Chiral separation using molecularly imprinted heteroaromatic polymers

Novel molecularly imprinted polymer systems utilizing 4-vinylpyridine and 1-vinylimidazole as functional monomers have been developed for enantioselective recognition of carboxylic and N-protected amino acids. Non-covalent interactions between the functional monomers and the template molecules were the source of the subsequent recognition sites in the resultant polymers. The capacity of the polymers for molecular recognition was investigated by using them as stationary phases in the HPLC mode. Polymers prepared with 4-vinylpyridine were found to be more efficient in racemic resolution than those prepared with 1-vinylimidazole. When applying a racemic mixture of the template molecule, the polymers showed highest affinity for the enantiomer used as template. Imprints of a racemic template molecule, as expected, did not exhibit enantioselectivity. The optimal molar ratio of 4-vinylpyridine to the template Cbz-L -Asp-OH in the polymerization mixture was determined to be 12:1. In addition to enantioselectivity, the investigated polymers demonstrated ‘ligand selectivity’ e.g., a Cbz-L -Asp-OH-imprinted polymer was able to separate Cbz-D ,L -Asp-OH, but was unable to separate Cbz-D ,L -Glu-OH.     

应用痘苗病毒为载体表达人白细胞介素4的研究

在真核细胞中表达近似于人类天然糖基化的白细胞介素４（ｈＩＬ－４）,为重要的研究课题。利用基因工程技术,以痘苗病毒作为载体,使之在真核细胞中表达ｈＩＬ－４,为生产糖基化的ｈＩＬ－４开辟新的可能途径。在痘苗病毒表达载体ｐＪ１２０质粒的基础上,组建了含有ｈＩＬ－４基因的ｐＪｌ２０／ｈＩＬ－４重组质粒,该质粒以痘苗病毒的胸腺嘧啶核苷激酶（ＴＫ）基因为侧翼序列,中间插入痘苗病毒的Ｐ１１和Ｐ２５两个转录方向相反的启动子,Ｐ２５的下游连接β－半乳糖苷酶（ＬａｃＺ）基因,ｈＩＬ－４基因在痘苗病毒启动子Ｐ１１控制下进行表达,将ＰＪ１２０／ｈＩＬ－４质粒ＤＮＡ用脂质体转染法导入ＴＫ阴性的骨髓瘤细胞（ＴＫ１４３细胞）,与预先感染的痘苗病毒在细胞内进行同源序列重组,并且用病毒的核酸杂交鉴定证实,ｈＩＬ－４基因已重组到痘苗病毒基因组中,用ｈＩＬ－４应答细胞株检测病毒感染细胞上清,发现重组病毒感染的细胞上清中含有高滴度的ｈＩＬ－４活性,并对此种情况下ｈＩＬ－４产生的条件进行了动态观察。     

Affinity precipitation of alpha-amylase inhibitor from wheat meal by metal chelate affinity binding using cu(II)-loaded copolymers of 1-vinylimidazole with N-isopropylacrylamide

A method for purifying alpha-amylase inhibitor from wheat meal based on immobilized metal affinity with a thermosensitive copolymer is developed. The studies represent the thermoprecipitation properties of the copolymers of N-isopropylacrylamide (NIPAM) with iminodiacetic acid (IDA) and 1-vinylimidazole (VI), respectively. The polymer which is obtained by the copolymerization of 1-vinylimidazole and N-isopropylacrylamide, charged with Cu(II), exhibited specific interaction of the metal ions to the protein inhibitor. The precipitation was induced by salt and the recovery of the amylase inhibitor was achieved by dissolving the inhibitor-polymer complex in imidazole buffer and subsequent precipitation of the polymer. A single family of the alpha-amylase inhibitor was recovered from the polymer with 89% yield and about fourfold purification. The SDS-PAGE pattern showed significant purification of the inhibitor. The binding of the inhibitor to the Cu(II)-polymer conjugate depends upon the Cu(II) concentration in the copolymer and also upon the concentration of the protein. The recovered polymer could be reused with reasonable efficiency. Copyright 1998 John Wiley & Sons, Inc.     

Reversibility of IUdR resistance to sensitivity to an HSV1 strain in experimental keratitis in rabbits]

During 7 serial passages of Herpes Simplex Virus (HSV1) in rabbit cornea treated with idoxuridine (IUdR) (P1 to P7), the emergence of resistance had been obtained from P3. The reversion towards IUdR sensitivity has been investigated from either viral population P3 or P6 by 6 serial passages in rabbit cornea treated by Vaseline (V1 to V6). From viral population P3, the reversion to IUdR sensitivity has been obtained at V4. In contrast, from viral population P6, the IUdR resistance was conserved from V1 to V6. In vitro, on Vero cells, the effective doses 50% (ED50) and 90% (ED90), determined by dye uptake assay and plaque reduction assay, confirmed the reversibility towards IUdR sensitivity obtained from P3 and the stability of IUdR resistance from P6.     

The molecular reaction vessels for a transesterification process created by molecular imprinting technique

A polymeric catalyst was synthesized by co-polymerizing 4(5)-vinylimidazole and itaconic acid with trimethylpropanol trimethacrylate micro spheres. The catalyst obtained catalysed the transesterification process between p-nitrophenyl acetate and hexanol with maximal initial velocity(nu(max)) of 4.73 +/- 0.47 microM min(-1) mg(-1), and turnover rate (k(cat)) of 8.67 min(-1). Using p-nitrophenyl acetate as template, molecular imprinting process enhanced the nu(max) of the polymeric catalyst 3-fold. Each imprinted site can be considered as a single molecular reaction vessel, and achieved a k(cat) of 169 min(-1) towards the conversion of p-nitrophenyl acetate in hexanol.     

Superoxide generated by glutathione reductase initiates a vanadate-dependent free radical chain oxidation of NADH.

Vanadate V(V) markedly stimulated the oxidation of NADPH by GSSG reductase and this oxidation was accompanied by the consumption of O2 and the accumulation of H2O2. Superoxide dismutases completely eliminated this effect of V(V), whereas catalase was without effect, as was exogenous H2O2 added to 0.1 mM. These effects could be seen equally well in phosphate- or in 4-(2-hydroxyethyl)1-piperazineethanesulfonic acid-buffered solutions. Under anaerobic conditions there was no V(V)-stimulated oxidation of NADPH. Approximately 4% of the electrons flowing from NADPH to O2, through GSSG reductase, resulted in release of O2-. The average length of the free radical chains causing the oxidation of NADPH, initiated by O2- plus V(V), was calculated to be in the range 140-200 NADPH oxidized per O2- introduced. We conclude that GSSG reductase, and by extension other O2(-)-producing flavoprotein dehydrogenases such as lipoyl dehydrogenase and ferredoxin reductase, catalyze V(V)-stimulated oxidation of NAD(P)H because they release O2- and because O2- plus V(V) initiate a free radical chain oxidation of NAD(P)H. There is no reason to suppose that these enzymes can act as NAD(P)H:V(V) oxidoreductases.     

Human parainfluenza virus type 4 is incapable of evading the interferon-induced antiviral effect

The V proteins of some paramyxoviruses have developed the ability to efficiently inactivate STAT protein function as a countermeasure for evading interferon (IFN) responses. Human parainfluenza virus type 4 (hPIV4) is one of the rubulaviruses, which are members of the family Paramyxoviridae, and has a V protein with a highly conserved cysteine-rich domain that is the hallmark of paramyxovirus V proteins. In order to study the function of the hPIV4 V protein, we established HeLa cells expressing the hPIV4A V protein (HeLa/FlagPIV4V). The hPIV4 V protein had no ability to reduce the level of STAT1 or STAT2, although it associated with STAT1, STAT2, DDB1, and Cul4A. It interfered with neither STAT1 and STAT2 tyrosine phosphorylation nor IFN-induced STAT nuclear accumulation. In addition, HeLa/FlagPIV4V cells are fully sensitive to both beta interferon (IFN-beta) and IFN-gamma, indicating that the hPIV4 V protein has no ability to block IFN-induced signaling. We further established HeLa cells expressing various chimeric proteins between the hPIV2 and hPIV4A V proteins. The lack of IFN-antagonistic activity of the hPIV4 V protein is caused by both the P/V common and V-specific domains. At least two regions (amino acids [aa] 32 to 45 and aa 143 to 164) of hPIV4 V in the P/V common domain and one region (aa 200 to 212) of the C terminus are involved in the inability to evade the IFN-induced signaling. Moreover, we established HeLa cells persistently infected with hPIV4 to make sure of the inability to escape IFN and confirmed that hPIV4 is the only paramyxovirus analyzed to date that can't evade the IFN-induced antiviral responses.     

Probing thrombin's ability to accommodate a V34F substitution within the factor XIII activation peptide segment (28-41).

In blood coagulation, thrombin helps to activate factor XIII (FXIII) by cleaving the activation peptide (AP) at the R37-G38 peptide bond. The common polymorphism V34L yields a FXIII that is more easily activated than the wild type enzyme. Peptides based on the FXIII (28-41) (28TVELQGVVPRGVNL41) sequence serve as an important model system to evaluate the substrate specificity of thrombin and thus how to regulate FXIII activation. Our previous kinetic and nuclear magnetic resonance (NMR) studies have suggested that the P4-P1 amino acids on this FXIII segment provide key anchors to the thrombin active site surface. Furthermore, the most effective amino acid to have at the P4 position is a leucine. In the current work, a peptide containing V34F was examined to probe the ability to accommodate an aromatic residue at this position. Kinetic parameters for thrombin-catalyzed hydrolysis of FXIII AP (28-41) V34F are comparable with that of the wild type V34. One-dimensional proton line-broadening studies reveal that the 34FVPR37 segment encompassing the P4-P1 positions makes the most contact with the thrombin surface. Two-dimensional transferred-nuclear overhauser effect spectroscopy (NOESY) studies indicate that when the peptide is bound to thrombin, the F34 aromatic ring is oriented to promote P4-P2 interactions with P36. This characteristic has been viewed as a hallmark for V34L. An ability to generate this interaction may promote the ability of FXIII AP (28-41) V34F to remain a viable substrate for thrombin.     

Sequence and Secondary Structure of the Mitochondrial Small-Subunit rRNA V4, V6, and V9 Domains Reveal Highly Species-Specific Variations within the Genus Agrocybe

A comparative study of variable domains V4, V6, and V9 of the mitochondrial small-subunit (SSU) rRNA was carried out with the genus Agrocybe by PCR amplification of 42 wild isolates belonging to 10 species, Agrocybe aegerita, Agrocybe dura, Agrocybe chaxingu, Agrocybe erebia, Agrocybe firma, Agrocybe praecox, Agrocybe paludosa, Agrocybe pediades, Agrocybe alnetorum, and Agrocybe vervacti. Sequencing of the PCR products showed that the three domains in the isolates belonging to the same species were the same length and had the same sequence, while variations were found among the 10 species. Alignment of the sequences showed that nucleotide motifs encountered in the smallest sequence of each variable domain were also found in the largest sequence, indicating that the sequences evolved by insertion-deletion events. Determination of the secondary structure of each domain revealed that the insertion-deletion events commonly occurred in regions not directly involved in the secondary structure (i.e., the loops). Moreover, conserved sequences ranging from 4 to 25 nucleotides long were found at the beginning and end of each domain and could constitute genus-specific sequences. Comparisons of the V4, V6, and V9 secondary structures resulted in identification of the following four groups: (i) group I, which was characterized by the presence of additional P23-1 and P23-3 helices in the V4 domain and the lack of the P49-1 helix in V9 and included A. aegerita, A. chaxingu, and A. erebia; (ii) group II, which had the P23-3 helix in V4 and the P49-1 helix in V9 and included A. pediades; (iii) group III, which did not have additional helices in V4, had the P49-1 helix in V9 and included A. paludosa, A. firma, A. alnetorum, and A. praecox; and (iv) group IV, which lacked both the V4 additional helices and the P49-1 helix in V9 and included A. vervacti and A. dura. This grouping of species was supported by the structure of a consensus tree based on the variable domain sequences. The conservation of the sequences of the V4, V6, and V9 domains of the mitochondrial SSU rRNA within species and the high degree of interspecific variation found in the Agrocybe species studied open the way for these sequences to be used as specific molecular markers of the Basidiomycota.     

Zinc-chelated imidazole groups for DNA polyion complex formation

This communication presents the pH-dependent chelation of Zn(2+) ions with the imidazole groups of poly(1-vinylimidazole) (PVIm) and DNA polyion complex formation with the zinc-chelated PVIm (PVIm-Zn) via chelated Zn(2+) ions. The resulting PVIm-Zn/DNA complexes exhibit no significant cytotoxicity and show outstanding gene expression.     

Middle cerebral artery blood velocity during a valsalva maneuver in the standing position.

Occasionally, lifting of a heavy weight leads to dizziness and even to fainting, suggesting that, especially in the standing position, expiratory straining compromises cerebral perfusion. In 10 subjects, the middle cerebral artery mean blood velocity (V(mean)) was evaluated during a Valsalva maneuver (mouth pressure 40 mmHg for 15 s) both in the supine and in the standing position. During standing, cardiac output decreased by 16 +/- 4 (SE) % (P < 0.05), and at the level of the brain mean arterial pressure (MAP) decreased from 89 +/- 2 to 78 +/- 3 mmHg (P < 0.05), as did V(mean) from 73 +/- 4 to 62 +/- 5 cm/s (P < 0.05). In both postures, the Valsalva maneuver increased central venous pressure by approximately 40 mmHg with a nadir in MAP and cardiac output that was most pronounced during standing (MAP: 65 +/- 6 vs. 87 +/- 3 mmHg; cardiac output: 37 +/- 3 vs. 57 +/- 4% of the resting value; P < 0.05). Also, V(mean) was lowest during the standing Valsalva maneuver (39 +/- 5 vs. 47 +/- 4 cm/s; P < 0.05). In healthy individuals, orthostasis induces an approximately 15% reduction in middle cerebral artery V(mean) that is exaggerated by a Valsalva maneuver performed with 40-mmHg mouth pressure to approximately 50% of supine rest.     

Design of Factor XIII V34X activation peptides to control ability to interact with thrombin mutants

Thrombin helps to activate Factor XIII (FXIII) by hydrolyzing the R37-G38 peptide bond. The resultant transglutaminase introduces cross-links into the fibrin clot. With the development of therapeutic coagulation factors, there is a need to better understand interactions involving FXIII. Such knowledge will help predict ability to activate FXIII and thus ability to promote/hinder the generation of transglutaminase activity. Kinetic parameters have been determined for a series of thrombin species hydrolyzing the FXIII (28-41) V34X activation peptides (V34, V34L, V34F, and V34P). The V34P substitution introduces PAR4 character into the FXIII, and the V34F exhibits important similarities to the cardioprotective V34L. FXIII activation peptides containing V34, V34L, or V34P could each be accommodated by alanine mutants of thrombin lacking either the W60d or Y60a residue in the 60-insertion loop. By contrast, FXIII V34F AP could be cleaved by thrombin W60dA but not by Y60aA. FXIII V34P is highly reliant on the thrombin W215 platform for its strong substrate properties whereas FXIII V34F AP becomes the first segment that can maintain its K(m) upon loss of the critical thrombin W215 residue. Interestingly, FXIII V34F AP could also be readily accommodated by thrombin L99A and E217A. Hydrolysis of FXIII V34F AP by thrombin W217A/E217A (WE) was similar to that of FXIII V34L AP whereas WE could not effectively cleave FXIII V34P AP. FXIII V34F and V34P AP show promise for designing FXIII activation systems that are either tolerant of or greatly hindered by the presence of anticoagulant thrombins.     

A molecularly imprinted catalyst designed by a computational approach in catalysing a transesterification process

A computational approach was developed to optimize the monomer formulation of molecularly imprinted catalysts. A virtual library of the intermediates of a lipase-catalysed transesterification process was constructed using Chem3D software with p-nitrophenyl acetate as substrate. The energies of the intermediates were minimized using the semi-empirical MOPAC method with the most stable intermediate expected to lead to a higher turn over rate. According to the optimization results, a MIC was prepared by co-polymerising 4(5)-vinylimidazole and itaconic acid with trimethylpropanol trimethacrylate micro spheres in the presence of p-nitrophenyl acetate. The MIC achieved of the transesterification process between p-nitrophenyl acetate and hexanol with a turn over rate of 26.2 min(-1), and showed substrate specificity towards its template with a 6.5-fold preference for p-nitrophenyl acetate over p-nitrophenyl salicylate.     

Plasma membrane depolarization reduces nitric oxide (NO) production in P388D.1 macrophage-like cells during Leishmania major infection

In the present study, we compare changes in host cell plasma membrane potential (V(m)), K(+) fluxes, and NO production during K(+) channel blockade with those changes that occur during infection with Leishmania major. Infection of P388D.1 cells with L. major promastigotes or treatment with K(+) channel blockers (either 1mM 4-AP, 10mM TEA, or 200 microM quinine) suppressed NO production. Inhibition of NO production correlated with depolarization of the P388D.1 cell V(m). Infection of P388D.1 cells with L. major increased the unidirectional influx of rubidium (86Rb), a tracer for K(+) flux, that was comparable to that induced by K(+) channel blockade by 1mM 4-AP. The similar effects of K(+) channel blockers and L. major on NO production, K(+) influx, and V(m) suggest that K(+) channel activity and the maintenance of V(m) is important for NO production in these cells. We suggest that intracellular parasites employ a strategy to inhibit NO production by disrupting V(m) during the invasion/infection process by altering host cell K(+) channel activity.     

Effects of variations in intragastric volume on blood pressure and splanchnic blood flow during intraduodenal glucose infusion in healthy older subjects

The postprandial reduction in blood pressure (BP) is triggered by the interaction of nutrients with the small intestine and associated with an increase in splanchnic blood flow. Gastric distension may attenuate the postprandial fall in BP. The aim of this study was to determine the effects of differences in intragastric volume, including distension at a low (100 ml) volume, on BP and superior mesenteric artery (SMA) blood flow responses to intraduodenal glucose in healthy older subjects. BP and heart rate (HR; automated device), SMA blood flow (Doppler ultrasound), mesenteric vascular resistance (MVR), and plasma norepinephrine of nine male subjects (65-75 yr old) were measured after an overnight fast on 4 separate days in random order. On each day, subjects were intubated with a nasoduodenal catheter, incorporating a duodenal infusion port, and orally with a second catheter, incorporating a barostat bag, positioned in the fundus. Each subject received a 60-min (t = 0-60 min) intraduodenal glucose infusion (3 kcal/min) and gastric distension at a volume of 1) 0 ml (V0), 2) 100 ml (V100), 3) 300 ml (V300), or 4) 500 ml (V500). Systolic BP fell (P < 0.05) during V0, but not during V100, V300, or V500. In contrast, HR (P < 0.01) and SMA blood flow (P < 0.001) increased and MVR decreased (P < 0.05) comparably on all 4 days. Plasma norepinephrine rose (P < 0.01) in response to intraduodenal glucose, with no difference between the four treatments. There was a relationship between the areas under the curve for the change in systolic BP from baseline with intragastric volume (r = 0.60, P < 0.001). In conclusion, low-volume (≤100 ml) gastric distension has the capacity to abolish the fall in BP induced by intraduodenal glucose in healthy older subjects without affecting SMA blood flow or MVR. These observations support the concept that nonnutrient gastric distension prior to a meal has potential therapeutic applications in the management of postprandial hypotension.     

山东昌乐早始新世五图组鼩形类(Soricomorpha,Insectivora, Mammalia)

山东早始新世五图组曾发现原始獏类,Homogalax wutunensis,最近又采到一批化石。这里仅记述其中的一种食虫类,可归入鼩形亚目的昌乐鼩(Changlelestes dissetiformis gen. et sp.nov.)。昌乐鼩与亚洲早第三纪食虫类(Ernosorex.Ictopidium和Tupaiodon)可能有较为密切的亲缘关系,本文将它们归入新科——Changlelestidae fam.nov.。昌乐鼩科与Plesiosoricidae和Soricidae有一些相同的进步特征,这些科与Nyctitheriidae有共同的差别。     

Why human cytochrome P450c21 is a progesterone 21-hydroxylase

Human cytochrome P450c21 (steroid 21-hydroxylase, CYP21A2) catalyzes the 21-hydroxylation of progesterone (P4) and its preferred substrate 17α-hydroxyprogestrone (17OHP4). CYP21A2 activities, which are required for cortisol and aldosterone biosynthesis, involve the formation of energetically disfavored primary carbon radicals. Therefore, we hypothesized that the binding of P4 and 17OHP4 to CYP21A2 restricts access of the reactive heme-oxygen complex to the C-21 hydrogen atoms, suppressing oxygenation at kinetically more favorable sites such as C-17 and C-16, which are both hydroxylated by cytochrome P450c17 (CYP17A1). We reasoned that expansion of the CYP21A2 substrate-binding pocket would increase substrate mobility and might yield additional hydroxylation activities. We built a computer model of CYP21A2 based principally on the crystal structure of CYP2C5, which also 21-hydroxylates P4. Molecular dynamics simulations indicate that binding of the steroid nucleus perpendicular to the plane of the CYP21A2 heme ring limits access of the heme oxygen to the C-21 hydrogen atoms. Residues L107, L109, V470, I471, and V359 were found to contribute to the CYP21A2 substate-binding pocket. Mutation of V470 and I471 to alanine or glycine preserved P4 21-hydroxylase activity, and mutations of L107 or L109 were inactive. Mutations V359A and V359G, in contrast, acquired 16α-hydroxylase activity, accounting for 40% and 90% of the P4 metabolites, respectively. We conclude that P4 binds to CYP21A2 in a fundamentally different orientation than to CYP17A1 and that expansion of the CYP21A2 substrate-binding pocket allows additional substrate trajectories and metabolic switching.     

Amino acid 305 determines catalytic center accessibility in CYP3A4

Site-directed mutagenesis has been used to replace alanine 305 with phenylalanine (A305F) and serine (A305S) in the active site of cytochrome P450 3A4 (CYP3A4). Enzyme kinetics for diazepam, erythromycin, nifedipine, and testosterone metabolism have been determined for both mutants and wild-type CYP3A4. The A305F mutation abolished diazepam oxidase activity and reduced the S(50) and V(max) for erythromycin N-demethylase activity from 17 to 10 microM and from 3.2 to 1.2 pmol product/min/pmol P450, respectively. The V(max) for testosterone 6beta-hydroxylase activity was also significantly reduced, from 2.3 to 0.6 pmol product/min/pmol P450, whereas the S(50) increased from 33 to 125 microM. The nifedipine oxidase activity was diminished to a lesser extent, down from 6.5 to 4.9 pmol product/min/pmol P450, whereas the S(50) increased from 9 to 42 microM. The K(i) for ketoconazole, a CYP3A4 selective inhibitor, was increased more than 10-fold from 0.050 to 0.55 microM, from 0.052 to 0.73 microM, and from 0.043 to 2.2 microM by the A305F mutation when measured against erythromycin, nifedipine, and testosterone metabolism activities, respectively. Similarly, the inhibition constants of the broader specificity inhibitors; clotrimazole, econazole, and miconazole were increased 3- to 15-fold by the A305F mutation. In contrast, the A305S mutation increased testosterone 6beta-hydroxylase (V(max) = 2.9 pmol product/min/pmol P450) and erythromycin N-demethylase (V(max) = 5.1 pmol product/min/pmol P450) activities, but reduced nifedipine oxidase activity (V(max) = 4.6 pmol product/min/pmol P450). K(i) values for ketoconazole and other azole inhibitors were unchanged by the A305S mutation. It is proposed that in CYP3A4, the mutagenesis of alanine 305 to a phenylalanine increases the steric hindrance of the catalytic center, thereby greatly reducing azole inhibitor binding affinity, but maintaining monoogygenase activity.     

Bacillus thuringiensis Cry4Aa insecticidal protein: Functional importance of the intrinsic stability of the unique α4–α5 loop comprising the Pro-rich sequence

The long loop connecting transmembrane α4 and α5 of the Bacillus thuringiensis Cry4Aa toxin possesses a unique feature with Pro-rich sequence (Pro¹⁹³Pro¹⁹⁴_Pro¹⁹⁶) which was shown to be crucial for toxicity. Here, the structural role in the intrinsic stability of the Pro-rich sequence toward toxin activity was investigated. Three Val-substituted mutants (P193V, P194V and P196V) and one Phe-substituted mutant (P193F) were generated and over-expressed in Escherichia coli as inclusions at levels equal to the wild-type. Bioassays demonstrated that all mutants, particularly P193V and P193F whose inclusions were hardly soluble in carbonate buffer (pH 9.0), exhibited reduced toxicity, suggesting an essential role in toxin function by the specific cyclic structure of individual Pro residues. Analysis of the 65-kDa Cry4Aa structure from 10-ns molecular dynamics (MD) simulations revealed that the α4–α5 loop is substantially stable as it showed low structural fluctuation with a 1.2-Å RMSF value. When the flexibility of the α4–α5 loop was increased through P193G, P194G and P196G substitutions, decreased toxicity was also observed for all mutants, mostly for the P193G mutant with low alkali-solubility, suggesting a functional importance of loop-rigidity attributed by individual Pro-cyclic side-chains, particularly Pro¹⁹³. Further MD simulations revealed that the most critical residue−Pro¹⁹³ for which mutations vastly affect toxin solubility and larval toxicity is in close contact with several surrounding residues, thus playing an additional role in the structural arrangement of the Cry4Aa toxin molecule. Altogether, our data signify that the intrinsic stability of the unique Cry4Aa α4–α5 loop structure comprising the Pro-rich sequence plays an important role in toxin activity.