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PubMed1997

Structural studies of the streptavidin binding loop.

The streptavidin-biotin complex provides the basis for many important biotechnological applications and is an interesting model system for studying high-affinity protein-ligand interactions. We report here crystallographic studies elucidating the conformation of the flexible binding loop of streptavidin (residues 45 to 52) in the unbound and bound forms. The crystal structures of unbound streptavidin have been determined in two monoclinic crystal forms. The binding loop generally adopts an open conformation in the unbound species. In one subunit of one crystal form, the flexible loop adopts the closed conformation and an analysis of packing interactions suggests that protein-protein contacts stabilize the closed loop conformation. In the other crystal form all loops adopt an open conformation. Co-crystallization of streptavidin and biotin resulted in two additional, different crystal forms, with ligand bound in all four binding sites of the first crystal form and biotin bound in only two subunits in a second. The major change associated with binding of biotin is the closure of the surface loop incorporating residues 45 to 52. Residues 49 to 52 display a 3(10) helical conformation in unbound subunits of our structures as opposed to the disordered loops observed in other structure determinations of streptavidin. In addition, the open conformation is stabilized by a beta-sheet hydrogen bond between residues 45 and 52, which cannot occur in the closed conformation. The 3(10) helix is observed in nearly all unbound subunits of both the co-crystallized and ligand-free structures. An analysis of the temperature factors of the binding loop regions suggests that the mobility of the closed loops in the complexed structures is lower than in the open loops of the ligand-free structures. The two biotin bound subunits in the tetramer found in the MONO-b1 crystal form are those that contribute Trp 120 across their respective binding pockets, suggesting a structural link between these binding sites in the tetramer. However, there are no obvious signatures of binding site communication observed upon ligand binding, such as quaternary structure changes or shifts in the region of Trp 120. These studies demonstrate that while crystallographic packing interactions can stabilize both the open and closed forms of the flexible loop, in their absence the loop is open in the unbound state and closed in the presence of biotin. If present in solution, the helical structure in the open loop conformation could moderate the entropic penalty associated with biotin binding by contributing an order-to-disorder component to the loop closure.

Allosteric Regulation↗

PubMed2005

Protein loop structure prediction with flexible stem geometries.

The structure prediction of loops with flexible stem residues is addressed in this article. While the secondary structure of the stem residues is assumed to be known, the geometry of the protein into which the loop must fit is considered to be unknown in our methodology. As a consequence, the compatibility of the loop with the remainder of the protein is not used as a criterion to reject loop decoys. The loop structure prediction with flexible stems is more difficult than fitting loops into a known protein structure in that a larger conformational space has to be covered. The main focus of the study is to assess the precision of loop structure prediction if no information on the protein geometry is available. The proposed approach is based on (1) dihedral angle sampling, (2) structure optimization by energy minimization with a physically based energy function, (3) clustering, and (4) a comparison of strategies for the selection of loops identified in (3). Steps (1) and (2) have similarities to previous approaches to loop structure prediction with fixed stems. Step (3) is based on a new iterative approach to clustering that is tailored for the loop structure prediction problem with flexible stems. In this new approach, clustering is not only used to identify conformers that are likely to be close to the native structure, but clustering is also employed to identify far-from-native decoys. By discarding these decoys iteratively, the overall quality of the ensemble and the loop structure prediction is improved. Step (4) provides a comparative study of criteria for loop selection based on energy, colony energy, cluster density, and a hybrid criterion introduced here. The proposed method is tested on a large set of 3215 loops from proteins in the Pdb-Select25 set and to 179 loops from proteins from the Casp6 experiment.

Cluster Analysis↗

PubMed1997

Nicked multifunctional loop of glutathione synthetase still protects the catalytic intermediate.

A derivative of glutathione synthetase (GSHase) with the multifunctional loop cleaved (nicked GSHase) was compared to both a deletion mutant of the loop (loopless GSHase) and wild-type with the intact loop (wild-type GSHase). The loop had been shown to be in a closed state in order to protect a catalytic intermediate and accelerate the reaction. Data indicated that cleavage of the loop resulted in a drastic decrease in glutathione synthetic activity which was similar to the results for the loop deletion. Kinetic analyses indicated that the manipulations of the loop impaired the substrate affinity, especially for glycine, and also catalytic efficiency. The nicked loop did not accelerate the reaction as fast as the intact loop; however, the catalytic intermediate was protected from hydrolysis by the cleaved loop as effectively as by the intact loop. These results suggest that the fragmental loop assumed the closed state. High concentrations of ATP showed some inhibitory effects on wild-type GSHase, while both nicked and loopless GSHase were not inhibited, indicating that the fragments of the nicked loop functioned independently. In conclusion, it is postulated that the two fragments of the nicked loop independently assumed the closed state to protect the catalytic intermediate and have lost the ability to accelerate glutathione synthesis.

Adenosine Triphosphate↗

PubMed1998

Automated classification of antibody complementarity determining region 3 of the heavy chain (H3) loops into canonical forms and its application to protein structure prediction.

A computer-based algorithm was used to cluster the loops forming the complementarity determining region (CDR) 3 of the heavy chain (H3) into canonical classes. Previous analyses of the three-dimensional structures of CDR loops (also known as the hypervariable regions) within antibody immunoglobulin variable domains have shown that for five of the six CDRs there are only a few main-chain conformations (known as canonical forms) that show clear relationships between sequence and structure. However, the larger variation in length and conformation of loops within H3 has limited the classification of these loops into canonical forms. The clustering procedure presented here is based on aligning the Ramachandran-coded main-chain conformation of the residues using a dynamic algorithm that allows the insertion of gaps to obtain an optimum alignment. A total of 41 H3 loops out of 62 non-identical loops, extracted from the Brookhaven Protein Data Bank, have been automatically grouped into 22 clusters. Inspection of the clusters for consensus sequences or intra-loop interactions or invariant conformation led to the proposal of 13 canonical forms representing 31 loops. These canonical forms include a consideration of the geometry of both the take-off region adjacent to the bracing beta-strands and the remaining loop apex. Subsequently a new set of 15 H3 loops not included in the initial analysis was considered. The clustering procedure was repeated and nine of these 15 loops could be assigned to original clusters, including seven to canonical forms. A sequence profile was generated for each canonical form from the original set of loops and matched against the sequences of the new H3 loops. For five out of the seven new H3 loops that were in a canonical form, the correct form was identified at first rank by this predictive scheme.

Amino Acid Sequence↗

PubMed2001

Mechanism of Tet repressor induction by tetracyclines: length compensates for sequence in the alpha8-alpha9 loop.

Natural Tet repressor (TetR) variants are alpha-helical proteins bearing a large loop between helices 8 and 9, which is variable in sequence and length. We have deleted this loop consisting of 14 amino acid residues in TetR(D) and rebuilt it stepwise with up to 42 alanine residues. All except the mutant with the longest alanine loop show wild-type repression, but none is inducible with tetracycline. This demonstrates the importance of the alpha8-alpha9 loop and its amino acid sequence for induction. The induction efficiencies increase with loop length, when the more tightly binding inducer anhydrotetracycline is used. The largest increase of inducibility was observed for TetR mutants with loop lengths between eight and 17 alanine residues. Since loop residues Asp/Glu157 and Arg158 are conserved in the natural TetR sequence variants, we constructed a mutant in which all other residues of the loop were replaced by alanine. This mutant exhibits increased anhydrotetracycline induction compared to the corresponding alanine variant. Thus, these residues are important for induction. Binding constants for the anhydrotetracycline-TetR interaction are below the detection level of 10(5) M(-1) for the mutant with a loop of two alanine residues and increase sharply until a loop size of ten residues is reached. TetR variants with longer loops have similar anhydrotetracycline-binding constants, ranging between 2.6 x 10(9) M(-1) and 8.0 x 10(9) M(-1), about 500-fold lower than wild-type TetR. The increase of the affinity occurs at shorter loop lengths than that of inducibility. We conclude that the induction defect of the polyalanine variants arises from two increments: (i) the loop must have a minimal length-to allow efficient inducer binding; (ii) the loop must structurally participate in the conformational change associated with induction.

Amino Acid Sequence↗

PubMed1991

Genetic and cytogenetic analysis of the "Th-Ps" region of the Y chromosome of Drosophila hydei: evidence for dual functions of the lampbrush loop-forming fertility genes?

Two competing hypotheses have been proposed for the function of the Y chromosomal fertility factors in Drosophila, which form giant lampbrush loops during the primary spermatocyte stage. The first hypothesis suggests a conventional coding function, the second proposes an unconventional gene function mediated through protein binding by nascent transcripts. Therefore, we studied the genetics and cytogenetics of the two Y chromosomal fertility genes A and C of Drosophila hydei (which form the lampbrush loops threads and pseudonucleolus) in order to test the validity of these different hypotheses. Both lampbrush loops bind specific proteins, which are recognized by different antisera. Absence of either of the lampbrush loops does not interfere with the synthesis of the antigens but completely prevents the binding of the particular antigen to other lampbrush loops. Absence of the loops also does not interfere with the postmeiotic presence and localization of the particular antigen. Deletion (or inactivation) of either of the lampbrush loops threads or pseudonucleolus causes sterility of the male flies as do other male-sterile alleles of both fertility genes, which do not affect the morphology of the lampbrush loops. The phenotypic effects of these mutations on sperm morphogenesis are identical for all various male-sterile alleles of each of the fertility genes A and C, regardless of whether a particular allele leaves the loop intact, modifies that loop, or deletes (or inactivates) the loop completely. Finally, the isolation of fertile Y chromosomal mutations which modify the morphology of the lampbrush loops demonstrates that it is possible to uncouple loop morphology and genetic function. These findings do not support the hypothesis that the binding of proteins to a lampbrush loop has a substantial impact on spermiogenesis.

Animals↗

PubMed2004

Arthroscopic knots: determining the optimal balance of loop security and knot security.

PURPOSE: The purpose of this study was to determine the optimal knot configuration that maximized both knot and loop security when tied with 2 different types of nonabsorbable, braided suture. TYPE OF STUDY: In vitro biomechanical study. METHODS: Six commonly used arthroscopic sliding knots (Duncan loop, Nicky's knot, Tennessee slider, Roeder knot, SMC knot, Weston knot) with and without a series of 3 reversing half-hitches on alternating posts (RHAPs) as well as a static surgeon's knot were tied. Two different nonabsorbable, braided sutures were used, and a total of 7 knots were tied for each possible combination of knots and sutures, for a total of 182 knots. Each knot was tied around a 30-mm circumference post to assure a consistent loop circumference of 30 mm before "locking" the complex sliding knots by tensioning the wrapping limb of the suture. Each loop was mounted on a Material Testing System machine, and its circumference was measured at a 5-N preload to assess each knot's ability to maintain a tight suture loop without slippage (loop security). Knot security was measured as the maximum force to failure at 3 mm of crosshead displacement or suture breakage during single-pull load testing. RESULTS: The surgeon's knot provided the highest force to failure and the tightest loop circumference whether tied with No. 2 Ethibond (Ethicon, Somerville, NJ) or No. 2 Fiberwire (Arthrex, Naples, FL) suture. Among the sliding knots, the Roeder knot with 3 RHAPs showed the best balance of loop security and knot security when tied with No. 2 Ethibond or No. 2 Fiberwire. Sliding knots tied without RHAPs showed low force to failure and loose suture loops whether tied with Ethibond or Fiberwire. The addition of 3 RHAPs improved knot security and, in most cases, loop security of all the sliding knots. When tying a static surgeon's knot or a sliding knot with RHAPs, using No. 2 Fiberwire increased the force to failure over comparable knots tied with No. 2 Ethibond. All knots failed by a combination of knot slippage and suture stretch. When using No. 2 Ethibond, securing most sliding knots with 3 RHAPs or tying a surgeon's knot changed the failure mechanism from knot slippage to suture stretch, suggesting that the maximum knot holding capacity of No. 2 Ethibond had been achieved when tying these knot configurations. However, even at failure forces twice that achieved with No. 2 Ethibond, suture slippage continued to occur with sliding knots with 3 RHAPs using No. 2 Fiberwire. This indicates that the maximum knot-holding capacity of No. 2 Fiberwire had not been achieved, and that further knot configurations should be tested. CONCLUSIONS: (1) A static surgeon's knot provides the best balance of loop security and knot security within the knot configurations tested in this study. (2) A sliding knot without RHAPs has both poor loop security and knot security and should not be tied. (3) The addition of 3 RHAPs improves knot security of all sliding knots tested and improves loop security of most of the sliding knots tested. (4) The addition of 3 RHAPs improves the knot security of all sliding knots to adequately resist predicted in vivo loads. (5) The Roeder knot with 3 RHAPs provides the best balance of loop security and knot security within the sliding knot configurations tested in this study regardless of suture type. (6) Tying a surgeon's knot or a sliding knot with 3 RHAPS using No. 2 Fiberwire increases knot security over the same knot tied with No. 2 Ethibond. CLINICAL RELEVANCE: This study identifies the static and sliding configurations of commonly used arthroscopic knots in order to aid the surgeon in choosing the most biomechanically effective knot for use in arthroscopic surgery.

Arthroscopy↗

PubMed2004

Loop-contraction mutagenesis of type 1 copper sites.

The shortest known type 1 copper binding loop (that of amicyanin, Ami) has been introduced into three different cupredoxin beta-barrel scaffolds. All of the loop-contraction variants possess copper centers with authentic type 1 properties and are redox active. The Cu(II) and Co(II) sites experience only small structural alterations upon loop contraction with the largest changes in the azurin variant (AzAmi), which can be ascribed to the removal of a hydrogen bond to the coordinating thiolate sulfur of the Cys ligand. In all cases, loop contraction leads to an increase in the pK(a) of the His ligand found on the loop in the reduced proteins, and in the pseudoazurin (Paz) and plastocyanin (Pc) variants the values are almost identical to that of Ami ( approximately 6.7). Thus, in Paz, Pc, and Ami, the length of this loop tunes the pK(a) of the His ligand. In the AzAmi variant, the pK(a) is 5.5, which is considerably higher than the estimated value for Az (<2), and other controlling factors, along with loop length, are involved. The reduction potentials of the loop-contraction variants are all lower than those of the wild-type proteins by approximately 30-60 mV, and thus this property of a type 1 copper site is fine-tuned by the C-terminal loop. The electron self-exchange rate constant of Paz is significantly diminished by the introduction of a shorter loop. However, in PcAmi only a 2-fold decrease is observed and in AzAmi there is no effect, and thus in these two cupredoxins loop contraction does not significantly influence electron-transfer reactivity. Loop contraction provides an active site environment in all of the cupredoxins which is preferable for Cu(II), whereas previous loop elongation experiments always favored the cuprous site. Thus, the ligand-containing loop plays an important role in tuning the entatic nature of a type 1 copper center.

Azurin↗

PubMed1999

3-D experimental identification of force systems from orthodontic loops activated for first order corrections.

Intra-arch irregularities can be corrected using wire of low stiffness, wires of increasing stiffnesses, or by the activation of loops built into the appliance. While the orthodontist controls only the magnitude of force when leveling with continuous arches, the configuration and positioning of loops offer the possibility of controlling the type and direction of force. In the present study, force systems developed by the L-loop, the T-loop, and the rectangular (R-) loop were analyzed with respect to the force systems acting for first order irregularities, buccolingual movement, and rotation along the long axis of the tooth. An interbracket distance of 21 mm was chosen, and the loops were analyzed in a testing machine that made it possible to register forces and moments simultaneously in three planes of space. The activations included a symmetrical translation of 1 mm made in steps of .2 mm, corresponding to a buccolingual movement, and 10-degree rotations clockwise and counterclockwise in steps of one degree. Force systems were recorded during activation and deactivation. Loops made of TMA wire delivered 40% of the force delivered by the same loops made of stainless steel wire. The T-loop generated a force system that deviated qualitatively only slightly from that delivered by a straight wire. The L-loop generated a force system that was dependent on orientation; constancy was better corresponding to the anterior part of the loop. It was evident that the rectangular loop was capable of generating any desired moment-to-force ratio, and the R-loop demonstrated a high degree of constancy of the force system. Rectangular loops should, therefore, be preferred for making first order corrections.

Dental Alloys↗

PubMed2002

Role of loop structures of neuropsin in the activity of serine protease and regulated secretion.

Neuropsin involved in neural plasticity in adult mouse brain is a member of the S1 (clan SA) family of serine proteases and forms characteristic surface loops surrounding the substrate-binding site (Kishi, T., Kato, M., Shimizu, T., Kato, K., Matsumoto, K., Yoshida, S., Shiosaka, S., and Hakoshima, T. (1999) J. Biol. Chem. 274, 4220-4224). Little, however, is known about the roles of these loops. Thus, the present study investigated whether surface loop structures of neuropsin were essential for the generation of enzymatic activity and/or secretion of the enzyme via a regulated secretory pathway. The loops include those stabilized by six disulfide bonds or a loop C (Gly(69)-Glu(80)) and an N-glycosylated kallikrein loop (His(91)-Ile(103)) not containing a site linked by a disulfide bond. First, among the six disulfide bonds, only SS1 in loop E (Gly(142)-Leu(155)) and SS6 in loop G (Ser(185)-Gly(197)) were necessary for the catalytic efficiency of neuropsin. Second, disruptions of loop C and the N-linked oligosaccharide chain on the kallikrein loop affected the catalytic efficiency and P2 specificity, respectively. Alternatively, disruptions of loop C and the kallikrein loop enhanced the regulated secretion, whereas there was no one disruption that inhibited the secretion, indicating that there was no critical loop required for the regulated secretion among loops surrounding the substrate-binding site.

Animals↗

PubMed2000

Conformational changes in the unique loops bordering the ATP binding cleft of skeletal muscle myosin mediate energy transduction.

Myosin has three highly-conserved, unique loops [B (320-327), M (677-689), and N (127-136)] at the entrance of the ATP binding cleft, and we previously showed that the effects of actin are mediated by a conformational change in loop M [Maruta and Homma (1998) J. Biochem. 124, 528-533]. In the present study, loops M and N were photolabeled respectively with fluorescent probes Mant-8-N(3)-ADP and Mant-2-N(3)-ADP in order to study conformational changes in the loops related to energy transduction. The effect of actin on the conformation of loop N was examined by analyzing fluorescence polarization and acrylamide quenching; the results were then compared with those previously reported for loop M. In contrast to loop M, the fluorescence polarization and the value of K(sv) of the Mant-groups crosslinked to loop N were slightly affected by actin binding. To study conformational changes in loops M and N during the ATPase cycle, FRET was analyzed using TNP-ADP.BeFn and TNP-ADP. AlF(4)(-) as FRET acceptors of Mant fluorescence. The resultant estimated distances between loop M and the active site differed for the Mant-S1.TNP-ADP.BeFn and Mant-S1.TNP-ADP.AlF(4)(-) complexes, whereas the distances between loop N and the active site differed slightly. These findings indicate that the conformation of loop M changes during the ATPase cycle, suggesting that Loop M acts as a signal transducer mediating communication between the ATP- and actin-binding sites. Loop N, by contrast, is not significantly flexible.

Acrylamide↗

PubMed2000

Modeling of loops in protein structures.

Comparative protein structure prediction is limited mostly by the errors in alignment and loop modeling. We describe here a new automated modeling technique that significantly improves the accuracy of loop predictions in protein structures. The positions of all nonhydrogen atoms of the loop are optimized in a fixed environment with respect to a pseudo energy function. The energy is a sum of many spatial restraints that include the bond length, bond angle, and improper dihedral angle terms from the CHARMM-22 force field, statistical preferences for the main-chain and side-chain dihedral angles, and statistical preferences for nonbonded atomic contacts that depend on the two atom types, their distance through space, and separation in sequence. The energy function is optimized with the method of conjugate gradients combined with molecular dynamics and simulated annealing. Typically, the predicted loop conformation corresponds to the lowest energy conformation among 500 independent optimizations. Predictions were made for 40 loops of known structure at each length from 1 to 14 residues. The accuracy of loop predictions is evaluated as a function of thoroughness of conformational sampling, loop length, and structural properties of native loops. When accuracy is measured by local superposition of the model on the native loop, 100, 90, and 30% of 4-, 8-, and 12-residue loop predictions, respectively, had <2 A RMSD error for the mainchain N, C(alpha), C, and O atoms; the average accuracies were 0.59 +/- 0.05, 1.16 +/- 0.10, and 2.61 +/- 0.16 A, respectively. To simulate real comparative modeling problems, the method was also evaluated by predicting loops of known structure in only approximately correct environments with errors typical of comparative modeling without misalignment. When the RMSD distortion of the main-chain stem atoms is 2.5 A, the average loop prediction error increased by 180, 25, and 3% for 4-, 8-, and 12-residue loops, respectively. The accuracy of the lowest energy prediction for a given loop can be estimated from the structural variability among a number of low energy predictions. The relative value of the present method is gauged by (1) comparing it with one of the most successful previously described methods, and (2) describing its accuracy in recent blind predictions of protein structure. Finally, it is shown that the average accuracy of prediction is limited primarily by the accuracy of the energy function rather than by the extent of conformational sampling.

Models, Molecular↗

PubMed2001

Channel cytoplasmic loops alter voltage-dependent sodium channel activation in an isoform-specific manner.

1. The isoform-specific functional role of cytoplasmic structures of two voltage-gated sodium channel isoforms, the human cardiac channel (hH1) and the adult human skeletal muscle channel (hSkM1) was investigated through functional comparison of chimeras. 2. The voltage of half-activation (V(a)) for hH1 was shifted by > 20 mV in the hyperpolarised direction following internal papain treatment ('papain sensitive'), while V(a) for hSkM1 was unaffected ('papain insensitive'). 3. The hH1 region(s) responsible for this papain sensitivity was localised by testing a series of hH1/hSkM1 chimeras in which combinations of the large hH1 cytoplasmic loops joining the four transmembrane domains replaced analogous hSkM1 loops. Various chimeras were used to determine the smallest subset of loops that converted fully the papain-insensitive hSkM1 into a papain-sensitive channel. Then three converse chimeras were tested in which hSkM1 loops replaced hH1 loops to determine the smallest subset of loops necessary and sufficient to convert the papain-sensitive hH1 into a papain-insensitive channel. 4. Functional studies of this inclusive set of chimeras indicate that the first two cytoplasmic loops of the cardiac sodium channel that join domain I to II (loop A), and domain II to III (loop B), are both necessary, and together are sufficient to produce a papain-induced hyperpolarising shift in the voltage at which channels activate. When both loops are present (wild-type hH1 and the chimera hSkM1AB), V(a) for the channel shifts in the hyperpolarised direction by > 20 mV with papain treatment. When the analogous hSkM1 loops are present (wild-type hSkM1 and the chimera hH1AB), V(a) for the channel is not sensitive to treatment with papain. For channels that contain only one of the two hH1 loops, the effect of papain on V(a) is intermediary. 5. Experiments performed in the absence of papain showed that the activation voltages of the double loop chimeras, hSkM1AB and hH1AB, were shifted significantly from V(a) for hSkM1 and V(a) for hH1, respectively, indicating that these loops directly alter channel activation voltage. The resulting shifts in V(a) were in opposing directions, suggesting that cytoplasmic control of activation voltage is isoform specific. V(a) for hSkM1AB was about 20 mV more depolarised than V(a) for hSkM1, and V(a) for hH1AB was about 9 mV more negative than V(a) for hH1. 6. These data are the first to indicate isoform-specific cytoplasmic regions of the voltage-gated sodium channel that directly and differently alter the voltage of channel activation.

Animals↗

PubMed1980

Variables affecting local immune response in ileal loops: role of immunization schedule, bacterial flora, and postsurgical inflammation.

Several variables inherent in chronically isolated ileal (Thiry-Vella) loops in rabbits were studied for their effect on the local immune response of the intestine to live, locally invasive bacteria (Shigella X16). A much more vigorous local immunoglobulin A response to Shigella X16 was elicited when rabbits were immunized in their Thiry-Vella loops shortly after surgical creation of the loop than if a week were allowed to pass before they were immunized. Three major differences existed in Thiry-Vella loops on the day after surgery and a week later: (i) their microbial flora, (ii) nonspecific acute inflammation due to the surgery itself, and (iii) the histological appearance of the intestine. On day 1 after surgical creation of the Thiry-Vella loop, there were few bacteria in the loop, and the histology was that of normal small bowel except for mild acute inflammation due to the surgery. By day 6 after surgery, all loops contained large numbers of Pseudomonas aeruginosa and other aerobes, an atrophy of intestinal epithelium occurred, and the acute inflammation due to surgical trauma had subsided. By artificially colonizing Thiry-Vella loops with 10(8) or 10(10) live P. aeruginosa on the day of surgery, we found that the presence of these bacteria alone did not greatly diminish local immune responses to live Shigella. Furthermore, when the acute inflammation due to surgical trauma was recreated in loops 6 days old, no enhancement of the immune response was seen as compared to nontraumatized 6-day-old Thiry-Vella loops. The difference between immunization soon after surgery and a week later related to changes that occur in the loop itself with increased isolation. Finally, multiple immunizations of Thiry-Vella loops resulted in a more vigorous local immunoglobulin A response than a single immunization. These studies demonstrated that Thiry-Vella loop models can be useful in studying the kinetics of local immune responses by the intestine only if careful attention is paid to key variables inherent in the Thiry-Vella loop models themselves.

Animals↗

PubMed2005

Multiple loops of the dihydropyridine receptor pore subunit are required for full-scale excitation-contraction coupling in skeletal muscle.

Understanding which cytosolic domains of the dihydropyridine receptor participate in excitation-contraction (EC) coupling is critical to validate current structural models. Here we quantified the contribution to skeletal-type EC coupling of the alpha1S (CaV1.1) II-III loop when alone or in combination with the rest of the cytosolic domains of alpha1S. Chimeras consisting of alpha1C (CaV1.2) with alpha1S substitutions at each of the interrepeat loops (I-II, II-III, and III-IV loops) and N- and C-terminal domains were evaluated in dysgenic (alpha1S-null) myotubes for phenotypic expression of skeletal-type EC coupling. Myotubes were voltage-clamped, and Ca2+ transients were measured by confocal line-scan imaging of fluo-4 fluorescence. In agreement with previous results, the alpha1C/alpha1S II-III loop chimera, but none of the other single-loop chimeras, recovered a sigmoidal fluorescence-voltage curve indicative of skeletal-type EC coupling. To quantify Ca2+ transients in the absence of inward Ca2+ current, but without changing the external solution, a mutation, E736K, was introduced into the P-loop of repeat II of alpha1C. The Ca2+ transients expressed by the alpha1C(E736K)/alpha1S II-III loop chimera were approximately 70% smaller than those expressed by the Ca2+-conducting alpha1C/alpha1S II-III variant. The low skeletal-type EC coupling expressed by the alpha1C/alpha1S II-III loop chimera was confirmed in the Ca2+-conducting alpha1C/alpha1S II-III loop variant using Cd2+ (10(-4) M) as the Ca2+ current blocker. In contrast to the behavior of the II-III loop chimera, Ca2+ transients expressed by an alpha1C/alpha1S chimera carrying all tested skeletal alpha1S domains (all alpha1S interrepeat loops, N- and C-terminus) were similar in shape and amplitude to wild-type alpha1S, and did not change in the presence of the E736K mutation or in the presence of 10(-4) M Cd2+. Controls indicated that similar dihydropyridine receptor charge movements were expressed by the non-Ca2+ permeant alpha1S(E1014K) variant, the alpha1C(E736K)/alpha1S II-III loop chimera, and the alpha1C(E736K)/alpha1S chimera carrying all tested alpha1S domains. The data indicate that the functional recovery produced by the alpha1S II-III loop is incomplete and that multiple cytosolic domains of alpha1S are necessary for a quantitative recovery of the EC-coupling phenotype of skeletal myotubes. Thus, despite the importance of the II-III loop there may be other critical determinants in alpha1S that influence the efficiency of EC coupling.

Aniline Compounds↗

PubMed1995

Omega loops: nonregular secondary structures significant in protein function and stability.

Omega (omega-) loops, a nonregular secondary structure found in globular proteins, are characterized by a polypeptide chain that follows a loop-shaped course in three-dimensional space. They do not contain repeating backbone dihedral angles or regular patterns of hydrogen bonding; however, many omega-loops contain a large number of hydrogen bonds, therefore it is not correct to think of omega-loops as structures lacking in hydrogen bonds. omega-Loops are found almost exclusively at the protein surface and exhibit amino acid preferences consistent with this observation. Since the first description of omega-loops in 1986, experiments have been conducted to probe the role of these structures in protein function, stability, and folding. It has become clear that omega-loops are often involved in protein function and molecular recognition. One motif, an omega-loop lid, that is flexible and mobile until substrate or inhibitor is bound and which probably plays a role in one or more steps of enzymatic catalysis, has been described in a variety of enzymes. Because they lack the periodic hydrogen bonding patterns of the regular secondary structures, some omega-loops are well suited for such functional roles in proteins. However, loops with a higher-than-average number of hydrogen bonds or hydrophobic contacts may play roles in protein stability or folding. Rather than determining further geometric definitions of loops, it may be instructional to group them according to their roles in protein structure, i.e., as categories of functional omega-loops, stability omega-loops, and folding omega-loops.

Amino Acids↗

PubMed2025

Proteins driving liquid-liquid phase separation and histone modifications cooperatively associate with chromatin looping and transcriptional regulation.

BACKGROUND: Although liquid-liquid phase separation (LLPS) proteins are known to participate in genome organization and transcriptional regulation through the formation of biomolecular condensates, their functional interplay with other regulatory proteins and histone modifications in chromatin loop formation remains poorly characterized. By combining Hi-C chromatin interaction data with ChIP-seq profiles of 12, 27, and 24 LLPS proteins in GM12878, K562, and HepG2 cell lines, respectively, we identified chromatin loops associated with LLPS proteins and systematically analysed patterns of cooperative protein binding and histone modification enrichment within these loop-associated peaks. RESULTS: We identified 162, 313, and 431 chromatin loops associated with LLPS proteins in GM12878, K562, and HepG2 cell lines, respectively. These loops were relatively small in size and predominantly anchored at enhancer regions. Examination of cooperative binding of proteins within loop-associated peaks revealed that transcriptional repressor IKZF1, HDAC1, and SAP130 most frequently co-localized with LLPS proteins in GM12878, K562, and HepG2 cells, respectively. Further analysis of histone modification enrichment patterns revealed that active histone modifications, such as H3K4me2, H3K4me3, H3K9ac, and H3K27ac, co-localized at loop-associated peaks, with H3K4me1 exhibiting additional specific co-localization with these four histone modifications at enhancer-localized loop-associated peaks. Notably, bivalent chromatin domains where H3K27me3 co-localized with active histone modifications were identified at promoter-localized loop-associated peaks in HepG2 cells, and elevated H3K27me3 occupancy at these peaks was associated with transcriptional repression of target genes. Moreover, quantitative RNA-seq analysis revealed that the expression of target genes associated with enhancer-promoter loops was correlated with both the binding of LLPS proteins and the enrichment patterns of histone modifications within their ChIP-seq peaks at loop anchors. CONCLUSIONS: Our study suggests that LLPS proteins may cooperate with transcriptional repressors to facilitate chromatin looping. Furthermore, local enrichment of histone modifications at loop-associated peaks provides additional regulatory control over chromatin architecture and gene transcription.

Humans↗

PubMed1996

RNA loop structure prediction via bond scaling and relaxation.

We have developed a method for predicting the structure of small RNA loops that can be used to augment already existing RNA modeling techniques. The method requires no input constraints on loop configuration other than end-to-end distance. Initial loop structures are generated by randomizing the torsion angles, beginning at one end of the polynucleotide chain and correlating each successive angle with the previous. The bond lengths of these structures are then scaled to fit within the known end constraints and the equilibrium bond lengths of the potential energy function are scaled accordingly. Through a series of rescaling and minimization steps the structures are allowed to relax to lower energy configurations with standard bond lengths and reduced van der Waals clashes. This algorithm has been tested on the variable loops of yeast tRNA-Asp and yeast tRNA-Phe, as well as the sarcin-ricin tetraloop and the anticodon loop of yeast tRNA-Phe. The results indicate good correlation between potential energy and the loop structure predictions that are closest to the variable loop crystal structures, but poorer correlation for the more isolated stem loops. The number of stacking interactions has proven to be a good objective measure of the best loop predictions. Selecting on the basis of energy and stacking, we obtain two structures with 0.65 and 0.75 A all-atom rms deviations (RMSD) from the crystal structure for the tRNA-Asp variable loop. The best structure prediction for the tRNA-Phe variable loop has an all-atom RMSD of 2.2 A and a backbone RMSD of 1.6 A, with a single base responsible for most of the deviation. For the sarcin-ricin loop from 28S ribosomal RNA, the predicted structure's all-atom RMSD from the nmr structure is 1.0 A. We obtain a 1.8 A RMSD structure for the tRNA-Phe anticodon loop.

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