BF3 Complexing Phosphate/Phosphonate Ionic Liquids: Synthesis, Characterization and Thermophysical Properties.

A new group of BF3 complexing phosphate/phosphonate ionic liquids (ILs) [Emim][X(BF3)2] (X=dimethyl phosphate, diethyl phosphate, methyl phosphonate, and ethyl phosphonate) were synthesized and characterized. Key thermophysical properties of the new complex ionic liquids, including density, viscosity, conductivity, surface tension, solid-liquid phase transition, and thermal stability were determined and compared with those of [Emim][X]. Some other important thermophysical properties such as isobaric thermal expansion coefficient, molecular volume, standard molar entropy, and lattice potential energy were obtained from measured density data, and the free volume was estimated by a linear equation presented in this article, while critical temperature, normal boiling temperature, and enthalpy of vaporization were estimated from measured surface tension and density data. Furthermore, Fragility study shows that [Emim][X(BF3)2] should be considered as fragile liquids, while [Emim][X] could be considered as extremely fragile liquids. The ionicity of [Emim][X(BF3)2] was predicted by Walden rule, and the result shows that these ILs fit well with Walden law. The key features of these complex ILs are their extremely low glass transition (-95.33~-98.46 °C) without melting, considerably low viscosities (33.876~58.117 mPa ⋅ s), and high values of free volume fraction (comparable to [Omim][BF4], [Emim][NTf2], and [Emim][TCB]).

Synthesis, Characterization, and Physicochemical Properties of New [Emim][BF3X] Complex Anion Ionic Liquids.

A new series of complex anion ionic liquids (ILs) [Emim][BF3X] (X = CH3SO3, EtSO4, HSO4, Tosylate) were synthesized and characterized by nuclear magnetic resonance, elemental analysis, differential scanning calorimetry analysis, and thermogravimetry. The physicochemical properties of these ILs, such as density, viscosity, conductivity, and surface tension, were measured and correlated with thermodynamic and empirical equations in the temperature range of 293.15-358.15 K under ambient conditions, and the thermal expansion coefficient, standard molar entropy, lattice potential energy, viscosity activation energy, surface enthalpy, and surface entropy were further calculated from experimental values. According to the temperature-dependent viscosity and conductivity, [Emim][BF3X] ILs follow the Walden rule, and they are classified as "good (or super) ionic liquids".

Metal-free catalytic hydrocarboxylation of hexafluorobut-2-yne.

An efficient method for stereoselective synthesis of trifluorinated enol esters catalyzed by base was introduced. The DFT calculations and experimental results both supported the nucleophilic addition process. The protocol featured mild reaction conditions and showed a wide functional group tolerance. The one-pot simultaneous etherification and esterification of the salicylic acids further demonstrated the prospective synthetic application.

A Meta-Analysis of Patellar Replacement in Total Knee Arthroplasty for Patients With Knee Osteoarthritis.

BACKGROUND: This meta-analysis (MA) aims at comparing the clinical outcomes of resurfacing and nonresurfacing the patella in patients undergoing total knee arthroplasty in the treatment of knee osteoarthritis. METHODS: Randomized controlled trials were included by retrieving data from electronic English databases. Both fixed and random-effects models were employed, and standardized mean difference and 95% confidence intervals were calculated. Stata13.1 software was used for statistical analysis for all the studies included to compare the differences in improving Knee Society Clinical Score and Knee Society Function Score as well as the reduction in rates of infection, reoperation, and anterior knee pain. RESULTS: A total of 394 studies were initially included in this MA. About 20 randomized controlled trials which met the inclusion criteria were finally enrolled in this MA. The results of our MA showed that the reoperation rate of the patellar resurfacing group was lower than that of the nonresurfacing group. The subgroup analysis was performed according to the follow-up time and revealed that the increase in the Knee Society Clinical Score was higher in the patellar resurfacing group than that in the nonresurfacing group in the follow-up period of 1 to 2 years. The risk of reoperation rate was lower in the patellar resurfacing group than that in the nonresurfacing group, while there were no statistical differences in the follow-up time over 2 years. CONCLUSION: Our study suggests that during the follow-up of 1 to 2 years, patellar resurfacing can significantly increase the Knee Society Clinical Score and reduce the reoperative rates in patients with knee osteoarthritis.

Effect of autologous platelet-rich plasma on the chondrogenic differentiation of rabbit adipose-derived stem cells in vitro.

This study aimed to isolate rabbit adipose-derived stem cells (ADSCs) and explore the potential of platelet-rich plasma (PRP) in the chondrogenic differentiation of ADSCs, thereby potentially providing a new approach for the repair and regeneration of cartilage injury. Rabbit ADSCs were isolated and characterized by induction towards adipogenic, osteogenic and chondrogenic lineages in vitro. The isolated ADSCs were also cultured with or without 10% PRP. Immunofluorescence staining, toluidine blue staining and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were used to detect type II collagen (Col II) and aggrecan (AGC) expression. Col II immunofluorescence staining and toluidine blue staining indicated that following induction by autologous PRP, ADSCs manifested Col II and AGC expression. The expression of Col II and AGC mRNA was significantly upregulated in the PRP-treated cells when compared with that in control cells. Autologous PRP produced by laboratory centrifugation was able to promote the chondrogenic differentiation of rabbit ADSCs in vitro.

[Comparison of therapeutic effects between patella replacement and patella osteotomy in total knee arthroplasty: a case-control study].

OBJECTIVE: To compare therapeutic effects between patella replacement and patella osteotomy in total knee arthroplasty. METHODS: From April 2004 to April 2011, 52 patients (54 knees) were enrolled in the clinical trail of total knee arthroplasty, who received patella replacement (24 knees, including 13 males and 11 females,ranging in age from 53 to 78 years old or patella osteotomy (30 knees,including 16 males and 12 females,ranging in age from 55 to 79 years old. The average follow-up period was 56 months,ranging from 20 to 80 months. The American HSS Score for knee, the Feller score for patella, range of motion (ROM) for knee, patient satisfaction and complications related to the patella were used to evaluate therapeutic effects. RESULTS: In the patella replacement group,the preoperative and final follow-up HSS scores of patients were 38.4 +/- 8.2 and 91.2 +/- 8.6 respectively; Feller scores were 13.6 +/- 6.2 and 25.2 +/- 4.2; scores of anterior knee pain were 3.9 +/- 3.2 and 11.2 +/- 3.7; ROM were (78 +/- 26) degrees and(108 +/- 18) degrees. In the patella osteotomy group,the preoperative and final follow-up HSS scores of patients were 39.5 +/- 8.4 and 91.0 +/- 8.5 respectively;Feller scores were 13.4 +/- 6.5 and 25.6 +/- 4.0; scores of anterior knee pain were 3.7 +/- 3.1 and 11.3 +/- 3.6; ROM were (76 +/- 27) degrees and (110 +/- 19) degrees. In the patella replacement group,patient's satisfaction was 91%, and complication related to the patella was 16.7%; in the patella osteotomy group, patient's satisfaction was 89%, and complications related to the patella was 10.0%. There were no statistically significant differeneces in final follow-up HSS scores, Feller scores, scores of anterior knee pain and ROM between the two groups. However,there was no significant difference of patient's satisfaction between them. There was statistically significant differenece of patella-related complications between the two groups, and the complication rate in the patella replacement group was higher than that in the patella osteotomy group. CONCLUSION: Total knee arthroplasty with patella replacement or patella osteotomy dramatically relieves pain and improves the knee function. Patella-related complications are associated with its treatment methods, but post-operative anterior knee pain and patient's satisfaction are not related to treatment methods of the patella.

[Expression and immunological activity of an anti-CD3×VEGFR2 bispecific single-chain antibody].

AIM: To construct and express an anti-VEGFR2/anti-CD3 bispecific single-chain antibody (bscVEGFR2×CD3)and to identify its binding specificities to CD3 and VEGFR2. METHODS: The gene encoding anti-VEGFR2/anti-CD3 bispecific single-chain antibody was designed and synthesized. Bispecific single-chain antibody (bsc-Ab) DNA was subcloned into a eukaryotic expression vector pcDNA3.1(+), then transfected into Chinese hamster ovary (CHO) cells and stable expression cell lines were selected. Expressed Bsc-Ab was purified by His-tag affinity chromatography and confirmed by 120 g/L SDS-PAGE and Western blotting. Antigen binding activity of the bsc-Ab was analyzed by FACS. RESULTS: The plasmid DNA containing bispecific single-chain fragments were confirmed. BscVEGFR2×CD3 was secreted by CHO into the supernatant. Six stable expression cell lines were established. The molecular weight of bsc-Ab was correct indicated by SDS-PAGE and Western blotting. The bsc-Ab could specifically bind to CD3(+); jurkat cells and VEGFR2(+); A375 cells. CONCLUSION: An anti-VEGFR2/anti-CD3 bispecific single-chain antibody is successfully constructed and expressed, and the antibody has specific binding capacity to CD3 and VEGFR2.

Multiple affinity states of cGMP-specific phosphodiesterase for sildenafil inhibition defined by cGMP-dependent and cGMP-independent mechanisms.

cGMP-specific phosphodiesterase (PDE5) has become a target for drug development for the treatment of a number of physiological dysfunctions, affected by changes in the cGMP/cGMP-dependent protein kinase (PKG) signaling pathway. PDE5 has two highly homologous regulatory domains, GAF-A and GAF-B. We showed previously that PDE5 could be converted from a low-activity (nonactivated) state to a high-activity state upon cGMP binding to the GAF-A domain with higher sensitivities toward sildenafil (EMBO J 22:469-478, 2003). Here we investigated whether sildenafil sensitivity of PDE5 could be modified by cGMP-independent mechanisms. Individually expressed recombinant GAF-A and GAF-B proteins were tested for their ability to modulate full-length recombinant PDE5 affinity to sildenafil. The GAF-A domain protein had the most dramatic effect on the affinity of the nonactivated recombinant PDE5 for sildenafil, revealing much higher sensitivity to sildenafil inhibition. The apparent affinity for sildenafil increased from the nanomolar range to the picomolar range, providing evidence for the presence of a "super-high" sensitivity state of PDE5 for sildenafil inhibition. In human platelet, higher sensitivity of PDE5 for sildenafil inhibition has been detected after blocking cGMP-binding sites of the GAF-A domain. Thus, our data demonstrate that high sensitivity of PDE5 for sildenafil can be obtained not only through cGMP-induced activation of PDE, but also through cGMP-independent modulation of PDE5 in the nonactivated state, possibly through protein-protein interaction. Furthermore, data suggest that nonactivated PDE5 with "super-high" affinities for sildenafil inhibition may be responsible for therapeutic effects of long-term treatments with low doses of PDE5 inhibitors.

[Preparation, characterization and application of polyclonal antibody against human carboxylesterases-II].

AIM: To construct a prokaryotic plasmid expressing human carboxyles-terases-II (hCE-II ), purify the recombinant protein and investigate the rabbit polyclonal antibody against hCE-II . METHODS: A recombinant plasmid expressing pGEX-4T-1-hCE-II (hCE-II -GST) and pET-32a- hCE-II (hCE-II -His) was constructed and then it was expressed in E.coli BL21 induced by IPTG. The polyclonal antibody was prepared by immunizing the rabbits with the purified recombinant protein. The sensitivity and specificity of the antibody was detected using enzyme -linked immunosorbent assay, immunohistochem ical staining and Western blot analysis. RESULTS: The polyclonal antibody against hCE-II was successfully prepared. Western blot analysis showed that the antibody specifically reacted with the recombinant protein and natural human liver microsomal proteins. Immunohistochemis result demonstrated that the pAb could combine with liver cytoplasm protein but not vascular smooth muscle cell protein. CONCLUSION: The successful preparation of the pAb against hCE-II with high titer and specificity lays a foundation for the investigation of diagnosis kit of liver cancer.

Disease-causing mutation in GPR54 reveals the importance of the second intracellular loop for class A G-protein-coupled receptor function.

The G-protein-coupled receptor (GPCR) GPR54 is essential for the development and maintenance of reproductive function in mammals. A point mutation (L148S) in the second intracellular loop (IL2) of GPR54 causes idiopathic hypogonadotropic hypogonadism, a disorder characterized by delayed puberty and infertility. Here, we characterize the molecular mechanism by which the L148S mutation causes disease and address the role of IL2 in Class A GPCR function. Biochemical, immunocytochemical, and pharmacological analysis demonstrates that the mutation does not affect the expression, ligand binding properties, or protein interaction network of GPR54. In contrast, diverse GPR54 functional responses are markedly inhibited by the L148S mutation. Importantly, the leucine residue at this position is highly conserved among class A GPCRs. Indeed, mutating the corresponding leucine of the alpha(1A)-AR recapitulates the effects observed with L148S GPR54, suggesting the critical importance of this hydrophobic IL2 residue for Class A GPCR functional coupling. Interestingly, co-immunoprecipitation studies indicate that L148S does not hinder the association of Galpha subunits with GPR54. However, fluorescence resonance energy transfer analysis strongly suggests that L148S impairs the ligand-induced catalytic activation of Galpha. Combining our data with a predictive Class A GPCR/Galpha model suggests that IL2 domains contain a conserved hydrophobic motif that, upon agonist stimulation, might stabilize the switch II region of Galpha. Such an interaction could promote opening of switch II of Galpha to facilitate GDP-GTP exchange and coupling to downstream signaling responses. Importantly, mutations that disrupt this key hydrophobic interface can manifest as human disease.

Blood pressure is regulated by an alpha1D-adrenergic receptor/dystrophin signalosome.

Hypertension is a cardiovascular disease associated with increased plasma catecholamines, overactivation of the sympathetic nervous system, and increased vascular tone and total peripheral resistance. A key regulator of sympathetic nervous system function is the alpha(1D)-adrenergic receptor (AR), which belongs to the adrenergic family of G-protein-coupled receptors (GPCRs). Endogenous catecholamines norepinephrine and epinephrine activate alpha(1D)-ARs on vascular smooth muscle to stimulate vasoconstriction, which increases total peripheral resistance and mean arterial pressure. Indeed, alpha(1D)-AR KO mice display a hypotensive phenotype and are resistant to salt-induced hypertension. Unfortunately, little information exists about how this important GPCR functions because of an inability to obtain functional expression in vitro. Here, we identified the dystrophin proteins, syntrophin, dystrobrevin, and utrophin as essential GPCR-interacting proteins for alpha(1D)-ARs. We found that dystrophins complex with alpha(1D)-AR both in vitro and in vivo to ensure proper functional expression. More importantly, we demonstrate that knock-out of multiple syntrophin isoforms results in the complete loss of alpha(1D)-AR function in mouse aortic smooth muscle cells and abrogation of alpha(1D)-AR-mediated increases in blood pressure. Our findings demonstrate that syntrophin and utrophin associate with alpha(1D)-ARs to create a functional signalosome, which is essential for alpha(1D)-AR regulation of vascular tone and blood pressure.

[Preparation and characterization of antibodies against clusterin].

AIM: To prepare rabbit polyclonal antibodies (pAb) and mouse monoclonal antibodies (mAbs) against human clusterin(CLU) and characterize these antibodies' properties. METHODS: CLU fragment was amplified from human liver cDNA library, and recombinant expression vectors pGEX-4T-1-CLU and PET-32a-CLU were constructed. GST-CLU fusion protein was expressed in E.coli and then used as the immunogen. Properties of antiserum against human CLU were identified by ELISA, Western blot, and the mAbs against human CLU was characterized by Western blot, indirect immunofluorescent staining and immunohistochemistry staining. RESULTS: The GST-CLU fusion protein was highly expressed with a molecular weight of M(r)54,000. Western blot analysis proved that the rabbit pAb could specifically recognize 52,000 and 58,000 proteins in human liver total protein. All of the nine established mAbs recognized recombinant human CLU protein, two of which specifically bound to proteins in the cytoplasm of HepG2 cells and four of which specifically bound to proteins in the cytoplasm of adult liver tissue. CONCLUSION: pAb and mAbs against human CLU were successfully prepared, which will provide efficient tools for functional studies of CLU expressed in human tumors.

[Preparation and characterization of monoclonal antibody against human carboxylesterases-II].

AIM: To prepare monoclonal antibody(mAb) against human carboxylesterases-II (hCE-II) and characterize its properties. METHODS: BALB/c mice were immunized with human liver microsome protein which contained hCE-II. The mAb was prepared by hybridoma technique and purified by protein-G affinity chromatography. The titer and specificity of mAb was detected by ELISA and Western blot respectively. Tissue localization of antigen was detected by immunohistochemical staining. Antigen was appraised by peptide mass fingerprint (PMF) matched with Mascot human protein database. PMF was obtained by immunoprecipitation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). RESULTS: One clone of hybridoma secreting specific mAb against hCE-II was obtained. The Ig subclass of the mAb was IgG1(kappa). The titer of the mAb was 1 x 10(-7). Western blot analysis showed one clear belt in the Mr of 62,000. Immunohistochemistry demonstrated that the mAb had special combination with the liver cytoplasm protein, but not with the vascular smooth muscle cell protein. Immunoprecipitation showed one clear band in the Mr of 62,000, which was in conformity with the Mr of hCE-II and the antigen was confirmed to be hCE-II after being analyzed with mass spectrometry. CONCLUSION: The mAb against hCE-II with high titer and specificity has been obtained, which lays the foundation for investigation of hCE-II function and diagnosis and therapy of liver cancer.

ERK1/2 signaling pathway is involved in 15-hydroxyeicosatetraenoic acid-induced hypoxic pulmonary vasoconstriction.

Hypoxia-induced 15-hydroxyeicosatetraenoic acid (15-HETE) is an essential mediator to constrict pulmonary arteries (PA). The signaling pathway involved in 15-HETE-induced PA vasoconstriction remains obscure. The aim of the present study was to test the hypothesis that hypoxic PA constriction induced by 15-HETE was possibly regulated by the extracellular signal-regulated kinase-1/2 (ERK1/2) pathway. PA ring tension measurement, Western blot and immunocytochemistry were used in the study to determine the possible role of ERK1/2 in 15-HETE-induced PA vasoconstriction. The organ bath for PA rings tension study was employed. Adult male Wistar rats were raised in hypoxic environment with fractional inspired oxygen (FIO2, 0.12) for 9 d. PA 1~1.5 mm in diameter were dissected and cut into 3 mm long rings for tension study. ERK1/2 up-stream kinase (MEK) inhibitor PD98059, which blocks the activation of ERK1/2, was used. The results showed that pretreatment of PD98059 significantly blunted 15-HETE-induced PA vasoconstrictions in the rings from hypoxic rat. Moreover, in endothelium-denuded rings, PD98059 also significantly attenuated 15-HETE-induced vasoconstriction. Phosphorylation of ERK1/2 in pulmonary arterial smooth muscle cells (PASMCs) of rat was enhanced evidently when stimulated by 15-HETE. Thus, the data suggest that ERK1/2 signaling pathway is involved in 15-HETE-induced hypoxic pulmonary vasoconstriction.

15-hydroxyeicosatetraenoic acid depressed endothelial nitric oxide synthase activity in pulmonary artery.

15-hydroxyeicosatetraenoic acid (15-HETE) plays an important role in hypoxia-induced pulmonary vasoconstriction. Release of nitric oxide (NO) is apparently decreased and activity of endothelial nitric oxide synthase (eNOS) is impaired in chronic hypoxia. However, little is known whether 15-HETE contributes to eNOS/NO pathway in the constriction induced by 15-HETE. We examined the response of rat pulmonary artery (PA) rings to 15-HETE, the production of NO, total eNOS expression and the phosphorylation of eNOS in bovine pulmonary artery endothelial cells (BPAECs) stimulated by 15-HETE. Rat PA rings were divided into three groups: endothelium intact group, endothelium denuded group, and nitro-L-arginine methyl ester (L-NAME, 0.1 mmol/L, an inhibitor of eNOS) group. Constrictions to 15-HETE were significantly enhanced in endothelium denuded group and L-NAME group (both P< 0.05 vs endothelium intact group, n= 9); BPAECs were incubated in different conditions to test nitrite production by Greiss method. Nitrite production was significantly reduced by 1 mumol/L 15-HETE (P<0.05), and increased by the lipoxygenase inhibitors, 10 mumol/L cinnamyl 3,4- dihydroxy-[alpha] -cyanocinnamate (CDC, P< 0.05) and 0.1 mmol/L nordihydroguiairetic acid (NDGA, P< 0.01 ); Western blot analysis of extracts from BPAECs incubated with 15-HETE in different time was carried out to test total eNOS expression, and the expression was changed unobviously. Immunoprecipitation (IP) and Western blot analysis of cell extracts from BPAECs treated with 2 mumol/L 15-HETE in different length of time were accomplished, using phospo-eNOS-threonine 495 (Thr495, an inhibitory site) antibody for IP, and eNOS or 15-lipoxygenase (15-LO) antibodies for Western blot. 15-HETE depressed eNOS activity by increasing the levels of phospho-eNOS-Thr 495. The data suggest that eNOS/NO pathway is involved in PA constrictions induced by 15-HETE and that 15-HETE depresses eNOS activity by phosphorylation in Thr495 site. The protein interaction between phospho-eNOS (Thr495) and 15-LO is discovered for the first time.

Molecular determinants for cyclic nucleotide binding to the regulatory domains of phosphodiesterase 2A.

Binding of cGMP to the GAF-B domain of phosphodiesterase 2A allosterically activates catalytic activity. We report here a series of mutagenesis studies on the GAF-B domain of PDE2A that support a novel mechanism for molecular recognition of cGMP. Alanine mutations of Phe-438, Asp-439, and Thr-488, amino acids that interact with the pyrimidine ring, decrease cGMP affinity slightly but increase cAMP affinity by up to 8-fold. Each interaction is required to provide for cAMP/cGMP specificity. Mutations of any of the residues that interact with the phosphate-ribose moiety or the imidazole ring abolish cGMP binding. Thus, residues that interact with the pyrimidine ring collectively control cAMP/cGMP specificity, whereas residues that bind the phosphate-ribose moiety and imidazole ring are critical for high affinity binding. Similar decreases in binding were found for mutations made in a bacterially expressed GAF-A/B plus catalytic domain construct. Because these constructs had very high catalytic activity, it appears that these mutations did not cause a global denaturation. The affinities of cAMP and cGMP for wild-type GAF-B alone were approximately 4-fold greater than for the holoenzyme, suggesting that the presence of neighboring domains alters the conformation of GAF-B. More importantly, the PDE2A GAF-B, GAF-A/B, GAF-A/B+C domains, and holoenzyme all bind cGMP with much higher affinity than has previously been reported. This high affinity suggests that cGMP binding to PDE2 GAF-B activates the enzyme rapidly, stoichiometrically, and in an all or none fashion, rather than variably over a large range of cyclic nucleotide concentrations.

PDE5 is converted to an activated state upon cGMP binding to the GAF A domain.

cGMP-specific, cGMP-binding phosphodiesterase (PDE5) regulates such physiological processes as smooth muscle relaxation and neuronal survival. PDE5 contains two N-terminal domains (GAF A and GAF B), but the functional roles of these domains have not been determined. Here we show that recombinant PDE5 is activated directly upon cGMP binding to the GAF A domain, and this effect does not require PDE5 phosphorylation. PDE5 exhibited time- and concentration-dependent reversible activation in response to cGMP, with the highest activation (9- to 11-fold) observed at low substrate concentrations (0.1 micro M cGMP). A monoclonal antibody directed against GAF A blocked cGMP binding, prevented PDE5 activation and decreased basal activity, revealing that PDE5 in its non-activated state has low intrinsic catalytic activity. Activated PDE5 showed higher sensitivity towards sildenafil than non-activated PDE5. The stimulatory effect of cGMP binding on the catalytic activity of PDE5 suggests that this mechanism of enzyme activation may be common among other GAF domain-containing proteins. The data also suggest that development of agonists and antagonists of PDE5 activity based on binding to this site might be possible.

The two GAF domains in phosphodiesterase 2A have distinct roles in dimerization and in cGMP binding.

Cyclic nucleotide phosphodiesterases (PDEs) regulate all pathways that use cGMP or cAMP as a second messenger. Five of the 11 PDE families have regulatory segments containing GAF domains, 3 of which are known to bind cGMP. In PDE2 binding of cGMP to the GAF domain causes an activation of the catalytic activity by a mechanism that apparently is shared even in the adenylyl cyclase of Anabaena, an organism separated from mouse by 2 billion years of evolution. The 2.9-A crystal structure of the mouse PDE2A regulatory segment reported in this paper reveals that the GAF A domain functions as a dimerization locus. The GAF B domain shows a deeply buried cGMP displaying a new cGMP-binding motif and is the first atomic structure of a physiological cGMP receptor with bound cGMP. Moreover, this cGMP site is located well away from the region predicted by previous mutagenesis and structural genomic approaches.