Engineering proteins with enhanced mechanical stability by force-specific sequence motifs

Use of atomic force microscopy (AFM) has recently led to a better understanding of the molecular mechanisms of the unfolding process by mechanical forces; however, the rational design of novel proteins with specific mechanical strength remains challenging. We have approached this problem from a new perspective that generates linear physical–chemical properties (PCP) motifs from a limited AFM data set. Guided by our linear sequence analysis, we designed and analyzed four new mutants of the titin I1 domain with the goal of increasing the domain's mechanical strength. All four mutants could be cloned and expressed as soluble proteins. AFM data indicate that at least two of the mutants have increased molecular mechanical strength. This observation suggests that the PCP method is useful to graft sequences specific for high mechanical stability to weak proteins to increase their mechanical stability, and represents an additional tool in the design of novel proteins besides steered molecular dynamics calculations, coarse grained simulations, and ?‐value analysis of the transition state. Proteins 2012; © 2011 Wiley Periodicals, Inc.     

Base of the Measles Virus Fusion Trimer Head Receives the Signal That Triggers Membrane Fusion

The measles virus (MV) fusion (F) protein trimer executes membrane fusion after receiving a signal elicited by receptor binding to the hemagglutinin (H) tetramer. Where and how this signal is received is understood neither for MV nor for other paramyxoviruses. Because only the prefusion structure of the parainfluenza virus 5 (PIV5) F-trimer is available, to study signal receipt by the MV F-trimer, we generated and energy-refined a homology model. We used two approaches to predict surface residues of the model interacting with other proteins. Both approaches measured interface propensity values for patches of residues. The second approach identified, in addition, individual residues based on the conservation of physical chemical properties among F-proteins. Altogether, about 50 candidate interactive residues were identified. Through iterative cycles of mutagenesis and functional analysis, we characterized six residues that are required specifically for signal transmission; their mutation interferes with fusion, although still allowing efficient F-protein processing and cell surface transport. One residue is located adjacent to the fusion peptide, four line a cavity in the base of the F-trimer head, while the sixth residue is located near this cavity. Hydrophobic interactions in the cavity sustain the fusion process and contacts with H. The cavity is flanked by two different subunits of the F-trimer. Tetrameric H-stalks may be lodged in apposed cavities of two F-trimers. Because these insights are based on a PIV5 homology model, the signal receipt mechanism may be conserved among paramyxoviruses.     

Rational design of DNA sequence-specific zinc fingers

We developed a rational scheme for designing DNA binding proteins. The scheme was applied for a zinc finger protein and the designed sequences were experimentally characterized with high DNA sequence specificity. Starting with the backbone of a known finger structure, we initially calculated amino acid sequences compatible with the expected structure and the secondary structures of the designed fingers were then experimentally confirmed. The DNA-binding function was added to the designed finger by reconsidering a section of the amino acid sequence and computationally selecting amino acids to have the lowest protein-DNA interaction energy for the target DNA sequences. Among the designed proteins, one had a gap between the lowest and second lowest protein-DNA interaction energies that was sufficient to give DNA sequence-specificity.     

A preliminary biomechanical study of cyclic preconditioning effects on canine cadaveric whole femurs

Biomechanical preconditioning of biological specimens by cyclic loading is routinely done presumably to stabilize properties prior to the main phase of a study. However, no prior studies have actually measured these effects for whole bone of any kind. The aim of this study, therefore, was to quantify these effects for whole bones. Fourteen matched pairs of fresh-frozen intact cadaveric canine femurs were sinusoidally loaded in 4-point bending from 50?N to 300?N at 1?Hz for 25 cycles. All femurs were tested in both anteroposterior (AP) and mediolateral (ML) bending planes. Bending stiffness (i.e., slope of the force-vs-displacement curve) and linearity R(2) (i.e., coefficient of determination) of each loading cycle were measured and compared statistically to determine the effect of limb side, cycle number, and bending plane. Stiffnesses rose from 809.7 to 867.7?N/mm (AP, left), 847.3 to 915.6?N/mm (AP, right), 829.2 to 892.5?N/mm (AP, combined), 538.7 to 580.4?N/mm (ML, left), 568.9 to 613.8?N/mm (ML, right), and 553.8 to 597.1?N/mm (ML, combined). Linearity R(2) rose from 0.96 to 0.99 (AP, left), 0.97 to 0.99 (AP, right), 0.96 to 0.99 (AP, combined), 0.95 to 0.98 (ML, left), 0.94 to 0.98 (ML, right), and 0.95 to 0.98 (ML, combined). Stiffness and linearity R(2) versus cycle number were well-described by exponential curves whose values leveled off, respectively, starting at 12 and 5 cycles. For stiffness, there were no statistical differences for left versus right femurs (p?=?0.166), but there were effects due to cycle number (p?相似文献   

ISSR assay for ascertaining genetic fidelity of micropropagated plants of apple rootstock Merton 793

With the current trends in high density plantations of fruit trees, numerous clonal rootstocks of apple have been developed through various breeding programs. Among them, Merton 793 is the most popular in India because of the desirable traits of vigorous growth and resistance to woolly apple aphid and collar rot. The planting material of this rootstock cannot be multiplied at a desirable rate by means of conventional vegetative propagation methods, so micropropagation techniques are being explored to augment scarce planting material. Large number of plants can be produced in vitro under aseptic conditions, but there is always a danger of producing somaclonal variants by tissue culture technology. Thus, it is advisable to check the clonal fidelity of in vitro raised plants, especially of perennials prior to their field transplantation. The genetic stability of in vitro raised plants of apple rootstock Merton 793, multiplied through enhanced axillary bud proliferation up to 22 subculture passages, was tested by intersimple sequence repeat (ISSR) assay. Of 24 ISSR primers screened, 15 primers produced clear reproducible bands, resulting in a total of 134 distinct bands with an average of 8.9 bands per primer. Apple rootstock MM 111 and scion Jonathan, taken as outliers with tissue culture-raised progenies of Merton 793, ruled out the possibility that the invariant banding pattern occurred because of inefficiency of ISSR primers in detecting variations. The homogenous amplification profile observed for all the micropropagated plants compared to the donor plant confirmed the clonal fidelity of the tissue culture-raised Merton 793 plants. This suggests that axillary bud multiplication is the safest mode for multiplication of true-to-type plants. This is the first study that evaluates the applicability of ISSR markers in establishing clonal fidelity of tissue culture-raised apple plants.     

In vitro activation of the IkappaB kinase complex by human T-cell leukemia virus type-1 Tax

Human T-cell leukemia virus type-I expresses Tax, a 40-kDa oncoprotein that activates IkappaB kinase (IKK), resulting in constitutive activation of NFkappaB. Herein, we have developed an in vitro signaling assay to analyze IKK complex activation by recombinant Tax. Using this assay in combination with reporter assays, we demonstrate that Tax-mediated activation of IKK is independent of phosphatases. We show that sustained activation of the Tax-mediated activation of the NFkappaB pathway is dependent on an intact Hsp90-IKK complex. By acetylating and thereby preventing activation of the IKK complex by the Yersinia effector YopJ, we demonstrate that Tax-mediated activation of the IKK complex requires a phosphorylation step. Our characterization of an in vitro signaling assay system for the mechanism of Tax-mediated activation of the IKK complex with a variety of mutants and inhibitors results in a working model for the biochemical mechanism of Tax-induced activation.     

Hesperidin Produces Cardioprotective Activity via PPAR-γ Pathway in Ischemic Heart Disease Model in Diabetic Rats

The present study investigated the effect of hesperidin, a natural flavonoid, in cardiac ischemia and reperfusion (I/R) injury in diabetic rats. Male Wistar rats with diabetes were divided into five groups and were orally administered saline once daily (IR-sham and IR-control), Hesperidin (100 mg/kg/day; IR-Hesperidin), GW9962 (PPAR-γ receptor antagonist), or combination of both for 14 days. On the 15^th day, in the IR-control and IR-treatment groups, rats were subjected to left anterior descending (LAD) coronary artery occlusion for 45 minutes followed by a one-hour reperfusion. Haemodynamic parameters were recorded and rats were sacrificed; hearts were isolated for biochemical, histopathological, ultrastructural and immunohistochemistry. In the IR-control group, significant ventricular dysfunctions were observed along with enhanced expression of pro-apoptotic protein Bax. A decline in cardiac injury markers lactate dehydrogenase activity, CK-MB and increased content of thiobarbituric acid reactive substances, a marker of lipid peroxidation, and TNF-α were observed. Hesperidin pretreatment significantly improved mean arterial pressure, reduced left ventricular end-diastolic pressure, and improved both inotropic and lusitropic function of the heart (+LVdP/dt and –LVdP/dt) as compared to IR-control. Furthermore, hesperidin treatment significantly decreased the level of thiobarbituric acid reactive substances and reversed the activity of lactate dehydrogenase towards normal value. Hesperidin showed anti-apoptotic effects by upregulating Bcl-2 protein and decreasing Bax protein expression. Additionally, histopathological and ultrastructural studies reconfirmed the protective action of hesperidin. On the other hand, GW9662, selective PPAR-γ receptor antagonist, produced opposite effects and attenuated the hesperidin induced improvements. The study for the first time evidence the involvement of PPAR-γ pathway in the cardioprotective activity of hesperidin in I/R model in rats.     

<Emphasis Type="Italic">In vitro</Emphasis> selection and regeneration of chrysanthemum (<Emphasis Type="Italic">Dendranthema grandiflorum</Emphasis> Tzelev) plants resistant to culture filtrate of <Emphasis Type="Italic">Septoria obesa</Emphasis> Syd

Callus cultures derived from leaf segments of chrysanthemum cultivar ‘Snow Ball’ which was susceptible to Septoria obesa were successfully used for in vitro selection for resistance to this pathogenic fungus. Resistant cell lines were selected by culturing callus on growth medium containing various concentrations of S. obesa filtrate. Resistant calluses obtained after two cycles (30 d each cycle) of selection were used for plant regeneration. About 30% of the plants regenerated from the resistant calluses and 70–80% of the plants raised from cuttings had acquired considerable resistance against the pathogen in the field. No phenotypic variation was observed in the selected regenerates.     

Seed traits,fatty acid profile and genetic diversity assessment in <Emphasis Type="Italic">Pongamia pinnata</Emphasis> (L.) Pierre germplasm

Phenotypic variation of important seed traits like seed length, seed breadth, seed thickness, 100 seed weight and seed oil content were recorded in a total of 157 collected accessions of Pongamia. Out of these, fatty acid profiles of 38 accessions selected based on their high and low oil content was analyzed. Fatty acid profile revealed high variability in stearic, oleic and linoleic acid which varied from 0.42 to 10.61 %, 34.34 to 74.58 %, and 7.00 to 31.28 % respectively. Variations in palmitic and linolenic acid were small. Iodine value, saponification number and cetane number (CN) of fatty acid methyl esters (FAME) of seed oil ranges from 186.99 to 201.25, 81.13 to 108.19 and 46.16 to 56.47 respectively. Fatty acid compositions, degree of unsaturation and CN are the important parameters, which are used to determine quality of FAME were used as biodiesel. Some of the Pongamia accessions identified were higher in oil content while some accessions showed higher degree of unsaturation and a few of them had CN values higher than 55. Genetic diversity analysis with six TE-AFLP primers generated a total of 334 bands out of which 174 (52.10 %) were polymorphic. The genetic similarity ranged from 0.11 to 0.47. These findings clearly showed high level of genetic diversity and all economically desirable traits were not present in a single genotype of Pongamia. All these traits could be selected from these CPTs and transfer to a single elite variety through selection and breeding programme and could be utilized for large scale multiplication and plantation to produce high quantity and quality biodiesel in future.