An exact analysis of the sliding motion in contractile system: A simplified model of muscle system

A system involving two kinds of sliding filaments is analysed with special attention to the actomyosin system. Rigorous results are obtained about the statistical effect originating from many active sites distributed on both filaments. It is necessary for the occurrence of smooth motion in sliding filament that the spatial periods of active sites on both filaments are relatively incommensurable, and that the number of active sites on each filament is large enough. Sufficient conditions for smooth contraction are derived under the assumption that both filaments are rigid; this is called rigid rod approximation in the present paper. The elastic mode of the filaments, during the sliding process, is analysed by perturbation theory based on the rigid rod approximation. A stochastic theory is briefly discussed in reference to the cooperative generation of contractile force, which is concerned in Hill's relation of muscle contraction.     

Amino propynyl benzoic acid building block in rigid spacers of divalent ligands binding to the Syk SH2 domains with equally high affinity as the natural ligand

The construction of rigid spacers composed of amino propynyl benzoic acid building blocks is described. These spacers were used to link two phosphopeptide ligand sites towards obtaining divalent ligands with a high affinity for Syk tandem SH2 domains, which are important in signal transduction. The spacer containing two of those rigid building blocks led to a ligand which was as active as the natural ligand, indicating that this building block can be used in the design and synthesis of high affinity divalent constructs that can successfully interfere with crucial protein-protein interactions.     

Active TEM-1 beta-lactamase mutants with random peptides inserted in three contiguous surface loops

Engineering of alternative binding sites on the surface of an enzyme while preserving the enzymatic activity would offer new opportunities for controlling the activity by binding of non-natural ligands. Loops and turns are the natural substructures in which binding sites might be engineered with this purpose. We have genetically inserted random peptide sequences into three relatively rigid and contiguous loops of the TEM-1 beta-lactamase and assessed the tolerance to insertion by the percentage of active mutants. Our results indicate that tolerance to insertion could not be correlated to tolerance to mutagenesis. A turn between two beta-strands bordering the active site was observed to be tolerant to random mutagenesis but not to insertions. Two rigid loops comprising rather well-conserved amino acid residues tolerated insertions, although with some constraints. Insertions between the N-terminal helix and the first beta-strand generated active libraries if cysteine residues were included at both ends of the insert, suggesting the requirement for a stabilizing disulfide bridge. Random sequences were relatively well accommodated within the loop connecting the final beta-strand to the C-terminal helix, particularly if the wild-type residue was retained at one of the loops' end. This suggests two strategies for increasing the percentage of active mutants in insertion libraries. The amino acid distribution in the engineered loops was analyzed and found to be less biased against hydrophobic residues than in natural medium-sized loops. The combination of these activity-selected libraries generated a huge library containing active hybrid enzymes with all three loops modified.     

Role of active site rigidity in activity: MD simulation and fluorescence study on a lipase mutant

Relationship between stability and activity of enzymes is maintained by underlying conformational flexibility. In thermophilic enzymes, a decrease in flexibility causes low enzyme activity while in less stable proteins such as mesophiles and psychrophiles, an increase in flexibility is associated with enhanced enzyme activity. Recently, we identified a mutant of a lipase whose stability and activity were enhanced simultaneously. In this work, we probed the conformational dynamics of the mutant and the wild type lipase, particularly flexibility of their active site using molecular dynamic simulations and time-resolved fluorescence techniques. In contrast to the earlier observations, our data show that active site of the mutant is more rigid than wild type enzyme. Further investigation suggests that this lipase needs minimal reorganization/flexibility of active site residues during its catalytic cycle. Molecular dynamic simulations suggest that catalytically competent active site geometry of the mutant is relatively more preserved than wild type lipase, which might have led to its higher enzyme activity. Our study implies that widely accepted positive correlation between conformation flexibility and enzyme activity need not be stringent and draws attention to the possibility that high enzyme activity can still be accomplished in a rigid active site and stable protein structures. This finding has a significant implication towards better understanding of involvement of dynamic motions in enzyme catalysis and enzyme engineering through mutations in active site.     

Comparison of Efficency of Translation Between a Deformable Swimmer Versus a Rigid Body in a Bounded Fluid Domain: Consequences for Subcellular Transport

In this paper, we compare the translation efficiencies of a deformable circle that swims by means of low amplitude periodic tangential surface waves versus a rigid circle, moving in a bounded fluid domain. The swimmer is found to be much more efficient than the rigid body. We believe that this result gives some support to the active hypothesis of subcellular transport, where it is supposed that the organelle can generate by itself a propulsive flux, (by changes of form or metabolic activities) instead of just being carried by the motion of an external agent, like a molecular motor.     

Conformational dynamics of free and catalytically active thermolysin are indistinguishable by hydrogen/deuterium exchange mass spectrometry

Conformational dynamics are thought to be a prerequisite for the catalytic activity of enzymes. However, the exact relationship between structural fluctuations and function is not well understood. In this work hydrogen/deuterium exchange (HDX) and electrospray ionization mass spectrometry (ESI-MS) are used for exploring the conformational dynamics of thermolysin. Amide HDX reflects the internal mobility of proteins; regions that undergo frequent unfolding-refolding show faster exchange than segments that are highly stable. Thermolysin is a zinc protease with an active site that is located between two lobes. Substrate turnover is associated with hinge bending that leads to a closed conformation. Product release regenerates the open form, such that steady-state catalysis involves a continuous closing/opening cycle. HDX/ESI-MS with proteolytic peptide mapping in the absence of substrate shows that elements in the periphery of the two lobes are most mobile. A comparison with previous X-ray data suggests that these peripheral regions undergo quite pronounced structural changes during the catalytic cycle. In contrast, active site residues exhibit only a moderate degree of backbone flexibility, and the central zinc appears to be in a fairly rigid environment. The presence of both rigid and moderately flexible elements in the active site may reflect a carefully tuned balance that is required for function. Interestingly, the HDX behavior of catalytically active thermolysin is indistinguishable from that of the free enzyme. This result is consistent with the view that catalytically relevant motions preexist in the resting state and that enzyme function can only be performed within the limitations given by the intrinsic dynamics of the protein. The data presented in this work indicate the prevalence of stochastic elements in the function of thermolysin, rather than supporting a deterministic mechanism.     

A mechanical model to determine the influence of masses and mass distribution on the impact force during running

Simple spring-damper-mass models have been widely used to simulate human locomotion. However, most previous models have not accounted for the effect of non-rigid masses (wobbling masses) on impact forces. A simple mechanical model of the human body developed in this study included the upper and lower bodies with each part represented by a rigid and a wobbling mass. Spring-damper units connected different masses to represent the stiffness and damping between the upper and lower bodies, and between the rigid and wobbling masses. The simulated impact forces were comparable to experimentally measured impact forces. Trends in changes of the impact forces due to changes in touch-down velocity reported in previous studies could be reproduced with the model. Simulated results showed that the impact force peaks increased with increasing rigid or wobbling masses of the lower body. The ratio of mass distribution between the rigid and wobbling mass in the lower body was also shown to affect the impact force peak, for example, the impact force peak increased with increasing rigid contribution. The variation in the masses of upper body was shown to have a minimum effect on the impact force peak, but a great effect on the active force peak (the second peak in the ground reaction force). Future studies on the dynamics and neuro-muscular control of human running are required to take into consideration the influence of individual variation in lower body masses and mass distribution.     

Preparation and characterization of recombinant DNA-derived human interferon-gamma

An attempt was made to prepare a highly purified, active recombinant DNA-derived human interferon-gamma. When the protein was denatured in urea and refolded, gel filtration and sedimentation velocity experiments indicated the presence of two forms, which are different in size and are not in a rapid reversible equilibrium. The two forms could be chromatographically separated. Far-UV circular dichroic spectra indicated the presence of secondary structures for both forms. Near-UV circular dichroic spectra revealed that the smaller form is folded into a rigid tertiary structure. The antiviral activity of the two forms of interferon-gamma showed a significant difference, i.e. the smaller form was 4-8-fold more active than the larger form. A variety of experiments show that the smaller form is more active, homogeneous, soluble, and stable than the larger form.     

Extended conformation of mammalian translation elongation factor 1A in solution

The conformation of mammalian elongation factor eEF1A in solution was examined by the small angle neutron scattering and scanning microcalorimetry. We have found that in contrast to the bacterial analogue the eEF1A molecule has no fixed rigid structure in solution. The radius of gyration of the eEF1A molecule (5.2 nm) is much greater than that of prokaryotic EF1A. The specific heat of denaturation is considerably lower for eEF1A than for EF1A, suggesting that the eEF1A conformation is significantly more disordered. Despite its flexible conformation, eEF1A is found to be highly active in different functional tests. According to the neutron scattering data, eEF1A becomes much more compact in the complex with uncharged tRNA. The absence of a rigid structure and the possibility of large conformational change upon interaction with a partner molecule could be important for eEF1A functioning in channeled protein synthesis and/or for the well-known capability of the protein to interact with different ligands besides the translational components.     

Purification of a highly modified RNA-aptamer: Effect of complete denaturation during chromatography on product recovery and specific activity

To evaluate RNA-aptamers as potential drug candidates, efficient and scaleable purification protocols are needed. Because aptamers are highly structured and rigid molecules, denaturation during the purification process is a critical aspect to obtain a pure and active product. A two-step chromatographic procedure was developed to purify a synthetic anti-VEGF aptamer at the preparative scale. A reversed-phase chromatographic step was optimized with a highly hydrophobic ion pairing reagent, followed by ion-exchange chromatography in which heat and a chaotropic salt were used. Because of the presence of 2′-modified ribose, denaturation conditions had to be optimized in both chromatographic steps to achieve a fully active molecule.     

Role of the ribose residue of substrates in reactions catalyzed by ribonuclease

The rate constants and Km for the hydrolysis of the optically active nonglycosidic analogues of the CpA and C greater than p catalysed by RNase A and RNase BS-I were measured. The rate of hydrolysis of the model substrates in 10(5) and 10(3) slower that for the appropriate dinucleoside phosphate and nucleoside cyclophosphate. However, substitution of the relatively rigid ribofuranose ring with flexible alifatic chains is accompanied by little variation in binding constants. The analyses based on the single substrate system indicate that the observed difference in rate constants must be accounted for by a difference between the binding of the substrates in the transition state to the RNase active site. Consequently, the "rigidity" of the ribose rings in RNA leads to large decreases in the free energy of activation for the reactions catalysed by RNases.     

Modulation of HIV protease flexibility by the T80N mutation

The flexibility of HIV protease (HIVp) plays a critical role in enabling enzymatic activity and is required for substrate access to the active site. While the importance of flexibility in the flaps that cover the active site is well known, flexibility in other parts of the enzyme is also critical for function. One key region is a loop containing Thr 80, which forms the walls of the active site. Although not situated within the active site, amino acid Thr80 is absolutely conserved. The mutation T80N preserves the structure of the enzyme but catalytic activity is completely lost. To investigate the potential influence of the T80N mutation on HIVp flexibility, wide‐angle X‐ray scattering (WAXS) data was measured for a series of HIVp variants. Starting with a calculated WAXS pattern from a rigid atomic model, the modulations in the intensity distribution caused by structural fluctuations in the protein were predicted by simple analytic methods and compared with the experimental data. An analysis of T80N WAXS data shows that this variant is significantly more rigid than the WT across all length scales. The effects of this single point mutation extend throughout the protein, to alter the mobility of amino acids in the enzymatic core. These results support the contentions that significant protein flexibility extends throughout HIVp and is critical to catalytic function. Proteins 2015; 83:1929–1939. © 2014 Wiley Periodicals, Inc.     

Generation of 3D templates of active sites of proteins with rigid prosthetic groups

MOTIVATION: With the increasing availability of protein structures, the generation of biologically meaningful 3D patterns from the simultaneous alignment of several protein structures is an exciting prospect: active sites could be better understood, protein functions and protein 3D structures could be predicted more accurately. Although patterns can already be generated at the fold and topological levels, no system produces high-resolution 3D patterns including atom and cavity positions. To address this challenge, our research focuses on generating patterns from proteins with rigid prosthetic groups. Since these groups are key elements of protein active sites, the generated 3D patterns are expected to be biologically meaningful. RESULTS: In this paper, we present a new approach which allows the generation of 3D patterns from proteins with rigid prosthetic groups. Using 237 protein chains representing proteins containing porphyrin rings, our method was validated by comparing 3D templates generated from homologues with the 3D structure of the proteins they model. Atom positions were predicted reliably: 93% of them had an accuracy of 1.00 A or less. Moreover, similar results were obtained regarding chemical group and cavity positions. Results also suggested our system could contribute to the validation of 3D protein models. Finally, a 3D template was generated for the active site of human cytochrome P450 CYP17, the 3D structure of which is unknown. Its analysis showed that it is biologically meaningful: our method detected the main patterns of the cytochrome P450 superfamily and the motifs linked to catalytic reactions. The 3D template also suggested the position of a residue, which could be involved in a hydrogen bond with CYP17 substrates and the shape and location of a cavity. Comparisons with independently generated 3D models comforted these hypotheses. AVAILABILITY: Alignment software (Nestor3D) is available at http://www.kingston.ac.uk/~ku33185/Nestor3D.html     

Identification of a new tamoxifen-xanthene hybrid as pro-apoptotic anticancer agent

Breast cancer is the most diagnosed type of cancer among women for which an exhaustive cure has not been discovered yet. Nowadays, tamoxifen still represents the gold standard for breast cancer therapy; it acts on both estrogen receptor-positive and estrogen receptor-negative breast cancers. Unfortunately, its toxicity and the related chemoresistance undermine its antitumor potential. In this paper, new tamoxifen-based derivatives with a rigid structural motif in their structure were designed, synthesized, and evaluated to assess their antitumor behavior. All the tested compounds affected estrogen receptor-positive tumor (MCF-7) cell growth, even with different extents, among which, the most active ones proved also to induce mitochondria-mediated apoptosis through activation of PARP cleavage, decrease in Bax/Bcl-2 ratio and increase in Bim gene expression levels. Here we found that the compound 1, carrying a rigid xanthene core, turned out to be the most promising of the set showing an activity profile comparable to that of tamoxifen. Furthermore, a more favorable genotoxic profile than tamoxifen made compound 1 a promising candidate for further studies.     

Characterization of the glucose transport systems in Neurospora crassa sl.

Neurospora crassa sl, a mutant that lacks a rigid cell wall, exhibits transport systems for glucose similar to those of wild-type strain 1A. When the orgnism is grown in a medium containing 50 mM glucose as the carbon source, glucose is transported primarily by a glucose-facilitated diffusion system (GluI). When it is grown in a medium with little or no glucose present, a glucose active transport system (Glu II) is expressed. Both of these systems are similar kinetically to those in the wild type. Significant differences do exist between strains sl and 1A with respect to genetic regulation of the glucose active transport system.     

PROFbval: predict flexible and rigid residues in proteins

SUMMARY: The mobility of a residue on the protein surface is closely linked to its function. The identification of extremely rigid or flexible surface residues can therefore contribute information crucial for solving the complex problem of identifying functionally important residues in proteins. Mobility is commonly measured by B-value data from high-resolution three-dimensional X-ray structures. Few methods predict B-values from sequence. Here, we present PROFbval, the first web server to predict normalized B-values from amino acid sequence. The server handles amino acid sequences (or alignments) as input and outputs normalized B-value and two-state (flexible/rigid) predictions. The server also assigns a reliability index for each prediction. For example, PROFbval correctly identifies residues in active sites on the surface of enzymes as particularly rigid. AVAILABILITY: http://www.rostlab.org/services/profbval CONTACT: profbval@rostlab.org SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.     

Kinetics of structural changes in the relay loop and SH3 domain of myosin

The intrinsic fluorescence of smooth muscle myosin signals conformational changes associated with different catalytic states of the ATPase cycle. To elucidate this relationship, we have examined the pre-steady-state kinetics of nucleotide binding, hydrolysis, and product release in motor domain-essential light chain mutants containing a single endogenous tryptophan, either residue 512 in the rigid relay loop or residue 29 adjacent to the SH3 domain. The intrinsic fluorescence of W512 is sensitive to both nucleotide binding and hydrolysis, and appears to report structural changes at the active site, presumably through a direct connection with switch II. The intrinsic fluorescence of W29 is sensitive to nucleotide binding but not hydrolysis, and does not appear to be tightly linked with structural changes occurring at the active site. We propose that the SH3 domain may be sensitive to conformational changes in the lever arm through contacts with the essential light chain.     

Structural analysis of human glutamine:fructose-6-phosphate amidotransferase, a key regulator in type 2 diabetes

Glutamine:fructose-6-phosphate amidotransferase (GFAT) is a rate-limiting enzyme in the hexoamine biosynthetic pathway and plays an important role in type 2 diabetes. We now report the first structures of the isomerase domain of the human GFAT in the presence of cyclic glucose-6-phosphate and linear glucosamine-6-phosphate. The C-terminal tail including the active site displays a rigid conformation, similar to the corresponding Escherichia coli enzyme. The diversity of the CF helix near the active site suggests the helix is a major target for drug design. Our study provides insights into the development of therapeutic drugs for type 2 diabetes.