus duplexes could modulate both protein-nucleic acid interactions and nucleic acid self-associations. Third, perpendicular aromatic interactions of loop residues are observed in many of the computed hairpins. This sort of interaction might be important in the stabilization of non-hydrogen-bonded nucleic acid secondary and tertiary structures. The predicted structural features in the models help, in addition, to account for the unusual thermodynamic properties of DNA hairpin loops. Comparison of the theoretically-generated NOEs in different structures further reveals that very different molecular structures and interactions can, in principle, produce the same NOEs. The multistate description suggested by this observation differs from the conventional interpretation of DNA solution structure in terms of the fluctuations about a single preferred chain conformation. There is not necessarily only one set of closely related structures consistent with the observed data.

Base Sequence↗
PubMed1993

Structural features of an RNA containing the CUGGGA loop of the human immunodeficiency virus type 1 trans-activation response element.

A 19-nucleotide RNA containing the CUGGGA loop sequence corresponding to nucleotides 30-35 of the HIV-1 trans-activation response element (TAR) was synthesized in vitro and analyzed by biochemical methods and one- and two-dimensional NMR spectroscopy. Diagnostic RNase cleavage patterns were similar for the loops in the full-length HIV-1 TAR and the 19-nucleotide RNA, indicating that they are similar in structure. NMR data showed that the loop is stabilized by base-stacking interactions. The first loop nucleotide is stacked upon the A-helical stem, and the loop uridine is stacked upon this cytosine. On the opposite side of the loop, the third loop guanosine is stacked upon the adenosine, which is stacked upon the stem. No specific Watson-Crick or non-Watson-Crick base pairing across the loop was identified. Unusually short interribose distances indicate a significant distortion of the sugar-phosphate backbone centered at the adenosine. Relatively short NMR relaxation times for protons of the adenosine and its adjacent guanosine, as well as rapidly exchanging imino protons, provide evidence for dynamic processes occurring in the loop.

Base Sequence↗
PubMed2001

Solution structure of the second extracellular loop of human thromboxane A2 receptor.

Thromboxane A(2) receptor (TP receptor), a prostanoid receptor, belongs to the G protein-coupled receptor family, composed of three intracellular loops and three extracellular loops connecting seven transmembrane helices. The highly conserved extracellular domains of the prostanoid receptors were found in the second extracellular loop (eLP(2)), which was proposed to be involved in ligand recognition. The 3D structure of the eLP(2) would help to further explain the ligand binding mechanism. Analysis of the human TP receptor model generated from molecular modeling based on bacteriorhodopsin crystallographic structure indicated that about 12-14 A separates the N- and C-termini of the extra- and intracellular loops. Synthetic loop peptides whose termini are constrained to this separation are presumably more likely to mimic the native loop structure than the corresponding loop region peptide with unrestricted ends. To test this new concept, a peptide corresponding to the eLP(2) (residues 173-193) of the TP receptor has been made with the N- and C-termini connected by a homocysteine disulfide bond. Through 2D nuclear magnetic resonance (NMR) experiments, complete (1)H NMR assignments, and structural construction, the overall 3D structure of the peptide was determined. The structure shows two beta-turns at residues 180 and 185. The distance between the N- and C-termini of the peptide shown in the NMR structure is 14.2 A, which matched the distance (14.5 A) between the two transmembrane helices connecting the eLP(2) in the TP receptor model. This suggests that the approach using the constrained loop peptides greatly increases the likelihood of solving the whole 3D structures of the extra- and the intracellular domains of the TP receptor. This approach may also be useful in structural studies of the extramembrane loops of other G protein-coupled receptors.

Circular Dichroism↗
PubMed2001

Role of the flexible loop of hypoxanthine-guanine-xanthine phosphoribosyltransferase from Tritrichomonas foetus in enzyme catalysis.

The hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRTase), a type I PRTase, from Tritrichomonas foetus, is a potential target for antitritrichomonal chemotherapy. Structural data on all the type I PRTases reveal a highly flexible, 11-14-amino acid loop, presumably covering the active site. With the exception of a highly conserved Ser-Tyr dipeptide, the other amino acids constituting the loop vary widely among different PRTases. The roles of the conserved Ser73 and Tyr74 residues in the loop and the dynamics of the loop in T. foetus HGXPRTase were investigated using site-directed mutants, stop-flow kinetics, chemical modification, and two-dimensional (1)H-(15)N heteronuclear NMR relaxation experiments. S73A, Y74F, and Y74E mutants of HGXPRTase exhibited a 5-7-fold increase in K(m) for guanine and a 3-5-fold increase in K(m) for PRPP compared to that of the wild type, reflecting the decreased affinity of binding for the two substrates. The k(cat)'s for these mutant-catalyzed reactions, however, do not change appreciably from that of the wild-type enzyme. Stopped-flow fluorescence with a Y74W mutant showed no apparent quenching by adding either PRPP or GMP alone. When both PRPP and guanine were added together, however, the fluorescence was rapidly quenched, followed by a slow recovery as the enzyme-catalyzed reaction progressed, suggesting movement of the loop during catalysis. In the presence of 9-deazaguanine and PRPP, the rapidly quenched fluorescence was not recovered, suggesting a closed loop form. The accessibility of Trp74 in the flexible loop of the mutant enzyme was also analyzed using N-bromosuccinimide (NBS), which reacts specifically with the tryptophan residue. NBS reacted with the only tryptophan in the Y74W mutant enzyme and rendered the enzyme inactive. GMP or PRPP alone failed to protect the enzyme from NBS inactivation. However, the presence of both 9-deazaguanine and PPRP protected the enzyme, allowing it to retain up to 70% of its activity. An S75H mutant, labeled with [(15)N]histidine, was used in the (1)H-(15)N NMR study. Spectra obtained in the presence of enzyme substrates indicated an apparent stabilization of the loop only in the presence of 9-deazaguanine and PRPP. These experimental results thus clearly demonstrated stabilization of the flexible loop upon binding of both PRPP and guanine and suggested its involvement in enzyme catalysis.

Amino Acid Sequence↗