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PubMed1999

Dimerization of HIV-1 genomic RNA of subtypes A and B: RNA loop structure and magnesium binding.

Retroviruses encapsidate their genome as a dimer of homologous RNA molecules noncovalently linked close to their 5' ends. The dimerization initiation site (DIS) of human immunodeficiency virus type 1 (HIV-1) RNA is a hairpin structure that contains in the loop a 6-nt self-complementary sequence flanked by two 5' and one 3' purines. The self-complementary sequence, as well as the flanking purines, are crucial for dimerization of HIV-1 RNA, which is mediated by formation of a "kissing-loop" complex between the DIS of each monomer. Here, we used chemical modification interference, lead-induced cleavage, and three-dimensional modeling to compare dimerization of subtype A and B HIV-1 RNAs. The DIS loop sequences of these RNAs are AGGUGCACA and AAGCGCGCA, respectively. In both RNAs, ethylation of most but not all phosphate groups in the loop and methylation of the N7 position of the G residues in the self-complementary sequence inhibited dimerization. These results demonstrate that small perturbations of the loop structure are detrimental to dimerization. Conversely, methylation of the N1 position of the first and last As in the loop were neutral or enhanced dimerization, a result consistent with these residues forming a noncanonical sheared base pair. Phosphorothioate interference, lead-induced cleavage, and Brownian-dynamics simulation revealed an unexpected difference in the dimerization mechanism of these RNAs. Unlike subtype B, subtype A requires binding of a divalent cation in the loop to promote RNA dimerization. This difference should be taken into consideration in the design of antidimerization molecules aimed at inhibiting HIV-1 replication.

Base Sequence↗
PubMed2004

Phase-shift fiber-loop ring-down spectroscopy.

Fiber-loop ring-down spectroscopy (FLRDS) is a recently developed absorption spectroscopic technique suitable for very small liquid samples. It is based on measurements of the optical decay constant of laser intensity in a loop made of optical waveguide material. This decay constant changes as small liquid samples containing absorbing species are introduced into the loop. In this report, it is demonstrated that one can also obtain the optical decay constant using a continuous wave laser beam that is intensity modulated and then coupled into an optical fiber loop. The inherent exponential decay in the fiber loop introduces a phase shift of the light emitted from the loop with respect to the pumping beam. By measuring this phase shift, one can readily determine the concentration of the analyte introduced between the two fiber ends and a model is established to describe the relationship. It is demonstrated that this technique, dubbed phase-shift fiber-loop ring-down spectroscopy (PS-FLRDS), is well suited as an absorption detector for any flow system in which the optical absorption path is limited by the instrument architecture. By measuring the phase angle as a function of concentration of 1,1'-diethyl-4,4'-dicarbocyanine iodide in dimethyl sulfoxide, the detection limit was determined as approximately 6 microM for a 30-40-microm absorption path. A temporal resolution of approximately 100 ms was demonstrated by a rapid displacement of the solutions between the two fiber ends. Proof-of-principle use of the PS-FLRDS detection in capillary flow systems using a commercial four-way microcross established that the alignment of the fiber and the capillary can be made simple and effective, while retaining both a low detection limit and a fast response.

Journal Article↗
PubMed1995

Analysis of the role of the KMSKS loop in the catalytic mechanism of the tyrosyl-tRNA synthetase using multimutant cycles.

A mobile loop in tyrosyl-tRNA synthetase, which corresponds to the KMSKS signature sequence of class I aminoacyl-tRNA synthetases, destabilizes the E.Tyr.ATP complex but stabilizes the following E.[Tyr-ATP]not equal to transition state for the formation of E.Tyr-AMP. Three amino acid residues in the mobile loop, K230, K233, and T234, are known to be primarily responsible for these effects. We now analyze the network of interactions between these three amino acids using multiple mutant free energy cycles. The complete characterization of the coupling energies within the mobile loop allows each of the steps leading to the formation of the transition state complex to be dissected into its energetic components. In particular, it is found that, in the absence of a functional mobile loop, there is synergistic coupling between the tyrosine and ATP substrates (i.e., each enhances the binding affinity of the other) which stabilizes the E.Tyr.ATP intermediate preceding the transition state complex. Thus, the mobile loop disrupts the synergism between the ATP and tyrosine substrates, using the ATP binding energy to stabilize the transition state for the reaction. Whereas the net effect of the mobile loop in the E.Tyr.ATP complex results from several conflicting side chain interactions that tend to offset each other, conflicting interactions in the E.[Tyr-ATP]not equal to transition state complex have been minimized and stabilizing pairwise interactions between the K230, K233, and T234 side chains are optimized. The tight coupling between the side chains of K230, K233, and T234 suggests that the mobile loop adopts a highly constrained conformation during formation of the transition state complex. These results quantitatively demonstrate the importance of side chain interactions in enzyme catalysis and illustrate the use of binding energy to stabilize the transition state of a reaction and the presence of unfavorable interactions to destabilize the ground state.

Adenosine Triphosphate↗
PubMed2000

Factors affecting the thermodynamic stability of small asymmetric internal loops in RNA.

Optical melting experiments were used to determine the thermodynamic parameters for oligoribonucleotides containing small asymmetric internal loops. The results show a broad range of thermodynamic stabilities, which depend on loop size, asymmetry, sequence, closing base pairs, and length of helix stems. Imino proton NMR experiments provide evidence for possible hydrogen bonding in GA and UU mismatches in some asymmetric loops. The stabilizing effects of GA, GG, and UU mismatches on the thermodynamic stability of internal loops vary depending on the size and asymmetry of the loop. The dependence of loop stability on Watson-Crick closing base pairs may be explained by an account of hydrogen bonds. Models are presented for approximating the free energy increments of 2 x 3 and 1 x 3 internal loops.

Adenine↗
PubMed1993

Flexibility impaired by mutations revealed the multifunctional roles of the loop in glutathione synthetase.

The loop from Ile-226 to Arg-241 in the glutathione synthetase (GSHase) from Escherichia coli B is rich in glycine and alanine and too flexible to take a fixed conformation [Yamaguchi, H., Kato, H., Hata, Y., Nishioka, T., Kimura, A., Oda, J., & Katsube, Y. (1993) J. Mol. Biol. 229, 1083-1100; Tanaka, T., Kato, H., Nishioka, T., & Oda, J. (1992) Biochemistry 31, 2259-2265]. To restrict the flexibility, three residues in the loop, Pro-227, Gly-229, and Gly-240, were replaced with alanine and valine residues. Variability in conformations of the mutant loops and shifts in the distribution of conformers between the open and closed states were assessed by steady-state kinetics, X-ray crystallographic structure analysis, and proteolysis with arginyl endopeptidase. Mutant enzymes replaced with a valine residue at the basal positions of the loop (P227V, G240V, and P227V/G240V) were identical with the wild-type enzyme in their crystal structures, except the loop region. The mutant loops retained apparent conformational variability, so as to take the open and closed states and to protect the acyl phosphate intermediate from the decomposition uncoupled from glutathione synthesis, but lost the catalytic activity; Kmapp values for glycine and gamma-Glu-Cys were sensitive to the mutations and drastically increased, and the k0app value was fatally reduced in the P227V/G240V mutant enzyme. The present results suggest that adjustability of the loop to the closed state is required for the recognition of the substrates, gamma-Glu-Cys and glycine, and for the chemical interactions with the bound substrates.

Amino Acid Sequence↗
PubMed1992

Solution structure of a trinucleotide A-T-A bulge loop within a DNA duplex.

We have synthesized an oligodeoxynucleotide duplex, d(G-C-A-T-C-G-A-T-A-G-C-T-A-C-G).d(C-G-T-A-G-C-C-G-A-T-C-G), with a three-base bulge loop (A-T-A) at a central site in the first strand. Nuclear Overhauser experiments (NOESY) in H2O indicate that the GC base pairs flanking the bulge loop are intact between 0 and 25 degrees C. Nuclear Overhauser effects in both H2O and D2O indicate that all bases within the bulge loop are stacked into the helix. These unpaired bases retain an anti conformation about their glycosidic bonds as they stack within the duplex. The absence of normal sequential connectivities between the two cytosine residues flanking the bulge site on the opposite strand indicates a disruption in the geometry of this base step upon insertion of the bulged bases into the helix. This conformational perturbation is more akin to a shearing apart of the bases, which laterally separates the two halves of the molecule, rather than the "wedge" model often invoked for single-base bulges. Using molecular dynamics calculations, with both NOE-derived proton-proton distances and relaxation matrix-calculated NOESY cross peak volumes as restraints, we have determined the solution structure of an A-T-A bulge loop within a DNA duplex. The bulged bases are stacked among themselves and with the guanine bases on either side of the loop. All three of the bulged bases are displaced by 2-3 A into the major groove, increasing the solvent accessibility of these residues. The ATA-bulge duplex is significantly kinked at the site of the lesion, in agreement with previously reported electron microscopy and gel retardation studies on bulge-containing duplexes [Hsieh, C.-H., & Griffith, J. D. (1989) Proc. Natl. Acad. Sci. U.S.A 86, 4833-4837; Bhattacharyya, A., & Lilley, D. M. J. (1989) Nucleic Acids Res. 17, 6821-6840]. Bending occurs in a direction away from the bulge-containing strand, and we find a significant twist difference of 84 degrees between the two base pairs flanking the bulge loop site. This value represents 58% of the twist difference for base pairs four steps apart in B-DNA. These results suggest a structural mechanism for the bending of DNA induced by unpaired bases, as well as accounting for the effect bulge loops may have on the secondary and tertiary structures of nucleic acids.

Adenine↗
PubMed1992

Segmental movement: definition of the structural requirements for loop closure in catalysis by triosephosphate isomerase.

To determine what drives the closure of the active-site loop in the reaction catalyzed by triosephosphate isomerase, several residues involved in hydrogen bonding between the loop and the bulk of the protein have been altered. It was known from earlier work that the loop serves two functions: to stabilize the reaction intermediate (and the two transition states that flank it) and to prevent the loss of this unstable species into free solution. To discover what elements of the protein are necessary for proper closure of the loop, selective destabilization of the "open" and the "closed" forms of the enzyme with respect to one another has been attempted. The mutant Y164F isomerase has been prepared to evaluate the importance of the structure of the "open" form, and the mutant E129Q, Y208F, and S211A enzymes have allowed investigation of the "closed" form. The integrity of the loop itself has been destabilized by making the T172A isomerase. We have found that only those mutations that destabilize the "closed" form of the enzyme significantly perturb the catalytic properties of the isomerase. The second-order rate constants (kcat/Km) of the S211A and E129Q enzymes are reduced 30-fold, and that of the mutant Y208F enzyme is reduced 2000-fold, from the level of the wild-type enzyme. The dramatic drop in activity of the Y208F enzyme is accompanied by a 200-fold increase in the dissociation constant of the intermediate analogue phosphoglycolohydroxamate. The most important property of the mobile loop of triosephosphate isomerase lies, therefore, in the stability of the system when the active site contains ligand and the loop is closed.

Amino Acid Sequence↗
PubMed1994

Dynamics of a flexible loop in dihydrofolate reductase from Escherichia coli and its implication for catalysis.

Apo-dihydrofolate reductase from Escherichia coli samples two distinct environments slowly on the NMR time scale at room temperature. Several assigned resonances belong to residues in, or proximal to, a loop (loop I) which is comprised of residues 9-24. This exchange process was altered (either removed or made fast on the NMR time scale) by deleting three hairpin turn forming residues from the loop and filling the gap with a single glycine [Li, L., Falzone, C. J., Wright, P. E., & Benkovic, S. J. (1992) Biochemistry 31, 7826-7833]. An approximate value of 35 s-1 for the exchange rate associated with loop I in apo-DHFR was obtained in two-dimensional nuclear Overhauser spectra by analyzing the time dependence of the cross-peak volume for N epsilon H of Trp-22, a residue which is located in this loop and which has resolved cross-peaks. Owing to the critical role that this loop plays in catalysis, the correspondence between this rate of conformational exchange and off-rates for tetrahydrofolate and the reduced nicotinamide cofactor from product and substrate complexes suggests that loop movement may be a limiting factor in substrate turnover.

Catalysis↗
PubMed1991

Refolded HIV-1 tat protein protects both bulge and loop nucleotides in TAR RNA from ribonucleolytic cleavage.

Substantial evidence indicates that HIV-1 trans-activation by tat protein is mediated through the TAR RNA element. This RNA forms a stem-loop structure containing a three-nucleotide bulge and a six-nucleotide loop. Previous mutagenic analysis of TAR indicates that the bulge residues and a 4 bp segment of the stem constitute, in part, the tat binding site. However, there appears to be no sequence-specific contribution of the six-base loop. We have employed a ribonuclease protection technique to explore the interaction of tat with single-stranded regions of TAR. The results indicate that tat interacts with both the bulge and loop regions of TAR. Treatment of TAR RNA with RNase A results in cleavage at U23 and U31, located in the bulge and loop regions, respectively. High concentrations (approximately 2 microM) of Escherichia coli derived tat protein, prepared by standard procedures, gave complete protection of TAR RNA from RNase A cleavage. However, under these conditions, truncated TAR derivatives in which no stem-loop structure is expected to form were also protected, indicating nonspecific binding. In order to obtain a tat preparation with enhanced specificity toward TAR RNA, methods were developed for refolding the recombinant protein. This treatment enhanced the affinity of tat for TAR by approximately 30-fold [Kd(apparent) less than 25 nM] and markedly increased its specificity for the TAR. Again, tat protected TAR RNA from RNase A cleavage at both U23 and U31. Protection was also observed with RNase T1 which cleaves TAR RNA at three G residues in the six-base loop.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1988

Kinetics of disulfide exchange reactions of monomer and dimer loops of cysteine-valine-cysteine peptides.

Rate constants have been determined in 3 M guanidine hydrochloride for disulfide exchange reactions between glutathione and two synthetic peptides containing a cysteine-valine-cysteine region. Equilibrium experiments demonstrate the absence of noncovalent peptide aggregation in this solvent. Procedures are given for separating seven different components in quenched reactions, including the fully reduced cysteine cluster, the monomeric disulfide loop, parallel and antiparallel dimer loops, and the three monomers containing one or two mixed disulfides with glutathione. Intramolecular rate constants for (1) formation of a sterically strained monomer loop, (2) transfer of glutathione between the two cysteines on the same peptide chain, and (3) formation of unstrained dimer loops correspond to a series of processes forming rings of increasing size. In one sequence, these rate constants are 3, 6, and about 21 s-1, respectively. The larger loops are formed more easily. In the other sequence, rate constants for formation and opening of monomer loops are accelerated 180- and 1300-fold, respectively, relative to analogous reactions in a peptide containing eight residues between the two cysteines. This gives a 7-fold smaller equilibrium constant for ring closure in the cysteine cluster. Dimer formation occurs by a mechanism utilizing the accelerated opening of monomer loops. Results provide information assisting efforts to develop strategies for directing disulfide pairing in novel protein structures. Results also help define factors contributing to formation of undesired oligomers during efforts to refold cysteine-containing proteins obtained by bacterial expression of mammalian genes.

Chromatography, High Pressure Liquid↗
PubMed1989

Effects of base sequence on the loop folding in DNA hairpins.

High-resolution NMR and UV-melting experiments have been used to study the hairpin formation of partly self-complementary DNA fragments in an attempt to derive rules that describe the folding in these molecules. Earlier experiments on the hexadecanucleotide d(ATCCTA-TTTT-TAGGAT) had indicated that within the loop of four thymidines a wobble T-T pair is formed (Blommers et al., 1987). In the present paper it is shown that if the first and the last thymines of the intervening sequence are replaced by complementary bases, sometimes base pairs can be formed. Thus for the intervening sequences -CTTG- and -TTTA- with the pyrimidine in the 5'-position and the purine in the 3'-position, a base pair is formed leading to a loop consisting of two residues. For the intervening sequences -GTTC- and -ATTT- with the purine in the 5'-position and the pyrimidine in the 3'-position, this turns out not to be the case. It was found that it made no difference when the four-membered sequence was closed by a G-C base pair or an A-T base pair. Replacement of the two central thymidine residues by the more bulky adenine residues limits the hairpin to a four-membered loop scheme. Very surprisingly, it was found from 2D NOE experiments that the T-A base pair, formed in the loop consisting of the -TTTA- sequence, is a Hoogsteen pair. It is argued that the pairing of the bases in this scheme may facilitate the formation of a loop of two residues, since the distance of the C1' atoms in this base pair is 8.6 A instead of 10.4 A found in the canonical Watson-Crick base pair. Combination of the data obtained for the series of DNA fragments studied shows that the results can be explained by a simple, earlier proposed, loop folding principle which assumes that the folding of the four-membered loop is dictated by the stacking of the double-helical stem of the hairpin.

Base Sequence↗
PubMed2001

Thermodynamic stabilities of internal loops with GU closing pairs in RNA.

Many internal loops that form tertiary contacts in natural RNAs have GU closing pairs; examples include the tetraloop receptor and P1 helix docking site in group I introns. Thus, thermodynamic parameters of internal loops with GU closing pairs can contribute to the prediction of both secondary and tertiary structure. Oligoribonucleotide duplexes containing small internal loops with GU closing pairs were studied by optical melting, one-dimensional imino proton NMR, and one-dimensional phosphorus NMR. The thermodynamic stabilities of asymmetric internal loops with GU closing pairs relative to those of loops with GC closing pairs may be explained by hydrogen bonds. In contrast, the free energy increments for symmetric internal loops of two noncanonical pairs with GU closing pairs relative to loops with GC closing pairs show much more sequence dependence. Imino proton and phosphorus NMR spectra suggest that some GA pairs adjacent to GU closing pairs may form an overall thermodynamically stable but non-A-form conformation.

Base Pairing↗
PubMed2004

Functional role of loop 2 in myosin V.

Myosin V is molecular motor that is capable of moving processively along actin filaments. The kinetics of monomeric myosin V containing a single IQ domain (MV 1IQ) differ from nonprocessive myosin II in that actin affinity is higher, phosphate release is extremely rapid, and ADP release is rate-limiting. We generated two mutants of myosin V by altering loop 2, a surface loop in the actin-binding region thought to alter actin affinity and phosphate release in myosin II, to determine the role that this loop plays in the kinetic tuning of myosin V. The loop 2 mutants altered the apparent affinity for actin (K(ATPase)) without altering the maximum ATPase rate (V(MAX)). Transient kinetic analysis determined that the rate of binding to actin, as well as the affinity for actin, was dependent on the net positive charge of loop 2, while other steps in the ATPase cycle were unchanged. The maximum rate of phosphate release was unchanged, but the affinity for actin in the M.ADP.Pi-state was dramatically altered by the mutations in loop 2. Thus, loop 2 is important for allowing myosin V to bind to actin with a relatively high affinity in the weak binding states but does not play a direct role in the product release steps. The ability to maintain a high affinity for actin in the weak binding states may prevent diffusion away from the actin filament and increase the degree of processive motion of myosin V.

Actins↗
PubMed2004

Tyrosine phosphorylation of the Janus kinase 2 activation loop is essential for a high-activity catalytic state but dispensable for a basal catalytic state.

The phosphorylation of an "activation loop" within protein kinases is commonly associated with establishing catalytic competence, and phosphorylation of the Tyr(1007) residue in the activation loop of Janus kinase 2 (JAK2) has been shown to be essential for intracellular propagation of cytokine-initiated signaling. We provide evidence for the presence of a basal activity state of JAK2, which was observed in the absence of activation loop phosphorylation. Phosphorylation of the JAK2 activation loop was essential for conversion to the high-activity state, characterized by high-efficiency ATP utilization during autophosphorylation. Mutagenesis of activation loop tyrosine residues Tyr(1007/1008) to phenylalanine residues impaired, but did not abolish, the enzyme's ability to autophosphorylate. The activation loop mutant JAK2 could also transphosphorylate an inactive JAK2 fragment coexpressed in Sf21 cells, providing evidence of exogenous substrate phosphorylation. The mutant enzyme remained in a basal activity state characterized by low-efficiency ATP utilization during autophosphorylation. Mutagenesis of a critical Lys(882) residue to a glutamate residue abolished all evidence of kinase activity, confirming that the observed activity of Tyr-to-Phe mutants was not due to another kinase. Our data are consistent with the proposal that JAK2 is an inefficient but active enzyme in the absence of activation loop phosphorylation and is capable of conversion to a high-activity state by autophosphorylation under physiological ATP concentrations. This theoretically precludes the need for an upstream activating kinase. The activation process of JAK2 may be envisioned as a multistate process involving at least two kinetically distinct states of activity.

Adenosine Triphosphate↗
PubMed2004

Conformational changes in the active site loops of dihydrofolate reductase during the catalytic cycle.

Escherichia coli dihydrofolate reductase (DHFR) has several flexible loops surrounding the active site that play a functional role in substrate and cofactor binding and in catalysis. We have used heteronuclear NMR methods to probe the loop conformations in solution in complexes of DHFR formed during the catalytic cycle. To facilitate the NMR analysis, the enzyme was labeled selectively with [(15)N]alanine. The 13 alanine resonances provide a fingerprint of the protein structure and report on the active site loop conformations and binding of substrate, product, and cofactor. Spectra were recorded for binary and ternary complexes of wild-type DHFR bound to the substrate dihydrofolate (DHF), the product tetrahydrofolate (THF), the pseudosubstrate folate, reduced and oxidized NADPH cofactor, and the inactive cofactor analogue 5,6-dihydroNADPH. The data show that DHFR exists in solution in two dominant conformational states, with the active site loops adopting conformations that closely approximate the occluded or closed conformations identified in earlier X-ray crystallographic analyses. A minor population of a third conformer of unknown structure was observed for the apoenzyme and for the disordered binary complex with 5,6-dihydroNADPH. The reactive Michaelis complex, with both DHF and NADPH bound to the enzyme, could not be studied directly but was modeled by the ternary folate:NADP(+) and dihydrofolate:NADP(+) complexes. From the NMR data, we are able to characterize the active site loop conformation and the occupancy of the substrate and cofactor binding sites in all intermediates formed in the extended catalytic cycle. In the dominant kinetic pathway under steady-state conditions, only the holoenzyme (the binary NADPH complex) and the Michaelis complex adopt the closed loop conformation, and all product complexes are occluded. The catalytic cycle thus involves obligatory conformational transitions between the closed and occluded states. Parallel studies on the catalytically impaired G121V mutant DHFR show that formation of the closed state, in which the nicotinamide ring of the cofactor is inserted into the active site, is energetically disfavored. The G121V mutation, at a position distant from the active site, interferes with coupled loop movements and appears to impair catalysis by destabilizing the closed Michaelis complex and introducing an extra step into the kinetic pathway.

Catalytic Domain↗
PubMed2004

A duck delta1 crystallin double loop mutant provides insight into residues important for argininosuccinate lyase activity.

Delta-crystallin is directly related to argininosuccinate lyase (ASL), and catalyzes the reversible hydrolysis of argininosuccinate to arginine and fumarate. Two delta-crystallin isoforms exist in duck lenses, delta1 and delta2, which are 94% identical in amino acid sequence. Although the sequences of duck delta2-crystallin (ddeltac2) and duck delta1-crystallin (ddeltac1) are 69 and 71% identical to that of human ASL, respectively, only ddeltac2 has maintained ASL activity. Domain exchange experiments and comparisons of various delta-crystallin structures have suggested that the amino acid substitutions in the 20's (residues 22-31) and 70's (residues 74-89) loops of ddeltac1 are responsible for the loss of enzyme activity in this isoform. To test this hypothesis, a double loop mutant (DLM) of ddeltac1 was constructed in which all the residues that differ between the two isoforms in the 20's and 70's loops were mutated to those of ddeltac2. Contrary to expectations, kinetic analysis of the DLM found that it was enzymatically inactive. Furthermore, binding of argininosuccinate by the DLM, as well as the ddeltac1, could not be detected by isothermal titration calorimetry (ITC). To examine the conformation of the 20's and 70's loops in the DLM, and to understand why the DLM is unable to bind the substrate, its structure was determined to 2.5 A resolution. Comparison of this structure with both wild-type ddeltac1 and ddeltac2 structures reveals that the conformations of the 20's and 70's loops in the DLM mutant are very similar to those of ddeltac2. This suggests that the five amino acid substitutions in domain 1 which lie outside of the two loop regions and which are different in the DLM, and ddeltac2, must be important enzymatically. The structure of the DLM in complex with sulfate was also determined to 2.2 A resolution. This structure demonstrates that the conformational changes of the 280's loop and domain 3, previously observed in ddeltac1, also occur in the DLM upon sulfate binding, reinforcing the hypothesis that these events may occur in the active ddeltac2 protein during catalysis.

Amino Acid Sequence↗
PubMed2004

C-terminus loop 13 of Na+ glucose cotransporter SGLT1 contains a binding site for alkyl glucosides.

Recently, we identified the extramembranous C-terminus loop 13 of SGLT1 as a binding site for the aromatic glucoside phlorizin, which competitively inhibits sodium D-glucose cotransport. Alkyl glucosides are also competitive inhibitors of the transport. Therefore, in this study, we searched for potential binding sites for alkyl glucosides in loop 13. To this end, we synthesized a photoaffinity label (2'-Azi-n-octyl)-beta-D-glucoside and analyzed the region of attachment using MALDI mass spectrometry, producing wild-type recombinant truncated loop 13. Furthermore, we prepared four single-Trp mutants of the loop and determined their fluorescence, its change in the presence of alkyl glucosides, and their accessibility to acrylamide. Photolabeling of truncated loop 13 with (2'-Azi-n-octyl)-beta-D-glucoside revealed an attachment of the C2 group of the alkyl chain to Gly-Phe-Phe-Arg (amino acid residues 598-601). In the presence of n-hexyl-beta-D-glucoside, all mutants (R601W, D611W, E621W, and L630W) exhibited a significant decrease in Trp fluorescence with an apparent binding affinity of 8-14 microM. Only L630W exhibited a significant blue shift, and only in R601W was a change in acrylamide quenching (protection) observed. No quenching or protection was found for D-glucose; however, 1-hexanol produced the same results as n-hexyl-beta-D-glucoside. The interaction shows stereoselectivity for n-hexyl-beta-D-glucoside binding; the beta-configuration of the sugar moiety at C1, the cis conformation of the unsaturated alkenyl side chain in the C3-C4 bond, and the alkyl chain length of six to eight carbon atoms lead to an optimum interaction. A schematic two-dimensional model was derived in which C2 interacts with the region around residue 601, C3 and C4 interact with the region between residues 614 and 619, and C6-C8 interact with the region between residues 621 and 630. The data demonstrate that loop 13 provides binding sites for alkyl glucosides as well as for phlorizin; thus, loop 13 of SGLT1 seems to be a major binding domain for the aglucone residues of competitive D-glucose transport inhibitors.

Acrylamide↗
PubMed2004

Structural features and thermodynamics of the J4/5 loop from the Candida albicans and Candida dubliniensis group I introns.

The J4/5 loop of group I introns has tertiary interactions with the P1 helix that position the P1 substrate for the self-splicing reaction. The J4/5 loop of Candida albicans and Candida dubliniensis, 5'GAAGG3'/3'UAAUU5', potentially contains two A.A pairs flanked by one G.U pair on one side and two G.U pairs on the other side. Results from optical melting, nuclear magnetic resonance spectroscopy, and functional group substitution experiments with a mimic of the C. albicans and C. dubliniensis J4/5 loop are consistent with the adenosines forming tandem sheared A.A pairs with a cross-strand stack and only the G.U pair not adjacent to an A.A pair forming a static wobble G.U pair. The two G.U pairs adjacent to the tandem A.A pairs are likely in a dynamic equilibrium between multiple conformations. Although Co(NH(3))(6)(3+) stabilizes the loop by several kilocalories per mole at 37 degrees C, addition of Mg(2+) or Co(NH(3))(6)(3+) has no effect on the structure of the loop. The tandem G.U pairs provide a pocket of negative charge for Co(NH(3))(6)(3+) to bind. The results contribute to understanding the structure and dynamics of purine-rich internal loops and potential G.U pairs adjacent to internal loops.

Base Pairing↗