Evidence for Chiral Wobbler in Nuclei.

Three ΔI=1 bands with the πg_{9/2}⊗νg_{9/2} configuration have been identified in _{35}^{74}Br_{39}. Angular distribution, linear polarization, and lifetime measurements were performed to determine the multipolarity, type, mixing ratio, and absolute transition probability of the transitions. By comparing these experimental observations with the corresponding fingerprints and the quantum particle rotor model calculations, the second and third lowest bands are, respectively, suggested as the chiral partner and one-phonon wobbling excitation built on the yrast band. The evidence indicates the first chiral wobbler in nuclei.

[Incidence and predictors of 90-day poor clinical outcome after successful endovascular treatment for acute basilar artery occlusion].

Objective: To investigate the incidence and predictors of 90-day poor clinical outcome after successful endovascular treatment for acute basilar artery occlusion. Methods: Patients were selected from the Acute Ischemic Stroke Cooperation Group of Endovascular Treatment (ANGEL) registry, which was a prospective, multicenter registry study between June 2015 and December 2017. The demographic characteristics, past history, personal history, vital signs, National Institutes of Health Stroke Scale (NIHSS) score, imaging examination, onset/admission/puncture/end of operation, operation-related variables, medication during operation, patency of occluded blood vessels after operation, etiology classification, and 90-day modified Rankin scale (mRS) score were collected. Successful endovascular treatment was defined as modified thrombolysis in cerebral infarction (mTICI) 2b-3. Poor outcome was defined as 90-day mRS 4-6. Multivariate logistic regression analysis was performed to analyze the predictors of poor clinical outcome after successful endovascular treatment. Results: A total of 170 (128 males and 42 females) acute basilar artery occlusion patients undergoing successful endovascular treatment were included in the analysis, with the median age of [M (Q1, Q3)] of 64 (55, 70) years. Poor clinical outcome occurred in 72 patients (42.4%). Multivariate logistic regression analysis revealed that high baseline NIHSS score (OR=1.166, 95%CI: 1.109-1.225, P<0.001) and high baseline systolic blood pressure (OR=1.032, 95%CI: 1.010-1.053, P=0.003) were the independent predictors of poor clinical outcome. Conclusions: The incidence of 90-day poor clinical outcome after successful endovascular treatment for acute basilar artery occlusion is 42.4%. High baseline NIHSS score and systolic blood pressure are associated with the poor clinical outcome.

[Observation and quantification of diabetic keratopathy in type 2 diabetes patients using in vivo laser confocal microscopy].

Objective: To study the diabetic keratopathy in type 2 diabetes patients with retinopathy by in vivo laser confocal microscopy. Methods: This was a case-control study. Ninety type 2 diabetes patients were involved in this study from May 2015 to December 2019 in Qingdao Eye Hospital. According to the diabetic retinopathy clinical stage, these patients were divided into the non-proliferative diabetic retinopathy (NPDR) group (30 cases), early stage proliferative diabetic retinopathy (PDR) group (30 cases) and intermediate to late stage PDR group (30 cases). Thirty non-diabetic healthy volunteers were included in the control group. The central cornea was observed with an in vivo laser confocal microscope. The corneal nerve fiber density, nerve fiber length, nerve branch density, and nerve fiber tortuosity were compared between groups. The corneal Langerhans cells, epithelial cells, stromal cells and endothelial cells were also compared. Results: There were more nerve fibers and branches in the control group than the other three diabetic groups. The nerve fiber length in the control group, NPDR group, early stage PDR group and intermediate to late stage PDR group was (21.55±2.57), (14.73±1.56), (11.23±1.40) and (8.02±1.33) mm/mm2, respectively, and there were statistically significant differences between the groups (F=316.17, P=0.00). In the nerve fiber density, nerve branch density and curvature, there were statistically significant differences between the groups (F=345.72, 479.46, 167.00, all P=0.00). The basal cell density in the control group, NPDR group, and two PDR groups was (5 761±303), (5 336±367), (4 146±379) and (3 658±365) cells/mm2, respectively, and there were statistically significant differences between the groups (F=234.94, P=0.00). The anterior stromal cell density in the four groups was (836±30), (727±57), (544±59) and (360±47) cells/mm2, respectively, and there were statistically significant differences between the groups (F=535.08, P=0.00). The hexagonal endothelium cell rate in the four groups was 62.0%±5.5%, 51.1%±3.7%, 40.2%±4.0% and 27.8%±3.9%, respectively, and the Langerhans cell density was (1.5±0.6), (4.2±1.3), (6.8±2.1) and (10.9±2.1) cells/mm2, respectively; there were statistically significant differences between the groups (F=342.28, 179.78, all P=0.00). There was no statistically significant difference between the groups in the corneal endothelial cell density (F=1.58, P=0.20). Conclusions: In type 2 diabetes patients with diabetic retinopathy, the corneal nerve fiber and branch density can be significantly reduced, and the density of the hexagonal corneal endothelial cells, epithelial basal cells and anterior stromal cells can also decrease. Langerhans cells may be involved in the development diabetic keratopathy. (Chin J Ophthalmol, 2020, 56: 754-760).

Evidence for Octupole Correlations in Multiple Chiral Doublet Bands.

Two pairs of positive-and negative-parity doublet bands together with eight strong electric dipole transitions linking their yrast positive- and negative-parity bands have been identified in ^{78}Br. They are interpreted as multiple chiral doublet bands with octupole correlations, which is supported by the microscopic multidimensionally-constrained covariant density functional theory and triaxial particle rotor model calculations. This observation reports the first example of chiral geometry in octupole soft nuclei.

[Genetic Polymorphisms of 24 Y-STR Loci in Nanjing Han Population].

OBJECTIVES: To investigate the genetic polymorphism of DYS391 and other 23 Y-STR loci and to explore its application value in forensic science. METHODS: Y-STRs loci of 580 unrelated Han males in Nanjing were amplified using AGCU Y-PLUS PCR （24） kit. The genetic parameters of 24 Y-STR loci such as gene frequency were calculated by software, and compared with the data of Hubei, Liao- ning, Guangdong, Beijing and Chengdu Han population. RESULTS: Total 580 haplotypes were detected among 24 Y-STR loci in 580 unrelated Han males in Nanjing. The genetic diversity （GD） of each locus was from 0.294 6 to 0.939 8, and the haplotypes diversity （HD） was 0.983 7. There was a significant difference between the GD of 6 areas. CONCLUSIONS: The 24 Y-STR loci such as DYS391 in Nanjing Han population have an application value in forensic science. They can also be used for cases testing and pedigree investigation.

Effects of intravenous analgesia with combined dezocine and butorphanol on postoperative cognitive function in elderly patients.

The aim of this study was to observe the analgesic effects of the combination of dezocine and butorphanol on postoperative cognitive function in elderly patients. Forty elderly patients undergoing upper abdominal surgeries or thoracotomies with general anesthesia were randomly divided into the dezocine and butorphanol group or the butorphanol group (20 patients per group). A visual analog scale was used to evaluate analgesia and the degree of malignant vomiting. The Ramsay scoring method was used to evaluate sedation. The Mini-Mental State Examination (MMSE) was used to evaluate cognitive function. Forty-eight hours after the operation, the pain score of the dezocine and butorphanol group (means ± SD, 1.75 ± 0.44) was lower than that of the butorphanol group (2.25 ± 0.79; P < 0.05), and the nausea and vomiting score of the dezocine and butorphanol group (0) was lower than that of the butorphanol group (0.70 ± 1.30; P < 0.05). Six hours after the operation, the sedative score of the butorphanol group (3.75 ± 0.79) was higher than that of the dezocine and butorphanol group (2.15 ± 0.75; P < 0.05). Compared to 1 day before the operation, the MMSE scores of both groups decreased 6 h after the operation, and the MMSE score of the butorphanol group (15.00 ± 2.00) was lower than that of the dezocine and butorphanol group (20.95 ± 1.54; P < 0.05). Dezocine and butorphanol analgesia had transient effects on postoperative cognitive function in elderly patients, and the effect of the combination was superior than butorphanol only.

Trends and predictors of outcomes after surgery for hepatocellular carcinoma: A nationwide population-based study in Taiwan.

BACKGROUND: Despite the huge and growing global burden of hepatocellular carcinoma (HCC), high-quality population-based studies of HCC prevalence and outcomes are scarce. PURPOSE: To analyze trends and predictors of hospital resource utilization and mortality rates in a population of patients who had received HCC surgery. PATIENTS AND MATERIALS: This population-based patient cohort study retrospectively analyzed 23,107 patients who had received surgical treatment for HCC from 1998 to 2009. RESULTS: The prevalence rate of surgical treatment in HCC patients significantly increased by 167.4% from 4.857 per 100,000 persons in 1998 to 12.989 per 100,000 persons in 2009 (P < 0.001). Age, gender, Deyo-Charlson co-morbidity index score, hospital volume, surgeon volume, digestive system disease, hepatitis type and liver cirrhosis were significantly associated with HCC surgical outcomes (P < 0.05). Over the 12-year period analyzed, the estimated mean hospital treatment cost increased 9.4% whereas mean length of stay (LOS) decreased 25.3%. The estimated mean overall survival time after HCC surgery was 40.9 months (SD 1.2 months), and the overall in-hospital 1-, 3-, and 5-year survival rates were 97.2%, 79.9%, 61.1%, and 54.6%, respectively. CONCLUSIONS: These population-based data reveal that the prevalence of HCC has increased, especially in older patients. Additionally, hospital treatment costs for HCC have increased despite decreases in LOS. These analytical results should be applicable to most countries with relatively small populations. Additionally, healthcare providers and patients should recognize that attributes of both the patient and the hospital may affect outcomes.

Effect of Tanshinone IIA intrathecal injections on pain and spinal inflammation in mice with bone tumors.

The study aimed to investigate the effect of intrathecal injections of Tanshinone IIA on thermal hyperalgesia in a mouse model of bone cancer-pain. Spinal IL-1ß, IL-6, TNF-α expression levels were analyzed. C3H/HeNCrlVr male mice were assigned to groups that either received dose-dependent injections of Tanshinone IIA, or the DMSO + Sham, Tanshinone IIA + Sham, DMSO + Tumor, and Control groups. Paw withdrawal thermal latency (PWTL) was measured with a radiant heat stimulus and mRNA expression levels were determined using real-time PCR. Fourteen days post-injection, PWTL in the DMSO + Tumor group was lower than that in the controls (P < 0.05). Twenty-one days post-injection, compared with the Control group, there was no significant difference in PWTL and IL-1ß, IL-6, and TNF-α expression levels between the Tanshinone IIA + Sham and DMSO + Sham groups (P > 0.05). PWTL in the DMSO + Tumor group was significantly lower than the Control group (P < 0.05), while the expression levels of IL-1ß, IL-6, and TNF-α were significantly higher than controls. Compared with the DMSO + Tumor group, PWTLs were higher in the Tanshinone IIA - 20-µg and 40-µg groups, while expression levels of IL-1ß, IL-6, and TNF-α were significantly lower (P < 0.05). These measures were not significantly different between the Tanshinone IIA 10 µg and the DMSO + Tumor groups (P > 0.05). In conclusion, Tanshinone IIA may inhibit the release of inflammatory cytokines, such as, IL-1 ß, IL-6 α, TNF-α.

Renal vein stenosis with transudative ascites from graft after renal transplantation with good response after percutaneous stent placement.

Ascites sometimes occurs as a result of technical complications of transplant surgery or other medical reasons, including hepatic, cardiac, or oncologic pathology. Renal vein stenosis after renal transplant resulting in transudative ascites is rare; thus there are few if any data on such cases. Stent implantation seems to be a safe and elective approach to treatment of this rare condition. We present the case of a 22-year-old woman in whom massive ascites developed 33 months after renal transplantation. After the analysis of the ascites fluid and exclusion of transplant artery stenosis, graft rejection, infection, portal hypertension, and other possible etiologies, the final diagnosis of graft renal vein stenosis with transudative ascites derived from the graft was made based on imaging studies, including Doppler ultrasonography and computed tomography. The patient underwent angiographic stent placement, and the ascites markedly improved after the procedure. Renal vein stenosis complicated with ascites after renal transplantation is highly unusual; the patient's response to angiographic stent placement was beneficial, with satisfactory resolution of the blockage and ascites.

Effect of addition of sodium alginate on bacterial cellulose production by Acetobacter xylinum.

Bacterial cellulose (BC) production by Acetobacter xylinum NUST4.1 was carried out in the shake flask and in a stirred-tank reactor by means of adding sodium alginate (NaAlg) into the medium. When 0.04% (w/v) NaAlg was added in the shake flask, BC production reached 6.0 g/l and the terminal yield of the cellulose was 27% of the total sugar initially added, compared with 3.7 g/l and 24% in the control, respectively. The variation between replicates in all determinations was less than 5%. During the cultivation in the stirred-tank reactor, the addition of NaAlg changed the morphology of cellulose from the irregular clumps and fibrous masses entangled in the internals to discrete masses dispersing into the broth, which indicates that NaAlg hinders formation of large clumps of BC, and enhances cellulose yield. Because the structure of cellulose is changed depending on the culture condition such as additives, structural characteristics of BC produced in the NaAlg-free and NaAlg medium are compared using scanning electron microscopy (SEM), fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD). SEM photographs show some differences in reticulated structures and ribbon width and FT-IR spectra indicate that there is the hydrogen bonding interaction between BC and NaAlg, then X-ray diffraction (XRD) analysis reveals that BC produced with NaAlg-added has a lower crystallinity and a smaller crystalline size. The results show that enhanced yields and modification of cellulose structure occur in the presence of NaAlg.

Interspecies hybrid HbS: complete neutralization of Val6(beta)-dependent polymerization of human beta-chain by pig alpha-chains.

Interspecies hybrid HbS (alpha(2)(P)beta(2)(S)), has been assembled in vitro from pig alpha-globin and human beta(S)-chain. The alpha(2)(P)beta(2)(S) retains normal tetrameric structure (alpha(2)beta(2)) of human Hb and an O(2) affinity comparable to that of HbS in 50 mM Hepes buffer; but, its O(2) affinity is slightly higher than that of HbS in the presence of allosteric effectors (chloride, DPG and phosphate). The (1)H-NMR spectroscopy detected distinct differences between the heme environments and alpha(1)beta(1) interfaces of pig Hb and HbS, while their alpha(1)beta(2) interfaces appear very similar. The interspecies hybrid alpha(2)(H)beta(2)(P) resembles pig Hb; the pig beta-chain dictated the conformation of the heme environment of the human alpha-subunit, and to the alpha(1)beta(1) interfaces of the hybrid. In the alpha(2)(P)beta(2)(S) hybrid, beta(S)-chain dictated the conformation of human heme environment to the pig alpha-chain in the hybrid; but the conformation of alpha(1)beta(1) interface of this hybrid is close to, but not identical to that of HbS. On the other hand, the alpha(1)beta(2) interface conformation is identical to that of HbS. More important, the alpha(2)(P)beta(2)(S) does not polymerize when deoxygenated; pig alpha-chain completely neutralizes the beta(S)-chain dependent polymerization. The polymerization inhibitory propensity of pig alpha-chain is higher when it is present in the cis alpha(P)beta(S) dimer relative to that in a trans alpha(P)beta(A) dimer. The semisynthetically generated chimeric pig-human and human-pig alpha-chains by exchanging the alpha(1-30) segments of human and pig alpha-chains have established that the sequence differences of pig alpha(31-141) segment can also completely neutralize the polymerization. Comparison of the electrostatic potential energy landscape of the alpha-chain surfaces of HbS and alpha(2)(P)beta(2)(S) suggests that the differences in electrostatic potential energy surfaces on the alpha-chain of alpha(2)(P)beta(2)(S) relative to that in HbS, particularly the ones involving CD region, E-helix and EF-corner of pig alpha-chain are responsible for the polymerization neutralization activity. The pig and human-pig chimeric alpha-chains can serve as blueprints for the design of a new generation of variants of alpha-chain(s) suitable for the gene therapy of sickle cell disease.

Correct assembly of human normal adult hemoglobin when expressed in transgenic swine: chemical, conformational and functional equivalence with the human-derived protein.

Structural and functional investigations of recombinant human hemoglobin A (HbA) isolated from the erythrocytes of transgenic swine coexpressing human alpha- and beta-globins have been carried out to authenticate its correct expression, post-translational processing and assembly. The HbA expressed in transgenic swine (TgHbA) is indistinguishable from the human-derived HbA in terms of its isoelectric pH, mass and elution pattern on a Mono S column. The chemical identity of the alpha- and beta-globin chains of TgHbA with the corresponding chains from human-derived HbA has been established by tryptic peptide mapping and amino acid sequencing. The proton NMR spectra of TgHbA have demonstrated that the conformational aspects of the protein around the heme pocket are indistinguishable from those of the control sample of HbA. The equivalence of the hydrogen bond pattern of TgHbA (in particular the inter-subunit surfaces) with that of authentic HbA has also been established by NMR studies. Consistent with these structural and conformational analyses, the TgHbA also exhibits complete functional equivalence with the human-derived HbA with respect to oxygen affinity, cooperativity, Bohr effect and allostery. Hence the studies presented here demonstrate that the transgenic swine system correctly transcribes the alpha- and beta-globin transgenes, translates the respective alpha- and beta-globin mRNA to generate the corresponding globin chains, carries out the correct cotranslational processing of the translated globin chains, inserts the heme into the globin chains in the same orientation as in the human-derived HbA and assembles the alpha- and beta-subunits into a functionally cooperative tetramer that exhibits a response to allosteric effectors identical with that of human-derived HbA. Thus, in the transgenic swine system, in vitro chemical manipulation steps such as those needed in the Escherichia coli and the yeast systems, to convert the rHbA expressed in these systems into forms functionally identical with that of the human-derived protein, are not needed. An additional advantage of the transgenic swine system is the stability of the transgenes over many generations. Hence the transgenic swine could serve as an excellent system for the production of human HbA (or its variants) for structure-function studies and for therapeutic applications.

Contribution of surface histidyl residues in the alpha-chain to the Bohr effect of human normal adult hemoglobin: roles of global electrostatic effects.

We have applied site-directed mutagenesis to our Escherichia coli hemoglobin expression plasmid and constructed five recombinant mutant hemoglobins (r Hbs): r Hb(alpha20His-->Gln or alpha:H20Q); r Hb(alpha:H50Q); r Hb(alpha:H72Q); r Hb(alpha:H89Q); and r Hb(alpha:H112Q). We have constructed these r Hbs to help us assess the contribution of the surface histidyl residues in the alpha-chain to the alkaline Bohr effect of human normal adult hemoglobin (Hb A). In our laboratory, we have monitored the variation of proton nuclear magnetic resonances arising from the C2 protons of the histidyl residues of Hb A as a function of pH and buffer conditions. Several of these resonances have been assigned to the individual histidyl residues on the surface of the hemoglobin molecule using naturally occurring mutant hemoglobins and chemically modified hemoglobins. In the present work, we have identified the C2 proton resonances of five surface histidyl residues of the alpha-chain, alpha20, alpha50, alpha72, alpha89, and alpha112, in both the carbonmonoxy and deoxy forms, by comparing the proton nuclear magnetic resonance spectra of Hb A with those of the r Hbs. For the assignment of the C2 proton resonances of alpha20His and alpha112His, we have used combinations of mutations to compensate for the spectral perturbations resulting from the single mutations, which obscure the resonance assignment. On the basis of the new findings, in solvent containing 0.1 M chloride, the overall contributions from surface histidyl residues of both the alpha- and beta-chain and from other previously identified alkaline Bohr groups account for approximately 75% of the observed Bohr effect at pH 7.3 (the maximum Bohr effect under the prescribed solvent conditions). Our results show that some histidyl residues contribute to the Bohr effect and some oppose the net Bohr effect. In some cases, the addition of anions can diminish or reverse the contributions of specific histidyl residues to the overall Bohr effect. Thus, the Bohr effect, a heterotropic effect, depends on the intricate arrangement and interactions of all hydrogen and anion binding sites in the hemoglobin molecule. It is an excellent example of global electrostatic effects in proteins.

Production of human normal adult and fetal hemoglobins in Escherichia coli.

A hemoglobin expression system in Escherichia coli is described. In order to produce authentic human hemoglobin, we need to co-express both methionine aminopeptidase and globin genes under the control of a strong promoter. We have constructed three plasmids, pHE2, pHE4 and pHE7, for the expression of human normal adult hemoglobin and a plasmid, pHE9, for the expression of human fetal hemoglobin, in high yields. The globin genes can be derived from either synthetic genes or human globin cDNAs. The extra amino-terminal methionine residues of the expressed globins can be removed by the co-expressed methionine aminopeptidase. The heme is inserted correctly into the expressed alpha-globin from our expression plasmids. A fraction (approximately 25%) of the heme is not inserted correctly into the expressed beta- or gamma-globin. However, the incorrectly inserted hemes can be converted into the correct conformation by carrying out a simple oxidation-reduction process on the purified hemoglobin molecule. We have investigated the functional properties of the expressed hemoglobins by measuring their oxygen-binding properties and their structural features by obtaining their 1H-NMR spectra. Our results show that authentic human normal adult and fetal hemoglobins can be produced from our expression plasmids in E. coli and in high yields. Our expression system allows us to design and to produce any recombinant hemoglobins needed for our research on the structure-function relationship in hemoglobin.

Roles of alpha 114 and beta 87 amino acid residues in the polymerization of hemoglobin S: implications for gene therapy.

Three novel recombinant mutants of sickle hemoglobin (Hb S, beta 6Glu-->Val) have been constructed to assess the role of proline at alpha 114 and threonine at beta 87 in the polymerization of deoxygenated Hb S. Using the hemoglobin expression system (pHE2) designed in our laboratory, four plasmids were expressed separately in Escherichia coli to produce the four recombinant hemoglobins: r Hb S (beta 6Glu-->Val); r Hb S-Chiapas (beta 6Glu-->Val, alpha 114Pro-->Arg); r Hb S-D-Ibadan (beta 6Glu-->Val, beta 87Thr-->Lys); and r Hb S-Chiapas-D-Ibadan (beta 6Glu-->Val, alpha 114Pro-->Arg, beta 87Thr-->Lys). The structural features of these four recombinant hemoglobins were analyzed by proton nuclear magnetic resonance spectroscopy, and were found to be similar to those of human normal adult hemoglobin (Hb A) under identical conditions. The recombinant hemoglobins were further investigated by measuring the oxygen-binding properties, which were found to be comparable to those of Hb A. Delay-time gelation studies of the three mutants of r Hb S were carried out in 1.8 M potassium phosphate (pH 7.34) by a temperature jump from 4 degrees C to 30 degrees C and an increase in delay time over that of r Hb S was observed, as well as an overall decrease in the polymerization of these three mutants of Hb S. A more detailed and quantitative investigation has also been carried out to determine the equilibrium solubility (Csat) in 0.1 M potassium phosphate (pH 7.35) at 25 degrees C of the three Hb S mutants as well as of mixtures of these mutants with Hb S versus mixtures of fetal hemoglobin (Hb F) and Hb A with Hb S. The inhibition of polymerization demonstrated in these experiments suggests that the interactions involving the two amino acid residues alpha 114Pro and beta 87Thr are very important to the formation of Hb S polymer, and modification of these amino acids results in an anti-sickling potential. Of particular interest is the inhibitory effect of alpha 114Pro-->Arg, which offers a novel opportunity to use an alpha-chain construct, in addition to a beta-chain construct in the same vector, in gene therapy for sickle cell anemia, with the objective of modifying a larger number of hemoglobin tetramers at a given level of expression.

A novel low oxygen affinity recombinant hemoglobin (alpha96val--> Trp): switching quaternary structure without changing the ligation state.

Using our Escherichia coli expression plasmid (pHE2) in which synthetic human alpha and beta-globin genes are coexpressed with the E. coli methionine aminopeptidase gene under the control of separate tac promoters, we have constructed a new artificial hemoglobin in which the valine residue at position 96 of the alpha chain, located in the alpha 1 beta 2 subunit interface, has been replaced by a tryptophan residue using site-directed mutagenesis. We have determined the oxygen-binding properties of this recombinant hemoglobin, r Hb (alpha 96Val-->Trp), and have used proton nuclear magnetic resonance spectroscopy to investigate its tertiary structure around the heme group and the quaternary structure in the alpha 1 beta 2 subunit interface. This artificial hemoglobin shows a low oxygen affinity, but high cooperativity in oxygen binding, and exhibits no unusual subunit dissociation when ligated. Molecular dynamics simulations suggest that the unique oxygen-binding property of r Hb (alpha 96Val-->Trp) may be due to an extra hydrogen bond between alpha 96Trp and beta 99Asp in the alpha 1 beta 2 subunit interface in the deoxy form. Despite the replacement of a small amino acid residue, valine, by a large tryptophan residue in the alpha 1 beta 2 subunit interface, this artificial hemoglobin shows very similar tertiary structure around the heme pockets and quaternary structure in the alpha 1 beta 2 subunit interface compared to those of human normal adult hemoglobin. Another unique feature of this artificial hemoglobin is that the ligated form, e.g. carbonmonoxy form, of this hemoglobin in the oxy-quaternary structure can be converted to the deoxy-like quaternary structure by the addition of an allosteric effector, inositol hexaphosphate, as well as by lowering the temperature in the absence of inositol hexaphosphate, without changing its ligation state. Thus, this recombinant hemoglobin can be used to gain new insights regarding the nature of subunit interactions in the alpha 1 beta 2 interface and the molecular basis for the allosteric mechanism of hemoglobin.

Restoring allosterism with compensatory mutations in hemoglobin.

Abnormal human hemoglobins (HBs) with amino acid substitutions in the alpha 1 beta 2 interface have very high oxygen affinity and greatly reduced cooperativity in O2 binding compared to normal human Hb. In such abnormal Hbs with mutations at position beta 99, the intersubunit hydrogen bonds between Asp-beta 99 and Tyr-alpha 42 and between Asp-beta 99 and Asn-alpha 97 are broken, thus destabilizing the deoxyquaternary structure of these Hbs. A molecular dynamics method has been used to design compensatory amino acid substitutions in these Hbs that can restore their allosteric properties. We have designed a compensatory mutation in a naturally occurring mutant Hb, Hb Kempsey (Asp-beta 99-->Asn), and have produced it using our Escherichia coli expression plasmid pHE2. We have determined the O2 binding properties of this recombinant double mutant Hb, Hb(Asp-beta 99-->Asn and Tyr-alpha 42-->Asp) and have used 1H NMR spectroscopy to investigate the tertiary structures around the heme groups and the quaternary structure in the alpha 1 beta 2 subunit interface. Our results clearly show that the Tyr-alpha 42-->Asp replacement can substantially compensate for the functional defect of Hb Kempsey caused by the Asp-beta 99-->Asn substitution. The structural and functional information derived from this recombinant Hb provides insights into the structural basis of allosterism and the design of compensatory amino acid substitutions to restore the functional properties of other abnormal HBs associated with hemoglobinopathies.

Contributions of engineered surface salt bridges to the stability of T4 lysozyme determined by directed mutagenesis.

Six designed mutants of T4 lysozyme were created in an attempt to create putative salt bridges on the surface of the protein. The first three of the mutants, T115E (Thr 115 to Glu), Q123E, and N144E, were designed to introduce a new charged side chain close to one or more existing charged groups of the opposite sign on the surface of the protein. In each of these cases the putative electrostatic interactions introduced by the mutation include possible salt bridges between residues within consecutive turns of an alpha-helix. Effects of the mutations ranged from no change in stability to a 1.5 degrees C (0.5 kcal/mol) increase in melting temperature. In two cases, secondary (double) mutants were constructed as controls in which the charge partner was removed from the primary mutant structure. These controls proteins indicate that the contributions to stability from each of the engineered salt bridges is very small (about 0.1-0.25 kcal/mol in 0.15 M KCl). The structures of the three primary mutants were determined by X-ray crystallography and shown to be essentially the same as the wild-type structure except at the site of the mutation. Although the introduced charges in the T115E and Q123E structures are within 3-5 A of their intended partner, the introduced side chains and their intended partners were observed to be quite mobile. It has been shown that the salt bridge between His 31 and Asp 70 in T4 lysozyme stabilizes the protein by 3-5 kcal/mol [Anderson, D. E., Becktel, W. J., & Dahlquist, F. W. (1990) Biochemistry 29, 2403-2408]. To test the effectiveness of His...Asp interactions in general, three additional double mutants, K60H/L13D, K83H/A112D, and S90H/Q122D, were created in order to introduce histidine-aspartate charge pairs on the surface of the protein. Each of these mutants destabilizes the protein by 1-3 kcal/mol in 0.15 M KCl at pH values from 2 to 6.5. The X-ray crystallographic structure of the mutant K83H/A112D has been determined and shows that there are backbone conformational changes of 0.3-0.6 A extending over several residues. The introduction of the histidine and aspartate presumably introduces strain into the folded protein that destabilizes this variant. It is concluded that pairs of oppositely charged residues that are on the surface of a protein and have freedom to adopt different conformations do not tend to come together to form structurally localized salt bridges. Rather, such residues tend to remain mobile, interact weakly if at all, and do not contribute significantly to protein stability. It is argued that the entropic cost of localizing a pair of solvent-exposed charged groups on the surface of a protein largely offsets the interaction energy expected from the formation of a defined salt bridge. There are examples of strong salt bridges in proteins, but such interactions require that the folding of the protein provides the requisite driving energy to hold the interacting partners in the correct rigid alignment.

Second-site revertants of an inactive T4 lysozyme mutant restore activity by restructuring the active site cleft.

Substitution of Thr26 by Gln in the lysozyme of bacteriophage T4 produces an enzyme with greatly reduced activity but essentially unaltered stability relative to wild type. Spontaneous second-site revertants of the mutant were selected genetically; two of them were chosen for structural and biochemical characterization. One revertant bears (in addition to the primary mutation) the substitution Tyr18----His, the other, Tyr18----Asp. The primary mutant and both revertant lysozyme genes were reconstructed in a plasmid-based expression system, and the proteins were produced and purified. The two revertant lysozymes exhibit enzymatic activities intermediate between wild type and the primary mutant; both also exhibit melting temperatures approximately 3 degrees C lower than either the wild type or the primary mutant. Crystals suitable for X-ray diffraction analysis were obtained from both revertant lysozymes, but not the primary mutant. Structures of the double mutant lysozymes were refined at 1.8-A resolution to crystallographic residuals of 15.1% (Tyr18----His) and 15.2% (Tyr18----Asp). Model building suggests that the side chain of Gln26 in the primary mutant is forced to protrude into the active site cleft, resulting in low catalytic activity. In contrast, the crystal structures of the revertants reveal that the double substitutions (Gln26 and His18, or Gln26 and Asp18) fit into the same space that is occupied by Thr26 and Tyr18 in the wild-type enzyme; the effect is a restructuring of the surface of the active site cleft, with essentially no perturbation of the polypeptide backbone. This restructuring is effected by a novel series of hydrogen bonds and electrostatic interactions that apparently stabilize the revertant structures.

Structural studies of mutants of T4 lysozyme that alter hydrophobic stabilization.

Multiple replacements at amino acid position 3 of bacteriophage T4 lysozyme have shown that the conformational stability of the protein is directly governed by the hydrophobicity of the residue substituted (Matsumura, M., Becktel, W. J., and Matthews, B. W. (1988) Nature 334, 406-410). Of the 13 mutant lysozymes made by site-directed mutagenesis, two variants, one with valine (I3V) and the other with tyrosine (I3Y), were crystallized and their structures solved. In this report we describe the crystal structures of these variants at 1.7 A resolution. While the structure of the I3V mutant is essentially the same as that of wild-type lysozyme, the I3Y mutant has substantial changes in its structure. The most significant of these are that the side chain of the tyrosine is not accommodated within the interior of the protein and the amino-terminal polypeptide (residues 1-9) moves 0.6-1.1 A relative to the wild-type structure. Using coordinates based on the wild-type and available mutant structures, solvent accessible surface area of residue 3 as well as the adjacent 9 residues in the folded form were calculated. The free energy of stabilization based on the transfer of these residues from a fully extended form to the interior to the folded protein was found to correlate well with the protein stability determined by thermodynamic analysis. The enhanced thermostability of the variant Ile-3----Leu, relative to wild-type lysozyme, can also be rationalized by surface-area calculations based on a model-built structure. Noncrystallization of most lysozyme variants at position 3 appears to be due to disruption of intermolecular contacts in the crystal. The Ile-3----Val variant is closely isomorphous with wild-type and maintains the same crystal contacts. In the Ile-3----Tyr variant, however, a new set of contacts is made in which direct protein-protein hydrogen bonds are replaced by protein-water-protein hydrogen bonds as well as a novel hydrogen bond involving the phenolic hydroxyl of the substituted tyrosine.