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PubMed1981

Transcriptionally active chromatin in loops of lampbrush chromosomes at physiological salt concentrations as revealed by electron microscopy of sections.

The structural organization of the transcribed loops of lampbrush chromosomes present in the vegetative nuclei of the green algae, Acetabularia mediterranea and A. crenulata, and in oocyte nuclei of the newt, Pleurodeles waltlii, has been studied by electron microscopy of relatively thick (100--200 nm) and ultrathin sections through chromosomes prepared and fixed at physiological salt concentrations. The procedure allows the direct comparison of the same chromosome or chromosome region by light and electron microscopy, that means identification of the transcriptional arrays in specific loops. After such preparations the loop axis reveals regions that are smoothly-contoured ("non-beaded") and only 4 to 7 nm thick and are clearly different from supranucleosomal forms of inactive chromatin fibrils as well as from extended filaments of nucleosomal granules examined in parallel. This indicates that the chromatin of the loops axis of intensely transcribed regions is in a structural form different from that of non-transcribed chromatin. A similarly thin axis has been identified in loops of chromosomes of nuclei fixed in situ. The lateral ribonucleoprotein (RNP) fibrils associated with transcribed loop regions appear as serial arrays of granules, often of regular size, which are smaller in the chromosomes of Acetabularia (mean diameter 18 nm) than in those of amphibia (mean diameter 28 nm). Discontinuous arrays of lateral RNP fibrils and fibril arrays with different polarity are found in some loops. Certain loops and loop regions are characterized by specific patterns of aggregation of such lateral RNP fibrils which appear to correspond to the "granules" visible in these loops in the light microscope. The observations show that arrays of transcriptional complexes in chromosome loops can be visualized in thin sections of material prepared at physiological ionic strength with similar resolution and clarity as in spread preparations of chromosomal material dispersed in extremely low salts buffers. The results are interpreted to approximate the organization of loop structures in vivo and to show that, at physiological ionic strength, the chromatin of the loop axis is organized in a form different from that characteristic of non-transcribed chromatin.

Chlorophyta↗
PubMed2026

Small molecule inhibition of CPSF3 may impact R-loop distribution and abundance.

R-loops are three-stranded nucleic acid structures consisting of an RNA/DNA hybrid and a displaced strand of DNA. These structures have been implicated in a variety of regulatory cellular processes. Their untimed or excess accumulation, however, can cause genomic instability and induce DNA damage. Most R-loops form co-transcriptionally when the nascent transcript reanneals to unwound DNA duplex. Changes in transcription rates have the potential to impact R-loop formation, and compounds that modulate R-loop formation would be useful molecular tools and therapeutic leads. Cleavage and Polyadenylation Specific Factor 3 (CPSF3) recognizes the pre-mRNA 3' cleavage site, cleaves the transcript prior to polyadenylation, and has been linked to R-loop formation. Inhibition of CPSF3 has been found to induce transcriptional readthrough and cell proliferation defects. A previous report suggested that inhibition of CPSF3 with a small molecule causes a global increase in R-loop formation. Here, we test the impact of YT-II-100, a novel inhibitor of CPSF3. We find that addition of YT-II-100 increases global R-loop formation but does not change R-loop formation at specific genes that are normally used as positive controls for R-loop formation. We performed parallel assays using a previously reported compound, JTE-607, and observed similar results. Our data emphasize the need for cautious interpretation of experiments using JTE-607 and YT-II-100. There may be different mechanisms of R-loop formation depending on gene loci, where the control of R-loop formation by agonists at certain genes may differ from the trends observed for impacts on global R-loop formation.

R-Loop Structures↗
PubMed2026

SSB deficiency-induced R-loop accumulation triggers podocyte inflammation in DKD.

INTRODUCTION: Diabetic kidney disease (DKD) is fundamentally a podocytopathy in which sterile inflammation plays a central pathogenic role, yet the upstream triggers that initiate inflammatory cascades in podocytes remain elusive. R-loops are critical regulators of genomic stability, and their pathological accumulation triggers DNA damage and innate immune activation. Whether R-loop dysregulation contributes to podocyte-driven inflammation in DKD is unknown. METHODS: We integrated single-cell transcriptomic profiling, dual machine learning algorithms, and functional experiments to dissect the R-loop regulatory network in the diabetic kidney. RESULTS: Integrated analysis of human diabetic kidney single-cell RNA-seq data revealed a globally compromised R-loop regulatory network selectively within podocytes. Intersection of podocyte-specific transcriptomic shifts with validated R-loop regulators identified 93 candidate genes, from which dual machine learning algorithms pinpointed SSB (Sjögren syndrome antigen B) as the principal podocyte-selective R-loop resolver and a superior diagnostic biomarker (AUC = 0.983). SSB expression was selectively downregulated in diabetic podocytes and showed the strongest positive correlation with the R-loop resolution module. Mechanistically, SSB loss impaired RNA splicing and stability pathways, leading to aberrant R-loop accumulation that activated the cGAS-dependent inflammatory signaling in podocytes. In two murine DKD models and high glucose-challenged podocytes, SSB was markedly reduced. Remarkably, SSB knockdown in podocytes alone sufficed to trigger R-loop accumulation and pro-inflammatory cytokine expression, whereas both RNase H1-mediated R-loop removal and cGAS co-depletion blunted this response. DISCUSSION: These findings suggest that an SSB-governed R-loop -cGAS -inflammatory signaling axis may link genomic instability to podocyte inflammation and contribute to DKD progression, nominating R-loop homeostasis as a previously unrecognized potential therapeutic target.

Podocytes↗
PubMed1983

Altered myoelectric activity in the experimental blind loop syndrome.

Nutrient malabsorption and diarrhea are characteristic of the blind loop syndrome. Alterations in motility have been implicated as a cause of bacterial overgrowth, but the possibility that altered motility may result from alterations in the flora has not been explored. The purpose of this study was to characterize the myoelectric activity of the small intestine in the blind loop rat model. Eight groups of rats were studied: rats with self-filling blind loops, which develop bacterial overgrowth; rats with self-emptying blind loops, which are surgical controls that do not develop overgrowth; unoperated litter mates; rats with self-filling blind loops and unoperated controls treated with chloramphenicol, 200 mg/d i.p.; rats with surgically removed self-filling blind loops; operated control rats; and gnotobiotic rats with self-filling blind loops. In the untreated rats with self-filling blind loops, there was altered myoelectric activity characterized by an increased percentage of slow waves occupied by action potentials and by organized activity similar to the migrating action potential complex. Migrating action potential complex activity and percentage of slow waves occupied by action potentials were significantly decreased with chloramphenicol therapy; that decrease correlated with a decrease in aerobes and anaerobes. Migrating action potential complex activity was abolished in rats with surgically removed self-filling blind loops; they also showed a significant decrease in percentage of slow waves occupied by action potentials. Gnotobiotic rats with self-filling blind loops showed no alteration in myoelectric activity. These data indicate: (a) bacterial overgrowth is associated with a significant increase in percentage of slow waves occupied by action potentials and migrating action potential complex activity; (b) chloramphenicol significantly reduced both percentage of slow waves occupied by action potentials and migrating action potential complex activity; and (c) surgical removal of the loop reduced the alterations in motor function. This study suggests that the altered myoelectric activity in this model of bacterial overgrowth was due, in part, to the abnormal bacterial flora and supports the concept that alterations in motility may contribute to the diarrhea that is characteristic of the blind loop syndrome.

Action Potentials↗
PubMed2001

Role of small loops in DNA melting.

Short melted regions less than 100 base pairs (bp) in length are rarely found in the differential melting curves (DMC) of natural DNAs. Therefore, it is supposed that their characteristics do not affect DNA melting behavior. However, in our previous study, a strong influence of the form of the entropy factor of small loops on melting of cross-linked DNAs was established (D. Y. Lando, A. S. Fridman et al., Journal of Biomolecular Structure and Dynamics, 1997, Vol. 15, pp. 141-150; Journal of Biomolecular Structure and Dynamics, 1998, Vol. 16, pp. 59-67). Quite different dependencies of the melting temperature on the relative concentration of interstrand cross-links were obtained for the loop entropy factors given by the Fixman-Freire (Jacobson-Stockmayer) and Wartell-Benight relations. In the present study, the influence of the entropy factor of small loops on the melting of natural DNAs, cross-linked DNAs and periodical double-stranded polynucleotides is compared using computer simulation. A fast combined computational method for calculating DNA melting curves was developed for this investigation. It allows us to assign an arbitrary dependence of the loop entropy factor on the length of melted regions for the terms corresponding to small loops (less than tau bp in length). These terms are calculated using Poland's approach. The Fixman-Freire approach is used for long loops. Our calculations have shown that the temperature dependence of the average length of interior melted regions (loops) has a maximum at T approximately T(m) (T(m) is the DNA melting temperature) in contrast to the dependence of the total average length of melted regions, which increases almost monotonously. Computer modeling demonstrates that prohibition of formation of loops less than tau base pairs in length does not markedly change the DMC for tau < 150 bp. However, the same prohibition strongly affects the average length of internal melted regions for much smaller tau's. The effect is already noticeable for tau = 1 bp and increases with tau. A tenfold increase in the entropy factor of all loops with length less than tau bp causes a noticeable alteration of the DMC for tau > or = 30 bp. It is shown that DMCs are identical for the Wartell-Benight and for the Fixman-Freire (Jacobson-Stockmayer) form of the loop entropy factor. However, for low degree of denaturation, the average length of internal melted regions is 40% lower for the Wartell-Benight form due to the fluctuational opening of short AT-rich regions less than 10 bp in length. The same calculations carried out for periodical polynucleotides demonstrate a much stronger difference in melting behavior for different forms of entropy factors of short loops. The strongest difference occurs if the length of stable GC-rich and unstable AT-rich stretches is equal to 30 bp. However, the comparison carried out in this work demonstrates that the entropy factor of short loops influences melting behavior of cross-linked DNA much stronger than of unmodified DNA with random or periodical sequences.

Cross-Linking Reagents↗
PubMed1989

Structural determinants of the conformations of medium-sized loops in proteins.

Loops are integral components of protein structures, providing links between elements of secondary structure, and in many cases contributing to catalytic and binding sites. The conformations of short loops are now understood to depend primarily on their amino acid sequences. In contrast, the structural determinants of longer loops involve hydrogen-bonding and packing interactions within the loop and with other parts of the protein. By searching solved protein structures for regions similar in main chain conformation to the antigen-binding loops in immunoglobulins, we identified medium-sized loops of similar structure in unrelated proteins, and compared the determinants of their conformations. For loops that form compact substructures the major determinant of the conformation is the formation of hydrogen bonds to inward-pointing main chain atoms. For loops that have more extended conformations, the major determinant of their structure is the packing of a particular residue or residues against the rest of the protein. The following picture emerges: Medium-sized loops of similar conformation are stabilized by similar interactions. The groups that interact with the loop have very similar spatial dispositions with respect to the loop. However, the residues that provide these interactions may arise from dissimilar parts of the protein: The conformation of the loop requires certain interactions that the protein may provide in a variety of ways.

Binding Sites↗
PubMed1996

Functional significance of loops in the receptor binding domain of Bacillus thuringiensis CryIIIA delta-endotoxin.

Analysis of the three surface loops in domain II of Bacillus thuringiensis CryIIIA delta-endotoxin has been carried out to assess their role in receptor binding and toxicity. Site-directed mutagenesis was used to convert loop residues to alanine and the mutant proteins were analyzed for structural stability, toxicity to beetle larvae (Tenebrio molitor), binding to receptors on T. molitor brush border membrane vesicles (Tm-BBMV) and insertion into BBMV, as measured by irreversible membrane receptor binding. This study demonstrates the functional significance of loops for binding and insertion. Alanine replacements in loop I resulted in disruption of receptor binding or structural instability. The double mutation, Y350A, Y351A, could be suppressed by replacing a nearby R345 with alanine, and the resultant mutant protein also showed reduced receptor binding. Substitution of N353 and D354 in loop I with alanine residues caused the loss of binding ability and toxicity. A loop II double mutant, P412A, S413A, had no effect on binding or toxicity. A block mutation of loop III residues to alanine had the effect of reducing receptor binding while concomitantly increasing toxicity by 2.4-fold. We compared this up-mutant to wild-type toxin in each step of physiological processing of protoxin: solubility, proteolytic activation, and insertion into the Tm-BBMV. The loop III block mutant showed increased membrane insertion, but was similar to wild-type toxin in other parameters. These results reveal that loop I and loop III in domain II of CryIIIA delta-endotoxin are involved in receptor binding. In addition, the direct correlation between toxicity and irreversible binding of the loop III block mutant (despite the indirect relationship to reversible binding) suggests that loop III may play a role in membrane insertion.

Animals↗
PubMed1996

The immune response of sheep surgically modified with intestinal loops to challenge with Trichostrongylus colubriformis.

Aspects of the local immune response to nematode challenge were investigated in vivo in isolated loops of the upper small intestine of mature sheep that were immunised by repeated infections with Trichostrongylus colubriformis infective larvae (L3). Groups of 3 sheep were challenged either through the loop (Group 1) or orally (Group 2) with T. colubriformis L3, the third group served as unchallenged controls (Group 3). Nematode specific antibody levels, mast cell proteinase levels (SMCP) and larval migration inhibition (LMI) activity were determined in loop secretions for 4 weeks after challenge. The intestinal loops remained functional throughout the experiment. Groups 1 and 2 were re-challenged 2 weeks after the first challenge, and all 3 Groups were slaughtered 2 weeks later. Histopathological examination showed elevated numbers of globule leukocytes (GL) in both the nematode-challenged loop and unchallenged small intestine of Group 1 and small intestine of Group 2 indicating that nematode infections induce the local appearance of large numbers of GL. Oral, but not loop challenge caused increased antibody levels in loop secretions when compared to unchallenged controls. Only loop-challenged sheep showed a peak in loop fluid SMCP levels 10-13 days after the first challenge which coincided with a peak in numbers of mucosal GL. The isolated loops of all 3 groups showed highly elevated numbers of eosinophils when compared to the intact small intestine. Loop fluid of all 3 groups showed a high level of LMI activity reflecting the high level of nematode-resistance induced by the immunisation procedures. Sheep in Groups 1 and 2 were both able to expel challenge infections, and when compared to Group 3, showed higher blastogenic activity of unstimulated cells derived from a mesenteric lymph node in the region of the challenged part of the intestine. The present experiment showed that surgically constructed intestinal loops provided a model system by which the substantial changes associated with the local intestinal immune response to challenge with T. colubriformis could be investigated.

Animals↗
PubMed1993

Expression and immunogenicity of the V3 loop from the envelope of human immunodeficiency virus type 1 in an attenuated aroA strain of Salmonella typhimurium upon genetic coupling to two Escherichia coli carrier proteins.

A peptide comprising residues glu293 to ser334 from the principal neutralization determinant (V3 loop) of the envelope of human immunodeficiency virus type 1 (HIV1 LAVBRU isolate) has been inserted within internal permissive sites of either LamB or MalE, two envelope proteins from Escherichia coli K12. The MalE hybrid protein (MalE133-V3 loop) was stably expressed in the periplasm of Escherichia coli K12, and the V3 loop peptide was detectable on the surface of the native protein by an anti-gp160 monoclonal antibody (mAb 110-A). The disulfide bridge between the two cysteines of the loop was formed. In contrast, genetic coupling to the outer membrane protein LamB did not allow the expression of a stable hybrid protein, and major proteolytic cleavage products of the LamB153-V3 loop were detected by mAb 110-A. The two plasmid-encoded hybrid genes were transferred to an aroA mutant of Salmonella typhimurium. Constitutive expression of the MalE133-V3 loop had no detectable effect on cell growth and on the survival in vivo of the recipient strain. The LamB153-V3 loop was not stably expressed in Salmonella, either in vitro or in vivo. Live recombinant salmonellas expressing MalE-V3 and LamB-V3 loop hybrids were used to immunize mice. The MalE-V3 loop hybrid induced anti-HIV1 envelope antibodies detectable by Western blot and ELISA, while the anti-HIV1 envelope antibodies induced by the LamB-V3 loop hybrid were only detectable by Western blot. In addition, purified MalE-V3 loop hybrid protein was able to stimulate in vitro and induce in vivo a V3 loop-specific T-cell proliferative response.

AIDS Vaccines↗
PubMed2025

Barrier effects on the kinetics of cohesin-mediated loop extrusion.

Chromosome organization mediated by structural maintenance of chromosome complexes is crucial in many organisms. Cohesin extrudes chromatin into loops that are thought to lengthen until it is obstructed by CTCF proteins. In complex cellular environments, the loop extrusion machinery may encounter other chromatin-binding proteins. How these proteins interfere with the cohesin-meditated extrusion process is largely unexplored, but recent experiments have shown that some proteins serve as physical barriers that block cohesin translocation. Other proteins containing a cohesin-interaction motif serve as chemical barriers to induce cohesin pausing through interactions with it. Here, we develop an analytically solvable approach for the loop extrusion model incorporating barriers to investigate the effect of the barrier on the passive extrusion process. To further quantify the impact of barriers, we calculate the mean looping time it takes for cohesin to translocate to form a stable loop before dissociation. Our finding reveals that the physical barrier can accelerate the loop formation, and the degree of acceleration is closely related to the impedance strength of the physical barrier. In particular, the synergy of the cohesin loading site and the physical barrier site accelerates loop formation more significantly. The proximity of the cohesin loading site to the barrier site facilitates the rapid formation of stable loops in long genomes, which implies loop extrusion and chromatin-binding proteins might shape functional genomic organization. Conversely, chemical barriers consistently impede loop formation, with increasing impedance strength of the chemical barrier leading to longer loop formation time. Our study contributes to a more comprehensive understanding of the complexity of the loop extrusion process, providing a new perspective on the potential mechanisms of gene regulation.

Cohesins↗
PubMed2005

Kinetics of loop formation and breakage in the denatured state of iso-1-cytochrome c.

The earliest events in protein folding involve the formation of simple loops. Observing the rates of loop closure under denaturing conditions can provide direct insight into the relative probability and sequence determinants for formation of loops of different sizes. The persistence of these initial contacts is equally important for efficient folding, so measurement of rates of loop breakage under denaturing conditions is also essential. We have used stopped-flow and continuous-flow methods to measure the rates of histidine-heme loop formation and breakage in the denatured state of iso-1-cytochrome c (in the presence of 3 M guanidine HCl). The data indicate that the mechanism for forming loops is a two-step process, the first step being the deprotonation of the histidine, and the second step being the binding of the histidine to the heme. This mechanism makes it possible to extract both the rate constants of formation, k(f), and breakage, k(b), of loops from the pH dependence of the observed rate constant, k(obs). To determine the dependence of k(f) and k(b) on loop size, we have carried out kinetic measurements for seven single surface histidine variants of iso-1-cytochrome c. A scaling factor (the dependence of k(f) on log[loop size]) of approximately -1.8 is observed for loop formation, similar to that observed in other systems. The magnitude of k(b) varies from 30 s(-1) to 300 s(-1), indicating that the stability of different loops varies considerably. The implications of the kinetics of loop formation and breakage in the denatured state for the mechanism of protein folding are discussed.

Cytochromes c↗
PubMed1997

The structure of an essential splicing element: stem loop IIa from yeast U2 snRNA.

BACKGROUND: Eukaryotic genes are usually transcribed as precursor mRNAs which are then spliced, removing introns to produce functional mRNAs. Splicing is performed by the spliceosome and provides an important level of post-translational control of gene expression. Stem loop IIa from U2 small nuclear (sn)RNA is required for the efficient association of the U2 small nuclear ribonuclear protein (snRNP) with the nascent spliceosome in yeast. Genetic analysis suggests that stem loop IIa is involved in RNA-protein interactions early in splicing, and it may also interact with other RNA sequences in U2. The sequence of loop IIa is well conserved, consistent with the idea that this loop is important for function. RESULTS: We have solved the structure of U2A, a 20-base analogue of stem loop IIa from Saccharomyces cerevisiae, using NMR and restrained molecular dynamics. In the process, we have demonstrated the efficacy of a new structure calculation protocol, torsion angle molecular dynamics. The structure that has emerged, which is consistent with the in vivo chemical protection data available for stem loop IIa in the context of intact U2 snRNA, contains a sheared GA pair followed by a U-turn in the loop. The U-turn conformation, which resembles the U-turns in tRNA anticodon loops, makes this stretch of U2 snRNA an obvious target for interactions with proteins and/or other RNA sequences. CONCLUSIONS: The phenotypes of many stem loop IIa mutants can be rationalized assuming that the U-turn conformation in the loop must be preserved for efficient splicing. This observation, combined with the phylogenetic conservation of its sequence, suggests that the conformation of the loop of stem loop IIa is essential for its function in pre-mRNA splicing.

Base Sequence↗
PubMed2004

Interstrand loops CD and EF act as pH-dependent gates to regulate fatty acid ligand binding in tear lipocalin.

Tear lipocalin (TL), a major component of human tears, shows pH-dependent endogenous ligand binding. The structural and conformational changes associated with ligand release in the pH range of 7.5-3.0 are monitored by circular dichroism spectroscopy and site-directed tryptophan fluorescence. In the transition from pH 7.5 to pH 5.5, the ligand affinity for 16-(9-anthroyloxy)palmitic acid (16AP) and 8-anilino-1-naphthalenesulfonic acid is reduced. At pH 4.0 these ligands no longer bind within the TL calyx. From pH 7.3 to pH 3.0, the residues on loops CD and EF, which overhang the calyx entrance, show reduced accessibility to acrylamide. In addition resonance energy transfer is enhanced between residues on the two loops; the distance between the loops narrows. These findings suggest that apposition of the loops at low pH excludes the ligand from the intracavitary binding site. The conformational changes observed in transition from pH 7.3 to pH 3.0 for loops CD and EF are quite different. The CD loop shows less population reshuffling than the EF loop with an acidic environment, probably because backbone motion is restrained by the adjacent disulfide bond. The Trp fluorescence wavelength maximum (lambda(max)) reflects internal electrostatic interactions for positions on loops CD and EF. The titration curves of lambda(max) for mutants on the EF loop fit the Hendersen-Hasselbalch equation for two apparent pK(a) values, while the CD loop positions fit satisfactorily with one pK(a) value. Midpoints of transition for the binding affinity of TL tryptophan mutants to 16AP occur at pH 5.5-6.1. Replacement of each amino acid on either loop by single tryptophan mutation does not disrupt the pH-dependent binding affinity to 16AP. Taken together the data suggest that pH-driven ligand release involves ionization changes in several titratable residues associated with CD and EF loop apposition and occlusion of the calyx.

Carrier Proteins↗
PubMed1997

The last two cytoplasmic loops in the lactose permease of Escherichia coli comprise a discontinuous epitope for a monoclonal antibody.

Monoclonal antibody (mAb) 4B11 binds to a conformational epitope in the lactose permease that is exposed on the cytoplasmic face of the membrane with a KD of 2.8 x 10(-7) M. By studying binding of 4B11 to permease mutants containing six contiguous His residues in each cytoplasmic loop, inserted factor Xa protease sites, or a C-terminal deletion, the cytoplasmic loops between helices VIII and IX (loop VIII/IX) and between helices X and XI (loop X/XI) are shown to comprise the epitope. Subsequently, Cys-scanning mutagenesis in conjunction with thiol modification was carried out in order to identify specific residues involved in 4B11 recognition. Glu342 and Arg344 in loop X/XI are primary determinants for 4B11 binding, while Ile283 in loop VIII/IX and Phe334 and Lys335 in loop X/XI are secondary determinants. Consistently, binding of avidin to biotinylated single-Cys replacements in loop VIII/IX or loop X/XI blocks 4B11 binding, but avidin binding to biotinylated Cys residues in other cytoplasmic loops or insertion of cytochrome b562 into cytoplasmic loop VI/VII has no significant effect. The studies demonstrate that the last two cytoplasmic loops in lactose permease comprise a discontinuous epitope for monoclonal antibody 4B11 and thereby provide independent evidence for the conclusion that helices VIII-XI are in close proximity.

Antibodies, Monoclonal↗
PubMed1997

Proximity of periplasmic loops in the lactose permease of Escherichia coli determined by site-directed cross-linking.

Out of over 60 single-Cys mutants in putative periplasmic loops in lactose permease, three mutants [Tyr101 --> Cys (loop III/IV), Leu313 --> Cys (loop IX/X), and Ser375 --> Cys (loop XI/XII)] spontaneously form disulfide-linked dimers, indicating that these loops are located on the periphery of the 12-helix bundle that comprises the permease. By using a permease construct with a factor Xa protease site in the middle cytoplasmic loop, cross-linking between paired-Cys residues in the N- and C-terminal halves of the permease was studied by spontaneous or copper-(1, 10-phenanthroline)3-catalyzed disulfide formation or by cross-linking with homo- or heterobifunctional reagents in which the distance between the reactive groups and the flexibility of the linker vary. The findings suggest that the longer loops are relatively flexible; however, cross-linking of residues between loops is specific, indicating that these domains are not simply flexible, hydrophilic connections between helices that interact randomly. More specifically, the findings indicate that the first periplasmic loop (loop I/II) is close to loops VII/VIII and XI/XII, placing helix XII in close proximity to helices II and XI. In addition, the observations are consistent with previous results [Wu, J., & Kaback, H. R. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 14498-502] demonstrating that helices I and II are close to helices VII and XI. Finally, evidence is presented indicating that conformational flexibility between loops I/II and XI/XII may be important for permease turnover.

Binding Sites↗
PubMed2000

Functions of fluctuation in the heme-binding loops of cytochrome b5 revealed in the process of heme incorporation.

Cytochrome b(5) (cyt b(5)) holds heme using two axial histidines, His63 and His39, that are located in the centers of the two heme-binding loops. The previous NMR study on the apo form of cyt b(5) (apocyt b(5)) revealed that the loop including His63 exhibits a larger fluctuation compared to the other loop including His39 [Falzone, C. J., Mayer, M. R., Whiteman, E. L., Moore, C. D., and Lecomte, J. T. (1996) Biochemistry 35, 6519-6526]. To understand the significance of the fluctuation, the heme association and dissociation rates of the two loops were compared using two mutants of cyt b(5) in which one of the axial histidines was replaced with leucine. It was demonstrated that the fluctuating loop possesses a significantly slower heme dissociation rate and a faster heme association rate than the other loop. To further verify the importance of the fluctuating loop, the heme association process of wild-type apocyt b(5) was investigated using optical absorption and CD spectroscopies. It was indicated that the process proceeds through the two pathways, and that the dominant pathway involves the initial coordination of His63 located in the fluctuating loop. The urea concentration dependency of the rate constants revealed that the folding of the fluctuating loop is associated with the coordination of His63. It was suggested that the fluctuation enables the loop to have a larger heme-loop contact in the heme-bound conformation. The fluctuating heme-binding loops might be useful for the artificial design of heme-binding proteins.

Animals↗
PubMed2003

Nick-dependent and -independent processing of large DNA loops in human cells.

DNA loop heterologies are products of normal DNA metabolism and can lead to severe genomic instability if unrepaired. To understand how human cells process DNA loop structures, a set of circular heteroduplexes containing a 30-nucleotide loop were constructed and tested for repair in vitro by human cell nuclear extracts. We demonstrate here that, in addition to the previously identified 5' nick-directed loop repair pathway (Littman, S. J., Fang, W. H., and Modrich, P. (1999) J. Biol. Chem. 274, 7474-7481), human cells can process large DNA loop heterologies in a loop-directed manner. The loop-directed repair specifically removes the loop structure and occurs only in the looped strand, and appears to require limited DNA synthesis. Like the nick-directed loop repair, the loop-directed repair is independent of many known DNA repair pathways, including DNA mismatch repair and nucleotide excision repair. In addition, our data also suggest that an aphidicolin-sensitive DNA polymerase is involved in the excision step of the nick-directed loop repair pathway.

Aphidicolin↗
PubMed1993

Solution structure of the V3 loop of a Thailand HIV isolate.

The principal neutralizing determinant (PND) of human immunodeficiency virus (HIV) is located inside the third variable loop (designated the V3 loop) of the envelope glycoprotein gp120. The V3 loop is typically 35 amino-acids long, and the 1st and the 35th residues in the loop are invariant cystines involved in a disulfide-bridge. Although PNDs from different HIV isolates contain a conserved GPG-sequence, the amino acids flanking the conserved sequence show hypervariability among HIV isolates; the GPG and the two flanking regions are collectively referred to as the GPG-crest or the PND. The amino acid sequence variability in the GPG-crest gives rise to different antigenic specificities for different PNDs from different HIV isolates. By combining two-dimensional nuclear magnetic resonance (2D NMR) and molecular modeling techniques, we have developed a method to study (1) the global tertiary fold of the V3 loops of HIV and (2) the local structure of the PND at the tip of the V3 loop. In this article, we report the results of our structural studies on the V3 loop of a Thailand HIV isolate. The sequential assignment is made by combining DQF-COSY, TOCSY, and NOESY/ROESY experiments. Various intra- and inter-residue inter-proton distances are estimated by full-matrix analyses of the NOESY data at 100 and 400 ms of mixing times and of the ROESY data at 60 and 200 ms of mixing times. 100 inter-residue distances are used as structural constraints in a simulated annealing procedure to derive energetically stable structures. Two functional motifs in the V3 loop, i.e., the glycosylation site and the GPG-crest, form defined structures: a turn is located at the glycosylation site, and the GPG-crest forms a protruding domain with a type-II GPGQ turn. The other regions of the V3 loop are rather flexible--especially the C-terminal DIRKAYC-stretch. These flexible regions of the V3 loop lead to conformational flexure of the entire V3 loop without altering the local structures of the glycosylation site or the GPG-crest. However, the ROESY experiments revealed no slow exchange among different V3 loop conformations, and therefore the flexible conformations are in fast exchange within the NMR time scale. The extent of this conformational flexibility is also discussed.

Amino Acid Sequence↗