A structural basis for the interaction of urea with lysozyme.

The effect of urea on the crystal structure of hen egg-white lysozyme has been investigated using X-ray crystallography. High resolution structures have been determined from crystals grown in the presence of 0, 0.7, 2, 3, 4, and 5 M urea and from crystals soaked in 9 M urea. All the forms are essentially isomorphous with the native type II crystals, and the derived structures exhibit excellent geometry and RMS differences from ideality in bond distances and angles. Comparison of the urea complex structures with the native enzyme (type II form, at 1.5 A resolution) indicates that the effect of urea is minimal over the concentration range studied. The mean difference in backbone conformation between the native enzyme and its urea complexes varies from 0.18 to 0.49 A. Conformational changes are limited to flexible surface loops (Thr 69-Asn 74, Ser 100-Asn 103), the active site loop (Asn 59-Cys 80), and the C-terminus (Cys 127-Leu 129). Urea molecules are bound to distinct sites on the surface of the protein. One molecule is bound to the active site cleft's C subsite, at all concentrations, in a fashion analogous to that of the N-acetyl substituent of substrate and inhibitor sugars normally bound to this site. Occupation of this subsite by urea alone does not appear to induce the conformational changes associated with inhibitor binding.     

Chimeric hemoglobins--hybrids of human and swine hemoglobin: assembly and stability of interspecies hybrids.

Transgenic swine expressing human HbA contained only one of two types of the anticipated interspecies hybrids, namely H alpha 2 P beta 2 (H = human, P = swine). In an attempt to establish whether the absence of the swine alpha and human beta (P alpha 2 H beta 2) hybrid in vivo is a reflection of the lack of complementarity between the interspecies chains to generate appropriate interfaces, we have undertaken the in vitro assembly of swine alpha and human beta chimeric tetramer. In contrast to the in vivo transgenic swine system, in vitro the hybrid of swine alpha human beta chain is assembled readily and the hybrid exhibits normal cooperative oxygen binding. Both the swine alpha human beta and the human alpha swine beta interspecies hybrids are stable around neutral pH and do not segregate into parent tetramers even when mixed together. On the other hand, nearly complete exchange of P alpha chain of P alpha 2 H beta 2 hybrid occurs in the presence of H alpha chain at pH 6.0 and room temperature, resulting in the formation of HbA. However, very little of such an exchange reaction takes place at pH 7.0. These results suggest that the thermodynamic stability of P alpha 2 H beta 2 hybrid is lower compared to that of HbA. In contrast, P beta chain of H alpha 2 P beta 2 hybrid is refractory to exchange with H beta chain at pH 7.0 as well as at pH 6.0, suggesting that the stability of H alpha 2 P beta 2 is higher compared to that of HbA (H alpha 2 H beta 2). The swine alpha human beta chimeric Hb undergoes subunit exchange reaction with human alpha-chain in the presence of 0.9 M MgCl2, at pH 7.0. This demonstrates the lower thermodynamic stability of the intradimeric interactions of the heterodimer even at neutral pH. A synergistic coupling of the intra- and interdimeric interactions of the swine alpha and human beta chain heterodimer is essential for the thermodynamic stability of the chimeric Hb under the physiological conditions. Accordingly, we speculate that the lower thermodynamic stability of P alpha H beta heterodimer (compared to the homodimers H alpha H beta and P alpha P beta) facilitates its segregation into the homodimers by subunit exchange reaction involving either H alpha or P beta. This molecular aspect by itself or possibly along with other cellular aspects of the swine system results in the absence of P alpha 2 H beta 2 hybrid in transgenic swine expressing HbA.     

N-acetyl-β-D-glucopyranosylamine: A potent T-state inhibitor of glycogen phosphorylase. A comparison with α-D-glucose

Structure-based drug design has led to the discovery of a number of glucose analogue inhibitors of glycogen phosphorylase that have an increased affinity compared to α-D-glucose (K_i = 1.7 mM). The best inhibitor in the class of N-acyl derivatives of β-D-glucopyranosylamine, N-acetyl-β-D-glucopyranosylamine (1-GlcNAc), has been characterized by kinetic, ultracentrifugation, and crystallographic studies. 1-GlcNAc acts as a competitive inhibitor for both the b (K_i = 32 μM) and the α (K_i = 35 μM) forms of the enzyme with respect to glucose 1-phosphate and in synergism with caffeine, mimicking the binding of glucose. Sedimentation velocity experiments demonstrated that 1-GlcNAc was able to induce dissociation of tetrameric phosphorylase α and stabilization of the dimeric T-state conformation. Co-crystals of the phosphorylase b-1-GlcNAc-IMP complex were grown in space group P4₃2₁2, with native-like unit cell dimensions, and the complex structure has been refined to give a crystallographic R factor of 18.1%, for data between 8 and 2.3 Å resolution. 1-GlcNAc binds tightly at the catalytic site of T-state phosphorylase b at approximately the same position as that of α-D-glucose. The ligand can be accommodated in the catalytic site with very little change in the protein structure and stabilizes the T-state conformation of the 280s loop by making several favorable contacts to Asn 284 of this loop. Structural comparisons show that the T-state phosphorylase b-1-GlcNAc-IMP complex structure is overall similar to the T-state phosphorylase b-α-D-glucose complex structure. The structure of the 1-GlcNAc complex provides a rational for the biochemical properties of the inhibitor.     

Structure and enzymic activity of ribonuclease-A esterified at glutamic acid-49 and aspartic acid-53

The dimethyl ester of bovine pancreatic ribonuclease-A (dimethyl RNAase-A), the initial product of esterification of RNAase-A in anhydrous methanolic HCl, was isolated in a homogeneous form. The two carboxy functions esterified in this derivative are those of glutamic acid-49 and aspartic acid-53. There were no changes in the u.v.-absorption spectral characteristics, the accessibility of the methionine residues, the resistance of the protein to proteolysis by trypsin and the antigenic behaviour of RNAase-A as a result of the esterification of these two carboxy groups. Dimethyl RNAase-A exhibited only 65% of the specific activity of RNAase-A, but still had the same K_m value for both RNA and 2′:3′-cyclic CMP. However, the V_max. was decreased by about 35%. On careful hydrolysis of the methyl ester groups at pH9.5, dimethyl RNAase-A was converted back into RNAase-A. Limited proteolysis of dimethyl RNAase-A by subtilisin resulted in the formation of an active RNAase-S-type derivative, namely dimethyl RNAase-S, which was chromatographically distinct from dimethyl RNAase-A and had very nearly the same enzymic activity as dimethyl RNAase-A. Fractionation of dimethyl RNAase-S by trichloroacetic acid yielded dimethyl RNAase-S-protein and dimethyl RNAase-S-peptide, both of which were inactive by themselves but regenerated dimethyl RNAase-S when mixed together. Dimethyl RNAase-A-peptide was identical with RNAase-S-peptide. RNAase-S-protein could be generated from dimethyl RNAase-S-protein by careful hydrolysis of the methyl ester groups at pH9.5. The interaction of dimethyl RNAase-S-protein with RNAase-S-peptide appears to be about 4-fold weaker than that between the RNAase-S-protein and RNAase-S-peptide. Conceivably, the binding of the S-peptide `tail' of dimethyl RNAase-A with the remainder of the molecule is similarly weaker than that in RNAase-A, and this brings about subtle changes in the geometrical orientation of the active-site amino acid residues of these modified methyl ester derivatives. It is suggested that these changes could be responsible for the generation of the catalytically less-efficient RNAase-A and RNAase-S molecules (dimethyl RNAase-A and dimethyl RNAase-S respectively).     

A study of renaturation of reduced hen egg white lysozyme. Enzymically active intermediates formed during oxidation of the reduced protein.

The material obtained from reduced hen egg white lysozyme after complete air oxidation at pH 8.0 and 37 degrees has yielded, by gel filtration on a Bio-Gel P-30 column, enzymically active species and an enzymically inactive form which eluted sooner than the active species but later than expected for a dimer of lysozyme. Reduced lysozyme also elutes at the same position as this inactive material. Examination of the fragments produced on CNBr cleavage of the inactive form indicates that at least 24% of the population contains incorrect disulfide bonds involving half-cystine residues 6, 30, 115, and 127. Tryptophan fluorescence and the intrinsic viscosity of the inactive form show an enlarged molecular domain with a disordered conformation. The yield of the inactive form increases as the oxidation of reduced lysozyme is accelerated using cupric ion. In the presence of 4 X 10(-5) M cupric ion, reduced lysozyme forms almost quantitatively the inactive form, which is almost completely converted to the native form by sulfhydryl-disulfide interchange catalyzed by thiol groups of either reduced lysozyme or beta-mercaptoethanol. The material trapped by alkylation of the free sulfhydryl groups with [1-14C]iodoacetic acid during the early stage of air oxidation of reduced lysozyme was fractionated by gel filtration to permit separation of the active species from the inactive form. Ion exchange chromatography of the active species yielded completely renatured lysozyme and three major enzymically active radioactive derivatives. Two of these derivatives contained approximately 2 mol of S-carboxymethylcysteine. Isolation and characterization of radioactive tryptic peptides from each of the three active forms, permitted the identification of Cys 6 and Cys 127, Cys 76 and 94, and Cys 80 as the sulfhydryl groups alkylated in these three incompletely oxidized, partially active forms. Thus, it appears that the interatomic interactions maintaining the compact three-dimensional structure of native lysozyme are operational even when one of these three native disulfide bonds between Cys 6 and Cys 127, Cys 76 and Cys 94, and Cys 64 and 80 is open.     

Uterine and placental 5-HT profile in different gestational period of albino rats

Possible role of 5-HT in pregnancy was investigated in albino rats by biological estimation of uterine and placental 5-HT contents in different periods of gestation in normal and drug treated rats. Uterine 5-HT level increased steadily from day-1 of gestation to reach the peak on day-7; thereafter, the level continued to decline throughout the period till day-20 when 5-HT level was lowest. From day-20, a mild secondary rise started and remained persistent even after parturition. The results show that a critical level of 5-HT in early gestational period is necessary for conception. Manipulation of endogenous 5-HT do not influence duration of gestation.     

Enhancing Data Reliability in TOMAHAQ for Large‐Scale Protein Quantification

The analytical scale of most mass‐spectrometry‐based targeted proteomics assays is usually limited by assay performance and instrument utilization. A recently introduced method, called triggered by offset, multiplexed, accurate mass, high resolution, and absolute quantitation (TOMAHAQ), combines both peptide and sample multiplexing to simultaneously improve analytical scale and quantitative performance. In the present work, critical technical requirements and data analysis considerations for successful implementation of the TOMAHAQ technique based on the study of a total of 185 target peptides across over 200 clinical plasma samples are discussed. Importantly, it is observed that significant interference originate from the TMTzero reporter ion used for the synthetic trigger peptides. This interference is not expected because only TMT10plex reporter ions from the target peptides should be observed under typical TOMAHAQ conditions. In order to unlock the great promise of the technique for high throughput quantification, here a post‐acquisition data correction strategy to deconvolute the reporter ion superposition and recover reliable data is proposed.     

In vitro and in vivo efficacy of anti‐chikungunya virus monoclonal antibodies produced in wild‐type and glycoengineered Nicotiana benthamiana plants

Chikungunya virus (CHIKV) is a mosquito‐transmitted alphavirus, and its infection can cause long‐term debilitating arthritis in humans. Currently, there are no licensed vaccines or therapeutics for human use to combat CHIKV infections. In this study, we explored the feasibility of using an anti‐CHIKV monoclonal antibody (mAb) produced in wild‐type (WT) and glycoengineered (?XFT) Nicotiana benthamiana plants in treating CHIKV infection in a mouse model. CHIKV mAb was efficiently expressed and assembled in plant leaves and enriched to homogeneity by a simple purification scheme. While mAb produced in ?XFT carried a single N‐glycan species at the Fc domain, namely GnGn structures, WT produced mAb exhibited a mixture of N‐glycans including the typical plant GnGnXF₃ glycans, accompanied by incompletely processed and oligomannosidic structures. Both WT and ?XFT plant‐produced mAbs demonstrated potent in vitro neutralization activity against CHIKV. Notably, both mAb glycoforms showed in vivo efficacy in a mouse model, with a slight increased efficacy by the ?XFT‐produced mAbs. This is the first report of the efficacy of plant‐produced mAbs against CHIKV, which demonstrates the ability of using plants as an effective platform for production of functionally active CHIKV mAbs and implies optimization of in vivo activity by controlling Fc glycosylation.     

Mechanics informed fluoroscopy of esophageal transport

Biomechanics and Modeling in Mechanobiology - Fluoroscopy is a radiographic procedure for evaluating esophageal disorders such as achalasia, dysphasia and gastroesophageal reflux disease. It...