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PubMed1999

Protein loops on structurally similar scaffolds: database and conformational analysis.

A general problem in comparative modeling and protein design is the conformational evaluation of loops with a certain sequence in specific environmental protein frameworks. Loops of different sequences and structures on similar scaffolds are common in the Protein Data Bank (PDB). In order to explore both structural and sequential diversity of them, a data base of loops connecting similar secondary structure fragments is constructed by searching the data base of families of structurally similar proteins and PDB. A total of 84 loop families having 2-13 residues are found among the well-determined structures of resolution better than 2.5 A. Eight alpha-alpha, 20 alpha-beta, 19 beta-alpha, and 37 beta-beta families are identified. Every family contains more than 5 loop motifs. In each family, no loops share same sequence and all the frameworks are well superimposed. Forty-three new loop classes are distinguished in the data base. The structural variability of loops in homologous proteins are examined and shown in 44 families. Motif families are characterized with geometric parameters and sequence patterns. The conformations of loops in each family are clustered into subfamilies using average linkage cluster analysis method. Information such as geometric properties, sequence profile, sequential and structural variability in loop, structural alignment parameters, sequence similarities, and clustering results are provided. Correlations between the conformation of loops and loop sequence, motif sequence, and global sequence of PDB chain are examined in order to find how loop structures depend on their sequences and how they are affected by the local and global environment. Strong correlations (R > 0.75) are only found in 24 families. The best R value is 0.98. The data base is available through the Internet.

Amino Acid Sequence↗
PubMed1995

Modeling protein loops using a phi i + 1, psi i dimer database.

We present an automated method for modeling backbones of protein loops. The method samples a database of phi i + 1 and psi i angles constructed from a nonredundant version of the Protein Data Bank (PDB). The dihedral angles phi i + 1 and psi i completely define the backbone conformation of a dimer when standard bond lengths, bond angles, and a trans planar peptide configuration are used. For the 400 possible dimers resulting from 20 natural amino acids, a list of allowed phi i + 1, psi i pairs for each dimer is created by pooling all such pairs from the loop segments of each protein in the nonredundant version of the PDB. Starting from the N-terminus of the loop sequence, conformations are generated by assigning randomly selected pairs of phi i + 1, psi i for each dimer from the respective pool using standard bond lengths, bond angles, and a trans peptide configuration. We use this database to simulate protein loops of lengths varying from 5 to 11 amino acids in five proteins of known three-dimensional structures. Typically, 10,000-50,000 models are simulated for each protein loop and are evaluated for stereochemical consistency. Depending on the length and sequence of a given loop, 50-80% of the models generated have no stereochemical strain in the backbone atoms. We demonstrate that, when simulated loops are extended to include flanking residues from homologous segments, only very few loops from an ensemble of sterically allowed conformations orient the flanking segments consistent with the protein topology. The presence of near-native backbone conformations for loops from five different proteins suggests the completeness of the dimeric database for use in modeling loops of homologous proteins. Here, we take advantage of this observation to design a method that filters near-native loop conformations from an ensemble of sterically allowed conformations. We demonstrate that our method eliminates the need for a loop-closure algorithm and hence allows for the use of topological constraints of the homologous proteins or disulfide constraints to filter near-native loop conformations.

Amino Acid Sequence↗
PubMed2003

Solvation parameters for predicting the structure of surface loops in proteins: transferability and entropic effects.

A new procedure for optimizing parameters of implicit solvation models introduced by us has been applied successfully first to cyclic peptides and more recently to three surface loops of ribonuclease A (Das and Meirovitch, Proteins 2001;43:303-314) using the simplified model E(tot) = E(FF)(epsilon = nr) + Sigma(i) sigma(i)A(i), where sigma(i) are atomic solvation parameters (ASPs) to be optimized, A(i) is the solvent accessible surface area of atom i, E(FF)(epsilon = nr) is the AMBER force-field energy of the loop-loop and loop-template interactions with a distance-dependent dielectric constant, epsilon = nr, where n is a parameter. The loop is free to move while the protein template is held fixed in its X-ray structure; an extensive conformational search for energy minimized loop structures is carried out with our local torsional deformation method. The optimal ASPs and n are those for which the structure with the lowest minimized energy [E(tot)(n,sigma(i))] becomes the experimental X-ray structure, or less strictly, the energy gap between these structures is within 2-3 kcal/mol. To check if a set of ASPs can be defined, which is transferable to a large number of loops, we optimize individual sets of ASPs (based on n = 2) for 12 surface loops from which an "averaged" best-fit set is defined. This set is then applied to the 12 loops and an independent "test" group of 8 loops leading in most cases to very small RMSD values; thus, this set can be useful for structure prediction of loops in homology modeling. For three loops we also calculate the free energy gaps to find that they are only slightly smaller than their energy counterparts, indicating that only larger n will enable reducing too large gaps. Because of its simplicity, this model allowed carrying out an extensive application of our methodology, providing thereby a large number of benchmark results for comparison with future calculations based on n > 2 as well as on more sophisticated solvation models with as yet unknown performance for loops.

Amino Acid Sequence↗
PubMed2004

Characterization of palindromic loop mismatch repair tracts in mammalian cells.

Single- and multi-base (loop) mismatches can arise in DNA by replication errors, during recombination, and by chemical modification of DNA. Single-base and loop mismatches of several nucleotides are efficiently repaired in mammalian cells by a nick-directed, MSH2-dependent mechanism. Larger loop mismatches (> or =12 bases) are repaired by an MSH2-independent mechanism. Prior studies have shown that 12- and 14-base palindromic loops are repaired with bias toward loop retention, and that repair bias is eliminated when five single-base mismatches flank the loop mismatch. Here we show that one single-base mismatch near a 12-base palindromic loop is sufficient to eliminate loop repair bias in wild-type, but not MSH2-defective mammalian cells. We also show that palindromic loop and single-base mismatches separated by 12 bases are repaired independently at least 10% of the time in wild-type cells, and at least 30% of the time in MSH2-defective cells. Palindromic loop and single-base mismatches separated by two bases were never repaired independently. These and other data indicate that loop repair tracts are variable in length. All tracts extend at least 2 bases, some extend <12 bases, and others >12 bases, on one side of the loop. These properties distinguish palindromic loop mismatch repair from the three known excision repair pathways: base excision repair which has one to six base tracts, nucleotide excision repair which has approximately 30 base tracts, and MSH2-dependent mismatch repair, which has tracts that extend for several hundred bases.

Animals↗
PubMed2005

Loop entropy and cytochrome c stability.

The loop entropy model proposes that loop closure in a protein becomes entropically more costly as the length of the loop increases. A model protein, cytochrome c, is composed of four loops connecting five helices surrounding a heme-containing core. To test the loop entropy model a series of mutant proteins are constructed with (Gly)n or (Thr)n segments (n = 4-20) inserted between Gly23 and Gly24 of omega loop A of a pseudo wild-type reference protein. Scanning calorimetry shows that protein stability decreases as n increases in the (Gly)n or (Thr)n segment. The dependence of stability on loop length is analyzed with the loop entropy model. Fitting to the model gives a quantitative description of stability differences for the mutant proteins, but with a smaller power-dependence of the probability of loop closure (c-value) than expected from polymer theory. A possible explanation for the discrepancy is that thermodynamically unfavorable loop entropy is partially offset by interactions between the inserted homopolymer and flanking heteropolymer portions of the unfolded protein. The interactions may involve molecular crowding that favors coalescence of the heteropolymer at the insert site and thus closure of the homopolymer loops, possibly as an aspect of the folding code. This may allow use of loop insert mutants to assess the strength of the heteropolymer-encoded folding signals that facilitate loop closure at the insert site.

Amino Acid Sequence↗
PubMed1997

Does conformational free energy distinguish loop conformations in proteins?

Limitations in protein homology modeling often arise from the inability to adequately model loops. In this paper we focus on the selection of loop conformations. We present a complete computational treatment that allows the screening of loop conformations to identify those that best fit a molecular model. The stability of a loop in a protein is evaluated via computations of conformational free energies in solution, i.e., the free energy difference between the reference structure and the modeled one. A thermodynamic cycle is used for calculation of the conformational free energy, in which the total free energy of the reference state (i.e., gas phase) is the CHARMm potential energy. The electrostatic contribution of the solvation free energy is obtained from solving the finite-difference Poisson-Boltzmann equation. The nonpolar contribution is based on a surface area-based expression. We applied this computational scheme to a simple but well-characterized system, the antibody hypervariable loop (complementarity-determining region, CDR). Instead of creating loop conformations, we generated a database of loops extracted from high-resolution crystal structures of proteins, which display geometrical similarities with antibody CDRs. We inserted loops from our database into a framework of an antibody; then we calculated the conformational free energies of each loop. Results show that we successfully identified loops with a "reference-like" CDR geometry, with the lowest conformational free energy in gas phase only. Surprisingly, the solvation energy term plays a confusing role, sometimes discriminating "reference-like" CDR geometry and many times allowing "non-reference-like" conformations to have the lowest conformational free energies (for short loops). Most "reference-like" loop conformations are separated from others by a gap in the gas phase conformational free energy scale. Naturally, loops from antibody molecules are found to be the best models for long CDRs (> or = 6 residues), mainly because of a better packing of backbone atoms into the framework of the antibody model.

Antibodies↗
PubMed1997

Is review of videotape necessary after review of digitized cine-loop images in stress echocardiography? A prospective study in 306 patients.

The interpretation of stress echocardiography has been made easier by the comparison of digitized prestress and poststress frame-grabbed images (cine-loops), each representing a portion of a single cardiac cycle. Often, review of these digitized images is substituted for review of the complete videotape record of the examination. An alternative is to review both the digitized images as well as the videotape record of the rest and stress images. To date, there has been insufficient documentation of whether these options (cine-loop images alone versus cine-loop images plus videotape) provide comparable or additive information. Therefore, we prospectively evaluated information obtained from review of cine-loop images versus combined review of cine-loop images and videotape records in 306 consecutive patients undergoing treadmill (213 patients, 70%) or dobutamine (93 patients, 30%) stress echocardiography. An experienced echocardiologist first reviewed the cine-loop images and scored the wall motion in 16 segments at rest and with stress. Next, the complete videotape record was reviewed with repeated wall motion scoring. A questionnaire comparing cine-loop and videotape images was completed at the end of each review. Digitization of images was technically inadequate in 14 patients (4%). In 116 (40%) of the other 292 patients, the regional wall motion assessment, after relying solely on cine-loop images, was modified with subsequent videotape review. In 40 patients (14%), these modifications resulted in a change in the final impression regarding whether the study result was normal or abnormal. In a multivariate analysis, age, gender, and type of stress echocardiography had no significant influence on discordance of the cine-loop image and combined cine-loop and video information. Stepwise logistic regression analysis identified poorer image quality (p < 0.0001) and regional wall motion abnormalities (p < 0.0001) as predictors of discordance between cine-loop and combined review. We conclude that relying solely on digitized cine-loop images representing a single cardiac cycle is not optimal, especially if the quality of the digitized images is suboptimal and if regional wall motion abnormalities are present. Thus we recommend a combined review of both cine-loop images and videotape images in the interpretation of stress echocardiography.

Aged↗
PubMed2001

Backbone dynamics in dihydrofolate reductase complexes: role of loop flexibility in the catalytic mechanism.

To elucidate the influence of local motion of the polypeptide chain on the catalytic mechanism of an enzyme, we have measured (15)N relaxation data for Escherichia coli dihydrofolate reductase in three different complexes, representing different stages in the catalytic cycle of the enzyme. NMR relaxation data were analyzed by the model-free approach, corrected for rotational anisotropy, to provide insights into the backbone dynamics. There are significant differences in the backbone dynamics in the different complexes. Complexes in which the cofactor binding site is occluded by the Met20 loop display large amplitude motions on the picosecond/nanosecond time scale for residues in the Met20 loop, the adjacent betaF-betaG loop and for residues 67-69 in the adenosine binding loop. Formation of the closed Met20 loop conformation in the ternary complex with folate and NADP(+), results in attenuation of the motions in the Met20 loop and the betaF-betaG loop but leads to increased flexibility in the adenosine binding loop. New fluctuations on a microsecond/millisecond time scale are observed in the closed E:folate:NADP(+) complex in regions that form hydrogen bonds between the Met20 and the betaF-betaG loops. The data provide insights into the changes in backbone dynamics during the catalytic cycle and point to an important role of the Met20 and betaF-betaG loops in controlling access to the active site. The high flexibility of these loops in the occluded conformation is expected to promote tetrahydrofolate-assisted product release and facilitate binding of the nicotinamide ring to form the Michaelis complex. The backbone fluctuations in the Met20 loop become attenuated once it closes over the active site, thereby stabilizing the nicotinamide ring in a geometry conducive to hydride transfer. Finally, the relaxation data provide evidence for long-range motional coupling between the adenosine binding loop and distant regions of the protein.

Binding Sites↗
PubMed2006

NMR structure of varkud satellite ribozyme stem-loop V in the presence of magnesium ions and localization of metal-binding sites.

In the Neurospora VS ribozyme, magnesium ions facilitate formation of a loop-loop interaction between stem-loops I and V, which is important for recognition and activation of the stem-loop I substrate. Here, we present the high-resolution NMR structure of stem-loop V (SL5) in the presence of Mg(2+) (SL5(Mg)) and demonstrate that Mg(2+) induces a conformational change in which the SL5 loop adopts a compact structure with most characteristics of canonical U-turn structures. Divalent cation-binding sites were probed with Mn(2+)-induced paramagnetic line broadening and intermolecular NOEs to Co(NH(3))(6)(3+). Structural modeling of Mn(H(2)O)(6)(2+) in SL5(Mg) revealed four divalent cation-binding sites in the loop. Sites 1, 3, and 4 are located in the major groove near multiple phosphate groups, whereas site 2 is adjacent to N7 of G697 and N7 of A698 in the minor groove. Cation-binding sites equivalent to sites 1-3 in SL5 are present in other U-turn motifs, and these metal-binding sites may represent a common feature of the U-turn fold. Although magnesium ions affect the loop conformation, they do not significantly change the conformation of residues 697-699 involved in the proposed Watson-Crick base pairs with stem-loop I. In both the presence and the absence of Mg(2+), G697, A698, and C699 adopt an A-form structure that exposes their Watson-Crick faces, and this is compatible with their proposed interaction with stem-loop I. In SL5(Mg), however, U700 becomes exposed on the minor groove face of the loop in the proximity of the bases of G697, A698, and C699, suggesting that the Mg(2+)-bound conformation of stem-loop V allows additional contacts with stem-loop I. These studies improve our understanding of the role of Mg(2+) in U-turn structures and in substrate recognition by the VS ribozyme.

Base Pairing↗
PubMed1996

Structure-function relationship of lipoprotein lipase-mediated enhancement of very low density lipoprotein binding and catabolism by the low density lipoprotein receptor. Functional importance of a properly folded surface loop covering the catalytic center.

We examined the structure-function relationship of human lipoprotein lipase (hLPL) in its ability to enhance the binding and catabolism of very low density lipoproteins (VLDL) in COS cells. Untransfected COS cells did not bind to or catabolize normal VLDL. Expression of wild-type hLPL by transient transfection enhanced binding, uptake, and degradation of the VLDL (a property of LPL that we call bridge function). Heparin pretreatment and a monoclonal antibody ID7 that blocks LDL receptor-binding domain of apoE both inhibited binding, and apoE2/E2 VLDL from a Type III hyperlipidemic subject did not bind. However, LDL did not reduce 125I-VLDL binding to the hLPL-expressing cells, whereas rabbit beta-VLDL was an effective competitor. By contrast, LDL reduced uptake and degradation of 125I-VLDL to the same extent as excess unlabeled VLDL or beta-VLDL. These data suggest that binding occurs by direct interaction of VLDL with LPL but the subsequent catabolism of the VLDL is mediated by the LDL receptor. Mutant hLPLs that were catalytically inactive, S132A, S132D, as well as the partially active mutant, S251T, and S172G, gave normal enhancement of VLDL binding and catabolism, whereas the partially active mutant S172D had markedly impaired capacity for the process; thus, there is no correlation between bridge function and lipolytic activity. A naturally occurring genetic variant hLPL, S447-->Ter, has normal bridge function. The catalytic center of LPL is covered by a 21-amino acid loop that must be repositioned before a lipid substrate can gain access to the active site for catalysis. We studied three hLPL loop mutants (LPL-cH, an enzymatically active mutant with the loop replaced by a hepatic lipase loop; LPL-cP, an enzymatically inactive mutant with the loop replaced by a pancreatic lipase loop; and C216S/C239S, an enzymatically inactive mutant with the pair of Cys residues delimiting the loop substituted by Ser residues) and a control double Cys mutant, C418S/C438S. Two of the loop mutants (LPL-cH and LPL-cP) and the control double Cys mutant C418S/C438S gave normal enhancement of VLDL binding and catabolism, whereas the third loop mutant, C216S/C239S, was completely inactive. We conclude that although catalytic activity and the actual primary sequence of the loop of LPL are relatively unimportant (wild-type LPL loop and pancreatic lipase loops have little sequence similarity), the intact folding of the loop, flanked by disulfide bonds, must be maintained for LPL to express its bridge function.

Animals↗
PubMed1997

Mapping the ends of transmembrane segments in a polytopic membrane protein. Scanning N-glycosylation mutagenesis of extracytosolic loops in the anion exchanger, band 3.

Band 3, the anion exchanger of human erythrocytes, contains up to 14 transmembrane (TM) segments and has a single endogenous site of N-glycosylation at Asn642 in extracellular (EC) loop 4. The requirements for N-glycosylation of EC loops and the topology of this polytopic membrane protein were determined by scanning N-glycosylation mutagenesis and cell-free translation in a reticulocyte lysate supplemented with microsomal membranes. The endogenous and novel acceptor sites located near the middle of the 35 residue EC loop 4 were efficiently N-glycosylated; however, no N-glycosylation occurred at sites located within sharply defined regions close to the adjacent TM segments. Acceptor sites located in the center of EC loop 3, which contains 25 residues, were poorly N-glycosylated. Expansion of this loop with a 4-residue insert containing an acceptor site increased N-glycosylation. Acceptor sites located in short (<10 residues) loops (putative EC loops 1, 2, 6, and 7) were not N-glycosylated; however, insertion of EC loop 4 into EC loops 1, 2, or 7, but not 6, resulted in efficient N-glycosylation. Acceptor sites in putative intracellular (IC) loop 5 exhibited a similar pattern of N-glycosylation as EC loop 4, indicating a lumenal disposition during biosynthesis. To be efficiently N-glycosylated, EC loops in polytopic membrane proteins must be larger than 25 residues in size, with acceptor sites located greater than 12 residues away from the preceding TM segment and greater than 14 residues away from the following TM segment. Application of this requirement allowed a significant refinement of the topology of Band 3 including a more accurate mapping of the ends of TM segments. The strict distance dependence for N-glycosylation of loops suggests that TM segments in polytopic membrane proteins are held quite precisely within the translocation machinery during the N-glycosylation process.

Amino Acid Sequence↗
PubMed2004

Interaction of nick-directed DNA mismatch repair and loop repair in human cells.

In human cells, large DNA loop heterologies are repaired through a nick-directed pathway independent of mismatch repair. However, a 3'-nick generated by bacteriophage fd gene II protein heterology is not capable of stimulating loop repair. To evaluate the possibility that a mismatch near a loop could induce both repair types in human cell extracts, we constructed and tested a set of DNA heteroduplexes, each of which contains a combination of mismatches and loops. We have demonstrated that a strand break generated by restriction endonucleases 3' to a large loop is capable of provoking and directing loop repair. The repair of 3'-heteroduplexes in human cell extracts is very similar to that of 5'-heteroduplex repair, being strand-specific and highly biased to the nicked strand. This observation suggests that the loop repair pathway possesses bidirectional repair capability similar to that of the bacterial loop repair system. We also found that a nick 5' to a coincident mismatch and loop can apparently stimulate the repair of both. In contrast, 3'-nick-directed repair of a G-G mismatch was reduced when in the vicinity of a loop (33 or 46 bp between two sites). Increasing the distance separating the G-G mismatch and loop by 325 bp restored the efficiency of repair to the level of a single base-base mismatch. This observation suggests interference between 3'-nick-directed large loop repair and conventional mismatch repair systems when a mispair is near a loop. We propose a model in which DNA repair systems avoid simultaneous repair at adjacent sites to avoid the creation of double-stranded DNA breaks.

Base Pair Mismatch↗
PubMed1988

Y chromosome loops in Drosophila melanogaster.

Primary spermatocyte nuclei of fixed testes of Drosophila melanogaster exhibit three large clusters of thread-like structures, each consisting of two long, continuous, loop-shaped filaments. No comparable intranuclear structures are observed in spermatogonia, secondary spermatocytes or spermatids. The threads begin to form in young spermatocytes, grow throughout spermatocyte development, reach their maximum size in mature spermatocytes and disintegrate prior to meiotic metaphase I. The presence of each cluster of threads depends upon the presence of a specific region of the Y chromosome; when this region is deleted the cluster is absent, and when it is duplicated the cluster is also duplicated. Together these observations strongly suggest that these structures represent giant Y chromosome lampbrush-like loops analogous to those described in Drosophila hydei. Two antibodies, one polyclonal and one monoclonal, differentially react with the three loops of D. melanogaster. Moreover, two of these loops are specifically stained by Giemsa at pH 10. By indirect immunofluorescence with these antibodies followed by Giemsa staining, each loop can be unambiguously identified and its presence and normality readily assessed. This enabled us to perform fine mapping experiments to determine the relationships between the Y chromosome fertility factors and the loops. The loop-forming sites map within the kl-5, kl-3 and ks-1 fertility factors. Regions h3 and h21 of the Y chromosome correspond to the loop-forming sites of kl-5 and ks-1, respectively. Each of these regions contains about 1300 kb of DNA and spans about one-third of its locus. The loop-forming site of the kl-3 locus is coextensive with region h7-h9 which contains about 4300 kb of DNA and corresponds to the minimum physical size of this locus. These data suggest that each loop is an integral part of a different fertility factor, representing the cytological manifestation of its activity in primary spermatocytes. The kl-2, kl-1 and ks-2 fertility regions do not produce any visible intranuclear structure and do not affect the kl-5, kl-3 and ks-1 loops. Thus, these loci may either not form loops at all or produce loop-like structures that we are unable to see because they are physically minute, destroyed by our fixation procedure, or both.

Animals↗
PubMed1997

Protein engineering with monomeric triosephosphate isomerase (monoTIM): the modelling and structure verification of a seven-residue loop.

Protein engineering experiments have been carried out with loop-1 of monomeric triosephosphate isomerase (monoTIM). Loop-1 of monoTIM is disordered in every crystal structure of liganded monoTIM, but in the wild-type TIM it is a very rigid dimer interface loop. This loop connects the first beta-strand with the first alpha-helix of the TIM-barrel scaffold. The first residue of this loop, Lys13, is a conserved catalytic residue. The protein design studies with loop-1 were aimed at rigidifying this loop such that the Lys13 side chain points in the same direction as seen in wild type. The modelling suggested that the loop should be made one residue shorter. With the modelling package ICM the optimal sequence of a new seven-residue loop-1 was determined and its structure was predicted. The new variant could be expressed and purified and has been characterized. The catalytic activity and stability are very similar to those of monoTIM. The crystal structure (at 2.6 A resolution) shows that the experimental loop-1 structure agrees well with the modelled loop-1 structure. The direct superposition of the seven loop residues of the modelled and experimental structures results in an r.m.s. difference of 0.5 A for the 28 main chain atoms. The good agreement between the predicted structure and the crystal structure shows that the described modelling protocol can be used successfully for the reliable prediction of loop structures.

Amino Acid Sequence↗
PubMedDate not supplied

Biomechanical evaluation of a crimp clamp system for loop fixation of monofilament nylon leader material used for stabilization of the canine stifle joint.

OBJECTIVE: To test a crimp clamp system designed to secure monofilament nylon leader (MNL) material commonly used as lateral fabellotibial sutures (LFS) in extra-articular stabilization of the canine stifle joint. STUDY DESIGN: In vitro biomechanical tests of MNL loops secured with either the crimp clamp system or knots were performed. Suture loops (n = 94) were created from 27.3 kg tensile strength MNL and fastened with knots or crimp clamps. Tests were conducted on steam-sterilized, ethylene-oxide-sterilized, and nonsterilized MNL sutures. Loops were evaluated in single load tests and cycled tests. Values for load to failure, initial loop tension, loop elongation, mode of failure, and point of failure were determined. RESULTS: Crimp-clamped loops were superior to knotted loops in all parameters tested in both cycled and noncycled tests. Loop failure generally occurred by breaking within 3 mm of the fixation in both clamped and knotted tests. Loop elongation after cycling was greater in the knotted loops compared with clamped loops (P < .001). Load to failure was greater in clamped tests than in knotted tests (P < .001), regardless of sterilization technique used. Significantly higher initial loop tension could be achieved with the clamp system compared with knot fixation (P < .001). CONCLUSIONS: The crimp clamp system provides superior in vitro loop fixation characteristics compared with knot fixation in 27.3 kg test MNL. CLINICAL RELEVANCE: Based on the results of biomechanical testing and the known biocompatibility of the system's implant components, clinical trials using the crimp clamp system are warranted.

Animals↗
PubMed1999

QT dispersion as an attribute of T-loop morphology.

BACKGROUND: The suggestion that increased QT dispersion (QTD) is due to increased differences in local action potential durations within the myocardium is wanting. An alternative explanation was sought by relating QTD to vectorcardiographic T-loop morphology. METHODS AND RESULTS: The T loop is characterized by its amplitude and width (defined as the spatial angle between the mean vectors of the first and second halves of the loop). We reasoned that small, wide ("pathological") T loops produce larger QTD than large, narrow ("normal") loops. To quantify the relationship between QTD and T-loop morphology, we used a program for automated analysis of ECGs and a database of 1220 standard simultaneous 12-lead ECGs. For each ECG, QT durations, QTD, and T-loop parameters were computed. T-loop amplitude and width were dichotomized, with 250 microV (small versus large amplitudes) and 30 degrees (narrow versus wide loops) taken as thresholds. Over all 1220 ECGs, QTDs were smallest for large, narrow T loops (54.2+/-27.1 ms) and largest for small, wide loops (69. 5+/-33.5 ms; P<0.001). CONCLUSIONS: QTD is an attribute of T-loop morphology, as expressed by T-loop amplitude and width.

Action Potentials↗
PubMed2005

Statistical-mechanical theory of DNA looping.

The lack of a rigorous analytical theory for DNA looping has caused many DNA-loop-mediated phenomena to be interpreted using theories describing the related process of DNA cyclization. However, distinctions in the mechanics of DNA looping versus cyclization can have profound quantitative effects on the thermodynamics of loop closure. We have extended a statistical mechanical theory recently developed for DNA cyclization to model DNA looping, taking into account protein flexibility. Notwithstanding the underlying theoretical similarity, we find that the topological constraint of loop closure leads to the coexistence of multiple classes of loops mediated by the same protein structure. These loop topologies are characterized by dramatic differences in twist and writhe; because of the strong coupling of twist and writhe within a loop, DNA looping can exhibit a complex overall helical dependence in terms of amplitude, phase, and deviations from uniform helical periodicity. Moreover, the DNA-length dependence of optimal looping efficiency depends on protein elasticity, protein geometry, and the presence of intrinsic DNA bends. We derive a rigorous theory of loop formation that connects global mechanical and geometric properties of both DNA and protein and demonstrates the importance of protein flexibility in loop-mediated protein-DNA interactions.

Binding Sites↗
PubMed1992

Human lipoprotein lipase: the loop covering the catalytic site is essential for interaction with lipid substrates.

Lipoprotein lipase (LPL), a key enzyme which initiates the hydrolysis of triglycerides present in chylomicrons and very low density lipoproteins, consists of multiple functional domains which are necessary for normal activity. The catalytic domain of LPL mediates the esterase function of the enzyme but separate lipid binding sites have been proposed to be involved in the interaction of LPL with emulsified lipid substrates at the water-lipid interface. Like pancreatic lipase (PL), LPL contains a surface loop covering the catalytic pocket that may modulate access of the substrate to the active site of the enzyme. Secondary structural analysis of this loop reveals a helix-turn-helix motif with two short amphipathic helices that have hydrophobic moments of 0.64 and 0.68. In order to investigate the role of the loop in the initial interaction of LPL with its substrate, we utilized site-directed mutagenesis to generate eight constructs in which the amphipathic properties of the loop were altered and expressed them in human embryonal kidney-293 cells. Reducing the amphiphilicity without changing the predicted secondary structure of the loop abolished the ability of the lipase to hydrolyze emulsified, long chain fatty acid triglycerides (triolein) but not the water soluble substrate tributyrin. Replacing the loop of LPL with the loop of hepatic lipase, which differs in 15 of 22 amino acids but is also amphiphilic, led to the expression of an enzyme that retained both triolein and tributyrin hydrolyzing activity. Substitution of the LPL loop by a short four amino acid peptide, which may allow more direct access to the active site than the 22 amino acid loop, enhanced hydrolysis of short chain fatty acid triglycerides by more than 2-fold, while the ability to hydrolyze emulsified substrates was abolished. Thus, disruption of the amphipathic structure of the LPL loop selectively decreases the hydrolysis of emulsified lipid substrate without affecting the esterase or catalytic function of the enzyme. These studies establish that the loop with its two amphipathic helices is essential for hydrolysis of long chain fatty acid substrate by LPL providing new insight into the role of the LPL loop in lipid-substrate interactions. We propose that the interaction between the lipoprotein substrates and the amphipathic helices within this loop may in part determine lipase substrate specificity.

Amino Acid Sequence↗