Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “looping”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 793 records · Page 44Linked to original sources
PubMed2003

Generation of fiber-modified adenovirus vectors containing heterologous peptides in both the HI loop and C terminus of the fiber knob.

BACKGROUND: Fiber-modified adenovirus (Ad) vectors can be effective in overcoming the limitations of conventional Ad vectors, specifically their inefficient gene transfer into cells lacking the primary receptor, the coxsackievirus and adenovirus receptor (CAR). Several types of fiber-modified Ad vectors have been developed. In this study, we evaluated the functionality of several fiber-modified Ad vectors. METHODS: We developed a simple method based on in vitro ligation to construct Ad vectors containing heterologous foreign peptides in both the HI loop and C terminus of the fiber knob. A functional comparison of Ad vectors containing RGD and/or K7 (KKKKKKK) peptide in the HI loop or C terminus of the fiber knob was performed in several types of human, mouse, and rat cells, including CAR-positive and -negative cells, and tumor cells in mice in vivo. RESULTS: In the case of the in vitro experiment, Ad vectors containing RGD peptide in the HI loop of the fiber knob showed a higher level of gene transfer than vectors containing RGD peptide at the C terminus of the fiber knob. Ad vectors containing K7 peptide at the C terminus of the fiber knob showed levels of gene transfer similar to those of Ad vectors containing RGD peptide in the HI loop of the fiber knob, depending on the cell type. Ad vectors containing both peptides in the HI loop or C terminus of the fiber knob showed the highest levels of gene transfer and a broader tropism. For gene transfer into tumor cells in vivo, the Ad vectors containing RGD peptide were the most efficient. CONCLUSIONS: In the experiment using cultured cells, Ad vectors containing both RGD and K7 peptides were the most efficient with a broader tropism. In contrast, in the experiment in vivo, Ad vectors containing RGD peptide in the HI loop of the fiber knob were more efficient than the vectors containing K7 peptide (including double-modified vectors containing both the RGD and K7 peptides). These comparative analyses could provide a systemic reference for the use of fiber-modified Ad vectors. Our simple method, in which the peptide of interest can be expressed in Ad vectors in either the HI loop or the C terminus of the fiber knob, or both, could be a powerful tool for gene transfer into mammalian cells in studies of gene function as well as in gene therapy.

Adenoviridae↗
PubMedDate not supplied

Structural requirements for and consequences of an antiviral porphyrin binding to the V3 loop of the human immunodeficiency virus (HIV-1) envelope glycoprotein gp120.

Several porphyrin derivatives were reported to have anti-HIV-1 activity. Among them, meso-teta(4-carboxyphenyl)porphine (MTCPP) and other carboxyphenyl derivatives were the most potent inhibitors (EC50 <0.7 mu M). MTCPP bound to the HIV-1 envelope glycoprotein gp120 and to full-length V3 loop peptides corresponding to several HIV-1 isolates but not to other peptides from gp120 + gp41. However, it remained possible that MTCPP bound to regions on gp120 which cannot be mimicked by peptides. Further characterization of the binding domain for MTCPP is important for understanding the antiviral activity of porphyrins and for the design of anti-HIV-1 drugs interfering with functions of the virus envelope. Results presented here show that: (i) deletion of the V3 loop from the gp120 sequence resulted in drastically diminished MTCPP binding, suggesting that the V3 loop is the dominant if not the only target site on gp120; (ii) this site was only partially mimicked by full-length V3 loop peptides; (iii) MTCPP binding to the gp120 V3 loop elicited allosteric effects resulting in decreased accessibility of the CD4 receptor binding site; (iv) the binding site for MTCPP lies within the central portion of the V3 loop (KSIHIGPGRAFY for the HIV-1 subtype B consensus sequence) and does not involve directly the GPG apex of the loop. These results may help in designing antiviral compounds with improved activity.

Allosteric Regulation↗
PubMed1993

The structure and function of omega loop A replacements in cytochrome c.

The structural and functional consequences of replacing omega-loop A (residues 18-32) in yeast iso-1-cytochrome c with the corresponding loop of Rhodospirillum rubrum cytochrome c2 have been examined. The three-dimensional structure of this loop replacement mutant RepA2 cytochrome c, and a second mutant RepA2(Val 20) cytochrome c in which residue 20 was back substituted to valine, were determined using X-ray diffraction techniques. A change in the molecular packing is evident in the RepA2 mutant protein, which has a phenylalanine at position 20, a residue considerably larger than the valine found in wild-type yeast iso-1-cytochrome c. The side chain of Phe 20 is redirected toward the molecular surface, altering the packing of this region of omega-loop A with the hydrophobic core of the protein. In the RepA2(Val 20) structure, omega-loop A contains a valine at position 20, which restores the original wild-type packing arrangement of the hydrophobic core. Also, as a result of omega-loop A replacement, residue 26 is changed from a histidine to asparagine, which results in displacements of the main-chain atoms near residue 44 to which residue 26 is hydrogen bonded. In vivo studies of the growth rate of the mutant strains on nonfermentable media indicate that the RepA2(Val 20) cytochrome c behaves much like the wild-type yeast iso-1 protein, whereas the stability and function of the RepA2 cytochrome c showed a temperature dependence. The midpoint reduction potential measured by cyclic voltammetry of the RepA2 mutant is 271 mV at 25 degrees C. This is 19 mV less than the wild-type and RepA2(Val 20) proteins (290 mV) and may result from disruption of the hydrophobic packing in the heme pocket and increased mobility of omega-loop A in RepA2 cytochrome c. The temperature dependence of the reduction potential is also greatly enhanced in the RepA2 protein.

Amino Acid Sequence↗
PubMed2002

Positioning of anchor groups in protein loop prediction: the importance of solvent accessibility and secondary structure elements.

The prediction of loop regions in the process of protein structure prediction by homology is still an unsolved problem. In an earlier publication, we could show that the correct placement of the amino acids serving as an anchor group to be connected by a loop fragment with a predicted geometry is a highly important step and an essential requirement within the process (Lessel and Schomburg, Proteins 1999; 37:56-64). In this article, we present an analysis of the quality of possible loop predictions with respect to gap length, fragment length, amino acid type, secondary structure, and solvent accessibility. For 550 insertions and 544 deletions, we test all possible positions for anchor groups with an inserted loop of a length between 3 and 12 amino acids. We could show that approximately 80% of the indel regions could be predicted within 1.5 A RMSD from a knowledge-based loop data base if criteria for the correct localization of anchor groups could be found and the loops can be sorted correctly. From our analysis, several conclusions regarding the optimal placement of anchor groups become obvious: (1) The correct placement of anchor groups is even more important for longer gap lengths, (2) medium length fragments (length 5-8) perform better than short or long ones, (3) the placement of anchor groups at hydrophobic amino acids gives a higher chance to include the best possible loop, (4) anchor groups within secondary structure elements, in particular beta-sheets are suitable, (5) amino acids with lower solvent accessibility are better anchor group. A preliminary test using a combination of the anchor group positioning criteria deduced from our analysis shows very promising results.

Algorithms↗
PubMed2003

Generation of accurate protein loop conformations through low-barrier molecular dynamics.

Prediction and refinement of protein loop structures are important and challenging tasks for which no general solution has been found. In addition to the accuracy of scoring functions, the main problems reside in (1) insufficient statistical sampling and (2) crossing energy barriers that impede conformational rearrangements of the loop. We approach these two issues by using "low-barrier molecular dynamics," a combination of energy smoothing techniques. To address statistical sampling, locally enhanced sampling (LES) is used to produce multiple copies of the loop, thus improving statistics and reducing energy barriers. We introduce a novel extension of LES that can improve local sampling even further through hierarchical subdivision of copies. Even though LES reduces energy barriers, it cannot provide for crossing infinite barriers, which can be problematic when substantial rearrangement of residues is necessary. To permit this kind of loop residue repacking, a "soft-core" potential energy function is introduced, so that atomic overlaps are temporarily allowed. We tested this new combined methodology to a loop in anti-influenza antibody Fab 17/9 (7 residues long) and to another loop in the antiprogesterone antibody DB3 (8 residues). In both cases, starting from random conformations, we were able to locate correct loop structures (including sidechain orientations) with heavy-atom root-mean-square deviation (fit to the nonloop region) of approximately 1.1 A in Fab 17/9 and approximately 1.8 A in DB3. We show that the combination of LES and soft-core potential substantially improves sampling compared to regular molecular dynamics. Moreover, the sampling improvement obtained with this combined approach is significantly better than that provided by either of the two methods alone.

Algorithms↗
PubMed2006

Accounting for loop flexibility during protein-protein docking.

Although reliable docking can now be achieved for systems that do not undergo important induced conformational change upon association, the presence of flexible surface loops, which must adapt to the steric and electrostatic properties of a partner, generally presents a major obstacle. We report here the first docking method that allows large loop movements during a systematic exploration of the possible arrangements of the two partners in terms of position and rotation. Our strategy consists in taking into account an ensemble of possible loop conformations by a multi-copy representation within a reduced protein model. The docking process starts from regularly distributed positions and orientations of the ligand around the whole receptor. Each starting configuration is submitted to energy minimization during which the best-fitting loop conformation is selected based on the mean-field theory. Trials were carried out on proteins with significant differences in the main-chain conformation of the binding loop between isolated form and complexed form, which were docked to their partner considered in their bound form. The method is able to predict complexes very close to the crystal complex both in terms of relative position of the two partners and of the geometry of the flexible loop. We also show that introducing loop flexibility on the isolated protein form during systematic docking largely improves the predictions of relative position of the partners in comparison with rigid-body docking.

Binding Sites↗
PubMed2006

Long loop prediction using the protein local optimization program.

We have developed an improved sampling algorithm and energy model for protein loop prediction, the combination of which has yielded the first methodology capable of achieving good results for the prediction of loop backbone conformations of 11 residue length or greater. Applied to our newly constructed test suite of 104 loops ranging from 11 to 13 residues, our method obtains average/median global backbone root-mean-square deviations (RMSDs) to the native structure (superimposing the body of the protein, not the loop itself) of 1.00/0.62 A for 11 residue loops, 1.15/0.60 A for 12 residue loops, and 1.25/0.76 A for 13 residue loops. Sampling errors are virtually eliminated, while energy errors leading to large backbone RMSDs are very infrequent compared to any previously reported efforts, including our own previous study. We attribute this success to both an improved sampling algorithm and, more critically, the inclusion of a hydrophobic term, which appears to approximately fix a major flaw in SGB solvation model that we have been employing. A discussion of these results in the context of the general question of the accuracy of continuum solvation models is presented.

Algorithms↗
PubMed2000

Linkage between the hormone binding site and the reactive center loop of thyroxine binding globulin.

Thyroxine binding globulin (TBG) is the major carrier of the thyroid hormones triiodothyronine (T3) and thyroxine (T4) in plasma. TBG is member of the serpin family of proteins although it has no proteinase inhibitory activity. In this study we show that TBG has properties typical of a metastable serpin and provide evidence that occupancy of the hormone binding site alters the conformation of the reactive center loop. After reactive center loop cleavage by endoproteinase Asp-N or neutrophil elastase the protein became more stable to guanidine hydrochloride denaturation compared to the native protein, as a result of loop insertion. In addition, incubation of the native protein with a reactive center loop peptide, caused a change in mobility on a native gel. This is consistent with the idea that thyroxine binding globulin is able to form a binary complex with the peptide as a result of beta-sheet A expansion. To assess the effect of cleavage and loop insertion on the hormone binding site we used the specific binding of a fluorophore, 1,8-anilinonaphthalene sulfonic acid (ANS). Loop insertion itself had no effect on ANS affinity, but cleavage with elastase at the P4'-P5' bond caused a reduction in affinity, presumably because this cleavage site is located within the hormone binding site. These data support the concept that cleavage of TBG by proteinases released in inflammation is a mechanism to deliver thyroid hormones to target tissues. A linkage between the occupancy state of the hormone binding site and the conformation of the reactive center loop was indicated by the observation that binding of T3 to native TBG reduced proteolytic susceptibility by both endoproteinase Asp-N and elastase.

Anilino Naphthalenesulfonates↗
PubMed2001

The correlation between colposcopically directed cervical biopsy and loop electrosurgical excision procedure pathology and the effect of time on that agreement.

OBJECTIVE: The goal of this study was to evaluate whether colposcopically directed cervical biopsy accurately predicts histopathology found on loop electrosurgical excision procedure specimen and to assess whether this correlation was affected by a delay in treatment of greater than 12 weeks. METHODS: A retrospective review was performed of all cervical biopsy and loop electrosurgical excision procedure pairs performed at University of Arizona Medical Center between March 1992 and March 2000. Agreement was assessed by kappa statistics and Spearman rho coefficients. A subgroup analysis was performed to assess the effect of a delay greater than 12 weeks between cervical biopsy and loop excision on the histopathologic correlation. RESULTS: An 84% agreement was found between cervical biopsy histopathology and loop electrosurgical excision procedure histopathology when compared to within 1 degree (kappa = 0.78, P < 0.0001; Spearman rho = 0.40, P < 0.0001). This agreement remained strong (87%) even when loop electrosurgical excision procedure was delayed greater than 12 weeks (kappa = 0.81, P = 0.01). CONCLUSION: Colposcopically directed cervical biopsy correlated strongly with loop specimen histopathology. A delay in loop electrosurgical excision procedure greater than 12 weeks does not negatively affect this correlation. This delay of 12 weeks may be used safely in the future to test chemotherapeutic dysplasia treatments.

Adolescent↗
PubMed1995

Characterization of the pre-mRNA binding site for yeast ribosomal protein L32: the importance of a purine-rich internal loop.

The structure of the RNA binding target for Saccharomyces cerevisiae ribosomal protein L32 was examined using chemical and enzymatic probes as well as thermodynamic methods. In vivo, the production of yeast RPL32 is regulated by a feedback mechanism whereby RPL32 binds to the 5' end of its transcript and inhibits splicing. The binding site of ribosomal protein L32 on the L32 RNA transcript can be reduced to fewer than 30 nucleotides which compromise a stem-internal loop-stem structural motif. The internal loop is closed by a potential G-U pair, is asymmetric and contains mostly purines. The existence of the two helical regions was confirmed by chemical and enzymatic probing. The reactivity of the loop region suggests a structure intermediate between that of single and double-stranded RNA. Base stacking continues into the loop, but two loop bases are extremely reactive to chemical agents. The interaction between the model RNA and the protein is specific and has a dissociation constant of approximately 10 nM. Several of the loop bases are critical for protein binding, as demonstrated by mutational data and chemical protection and modification interference studies. The internal loop destabilizes the RNA, and allows the RNA to melt in an all-or-none fashion.

Aniline Compounds↗
PubMed1996

Affinity maturation of a high-affinity human monoclonal antibody against the third hypervariable loop of human immunodeficiency virus: use of phage display to improve affinity and broaden strain reactivity.

The present study set out to investigate whether phage display could be used to improve the properties of a high-affinity human monoclonal antibody directed against the third hypervariable loop (V3 loop) of human immunodeficiency virus (HIV). The aim was to increase affinity through slowing the dissociation rate (off-rate constant of koff), whilst retaining the ability of this antibody to bind diverse V3 loop sequences. When reformatted as a scFv, the antibody fragment retained the properties of the parental IgG, including the ability to neutralise virus. Heavy and light chains were sequentially replaced with repertoires of variable domains from non-immunised human donors followed by selection on biotinylated synthetic peptide. All selected variants derived from the same germline as the parental antibody. Variants of the light chain provided little if any improvement, whereas two residue changes in VHCDR2 and one in VHFR3 resulted in a reduced koff from gp120 protein of the MN strain (MNgp120) and synthetic V3 loop peptides as measured by surface plasmon resonance using the BIAcore instrument (Pharmacia Biosensor). VHCDR3 was modified using synthetic oligonucleotides and several clones with reduced koff identified, a number of different substitutions occurring at a single residue position. The residues in the heavy chain identified as reducing koff were simultaneously randomised by site-directed mutagenesis, resulting in scFv variants with koff slowed up to sevenfold. Far from compromising recognition of variant loops, binding to these sequences was improved; the koff from synthetic peptides modelled on V3 loop variants being slowed to a degree similar to that observed with MNgp120. All four changes were located towards either extremes of CDRs 2 and 3, suggesting that the mechanism of improvement may be one of alternation of loop conformation. This work illustrates that phage display can be used to tailor the properties of a therapeutic monoclonal antibody in a predefined fashion.

Amino Acid Sequence↗
PubMed1998

A common motif organizes the structure of multi-helix loops in 16 S and 23 S ribosomal RNAs.

Phylogenetic and chemical probing data indicate that a modular RNA motif, common to loop E of eucaryotic 5 S ribosomal RNA (rRNA) and the alpha-sarcin/ricin loop of 23 S rRNA, organizes the structure of multi-helix loops in 16 S and 23 S ribosomal RNAs. The motif occurs in the 3' domain of 16 S rRNA at positions 1345-1350/1372-1376 (Escherichia coli numbering), within the three-way junction loop, which binds ribosomal protein S7, and which contains nucleotides that help to form the binding site for P-site tRNA in the ribosome. The motif also helps to structure a three-way junction within domain I of 23 S, which contains many universally conserved bases and which lies close in the primary and secondary structure to the binding site of r-protein L24. Several other highly conserved hairpin, internal, and multi-helix loops in 16 S and 23 S rRNA contain the motif, including the core junction loop of 23 S and helix 27 in the core of 16 S rRNA. Sequence conservation and range of variation in bacteria, archaea, and eucaryotes as well as chemical probing and cross-linking data, provide support for the recurrent and autonomous existence of the motif in ribosomal RNAs. Besides its presence in the hairpin ribozyme, the loop E motif is also apparent in helix P10 of bacterial RNase P, in domain P7 of one sub-group of group I introns, and in domain 3 of one subgroup of group II introns.

Catalysis↗
PubMed1999

Using loop length variants to dissect the folding pathway of a four-helix-bundle protein.

Rop is a four-helix-bundle protein formed by the association of two helix-loop-helix monomers. The short helix-connecting loop was replaced with a series of polyglycine linkers of increasing length. These mutant proteins all appear to fold via the same general mechanism as that of the wild-type protein, even at the longest loop lengths. Replacement of the wild-type two-residue loop (Asp-Ala) with a (Gly-Gly) linker accelerates both unfolding and refolding rates. These changes in folding and unfolding kinetics likely indicate an alteration in the energy of the transition state. As the length of the glycine linker is further increased, the unfolding rate increases while the refolding rates decrease. The influence of loop length is not limited to these rates, but also impacts upon the stability of the folding intermediate. These dependences underscore the importance of loop closure and help refine the model for Rop's folding, implicating a dimeric intermediate involving hairpin formation. These observations show that loop alteration may be useful as a general technique for dissecting protein folding pathways.

Escherichia coli↗
PubMed1999

Structure-activity relationships of omega-conotoxins MVIIA, MVIIC and 14 loop splice hybrids at N and P/Q-type calcium channels.

The omega-conotoxins are a set of structurally related, four-loop, six cysteine containing peptides, that have a range of selectivities for different subtypes of the voltage-sensitive calcium channel (VSCC). To investigate the basis of the selectivity displayed by these peptides, we have studied the binding affinities of two naturally occurring omega-conotoxins, MVIIA and MVIIC and a series of 14 MVIIA/MVIIC loop hybrids using radioligand binding assays for N and P/Q-type Ca2+channels in rat brain tissue. A selectivity profile was developed from the ratio of relative potencies at N-type VSCCs (using [125I]GVIA radioligand binding assays) and P/Q-type VSCCs (using [125I]MVIIC radioligand binding assays). In these peptides, loops 2 and 4 make the greatest contribution to VSCC subtype selectivity, while the effects of loops 1 and 3 are negligible. Peptides with homogenous combinations of loop 2 and 4 display clear selectivity preferences, while those with heterogeneous combinations of loops 2 and 4 are less discriminatory. 1H NMR spectroscopy revealed that the global folds of MVIIA, MVIIC and the 14 loop hybrid peptides were similar; however, several differences in local structure were identified. Based on the binding data and the 3D structures of MVIIA, GVIA and MVIIC, we have developed a preliminary pharmacophore based on the omega-conotoxin residues most likely to interact with the N-type VSCC.

Amino Acid Sequence↗
PubMed1994

Mapping of a strain-specific bactericidal epitope to the surface-exposed loop 5 on the P2 porin protein of non-typeable Haemophilus influenzae.

The P2 protein is the major outer-membrane protein of non-typeable Haemophilus influenzae (NTHI) and shows extreme heterogeneity among strains. Based on the analysis of antigenic structure, the P2 protein consists of eight potentially surface-exposed loops. Previous studies of monoclonal antibodies (mABs) to a single strain of NTHI showed that P2 contains potentially immunodominant epitopes in loop 5 of the molecule. The goal of the current work is to test the hypothesis that strain-specific and potentially immunodominant epitopes are located in loop 5 of P2 in other strains of NTHI as well. Gene fragments which encode peptides of loop 5 of strains 2019 and 5657 were cloned into an expression vector and subjected to immunoassays with mABs which recognize surface-exposed, bactericidal, strain-specific epitopes. Each mAB recognized loop 5 of the P2 protein of the homologous strain. Analysis of mutant clones with minor amino acid changes showed a loss of reactivity with the mABs. These observations indicate that loop 5 of the P2 molecule contains strain-specific, abundantly expressed surface-exposed epitopes. This further supports the hypothesis that loop 5 is an immunodominant region of the P2 molecule.

Adult↗
PubMed1999

Flexibility of the major antigenic loop of foot-and-mouth disease virus bound to a Fab fragment of a neutralising antibody: structure and neutralisation.

The interaction of foot-and-mouth disease virus (FMDV) serotype C (clone C-S8c1) with a strongly neutralising monoclonal antibody (MAb) 4C4 has been studied by combining data from cryoelectron microscopy and x-ray crystallography. The MAb 4C4 binds to the exposed flexible GH-loop of viral protein 1 (VP1), which appears to retain its flexibility, allowing movement of the bound Fab. This is in striking contrast to MAb SD6, which binds to the same GH-loop of VP1 but exhibits no movement of the bound Fab when observed under identical conditions. However, MAbs 4C4 and SD6 have very similar neutralisation characteristics. The known atomic structure of FMDV C-S8c1 and that of the 4C4 Fab cocrystallised with a synthetic peptide corresponding to the GH-loop of VP1 were fitted to the cryoelectron microscope density map. The best fit of the 4C4 Fab is compatible only with monovalent binding of the MAb in agreement with the neutralisation data on 4C4 MAbs, Fab2s, and Fabs. The position of the bound GH-loop is related to other known positions of this loop by a hinge rotation about the base of the loop. The 4C4 Fab appears to interact almost exclusively with the G-H loop of VP1, making no other contacts with the viral capsid.

Animals↗
PubMed2003

Immunogenicity and ability of variable loop-deleted human immunodeficiency virus type 1 envelope glycoproteins to elicit neutralizing antibodies.

It has been extremely difficult to elicit broadly cross-reactive neutralizing antibodies (Nabs) against human immunodeficiency virus type 1 (HIV-1). In this study, we compared the immunogenic properties of the wild-type and variable loop-deleted HIV-1 envelope glycoproteins. Mice were immunized with recombinant vaccinia viruses expressing either the wild-type or the variable loop-deleted (V1-2, V3, V4, and V1-3) HIV-1(DH12) gp160s. The animals were subsequently boosted with respective recombinant gp120s. All envelope constructs elicited similar levels of gp120-binding antibodies when analyzed by enzyme-linked immunosorbent assay (ELISA). However, the highest neutralizing activity was observed in sera from animals immunized with the wild-type envelope protein, followed by those immunized with DeltaV4 and DeltaV1-2. No neutralizing activity was detected in sera from animals immunized with DeltaV3 or DeltaV1-3. To identify immunogenic epitopes, ELISA was performed with overlapping 15-mer peptides that cover the entire length of gp120. For the wild-type gp120, the immunogenic epitopes mapped primarily to the variable loops V1-2 and to the conserved regions C1 and C5. When they were plotted onto known coordinates of gp120 core crystal structure, the epitopes in the conserved regions mapped predominantly to the inner domain of the protein. By immunizing with variable loop-deleted envelopes, the immune responses could be redirected to other regions of the protein. However, the newly targeted epitopes were neither on the exposed surface of the protein nor on the receptor binding regions. Interestingly, the removal of the V3 loop resulted in loss of immunoreactivity for both V3 and V1/V2 loops, suggesting structural interaction between the two regions. Our results suggest that obtaining broadly reactive Nabs may not be achieved simply by deleting the variable loops of gp120. However, the observation that the immune responses could be redirected by altering the protein composition might allow us to explore alternative strategies for modifying the antigenic properties of HIV-1 envelope glycoprotein.

Amino Acid Sequence↗
PubMed1996

Active site binding loop stabilization in the subtilisin inhibitor eglin c: structural and functional studies on specifically designed mutants in complex with subtilisin and the uncomplexed inhibitor.

As known from the x-ray crystal structure in complex with a proteinase and from NMR studies, the serine proteinase inhibitor eglin c has a wedge-like shape with a hydrophobic core and a solvent exposed active site binding loop which is stabilized by a network of non-covalent core-binding loop interactions. Previous studies implied a crucial role of the P1'-residue Asp-46 for binding loop stabilization and high inhibitory potency of eglin c towards serine proteinases such as subtilisin. In the present study, the formation of specific eglin core-binding loop interactions was modulated by replacing the wildtype Asp-46 by asparagine, glutarnate and glutamine. The x-ray crystal structures of these mutants were solved in complex with subtilisin, and the inhibitory potency towards this enzyme was determined. Our results imply a reduction of inhibitory potency with declining core-binding loop interactions. We succeeded in crystallizing free wildtype eglin c. The 1.95 angstroms x-ray crystal structure indicates that the transition from the free to the bound form of eglin is accompanied by a concerted conformational change in the binding loop, implying an induced fit to the accessible enzyme surface. Except for the binding loop domain and a few residues on the surface of eglin, the differences observed between the uncomplexed and bound form of the inhibitor are only small.

Binding Sites↗