Gram-negative bacteria expressing class A β-lactamases pose a serious health threat due to their ability to inactivate all β-lactam antibiotics. The acyl–enzyme intermediate is a central milestone in ...the hydrolysis reaction catalyzed by these enzymes. However, the protonation states of the catalytic residues in this complex have never been fully analyzed experimentally due to inherent difficulties. To help unravel the ambiguity surrounding class A β-lactamase catalysis, we have used ultrahigh-resolution X-ray crystallography and the recently approved β-lactamase inhibitor avibactam to trap the acyl–enzyme complex of class A β-lactamase CTX-M-14 at varying pHs. A 0.83-Å-resolution CTX-M-14 complex structure at pH 7.9 revealed a neutral state for both Lys73 and Glu166. Furthermore, the avibactam hydroxylamine-O-sulfonate group conformation varied according to pH, and this conformational switch appeared to correspond to a change in the Lys73 protonation state at low pH. In conjunction with computational analyses, our structures suggest that Lys73 has a perturbed acid dissociation constant (pKₐ) compared with acyl–enzyme complexes with β-lactams, hindering its function to deprotonate Glu166 and the initiation of the deacylation reaction. Further NMR analysis demonstrated Lys73 pKₐ to be ∼5.2 to 5.6. Together with previous ultrahigh-resolution crystal structures, these findings enable us to follow the proton transfer process of the entire acylation reaction and reveal the critical role of Lys73. They also shed light on the stability and reversibility of the avibactam carbamoyl acyl–enzyme complex, highlighting the effect of substrate functional groups in influencing the protonation states of catalytic residues and subsequently the progression of the reaction.
Molecular chaperones have an essential role for the maintenance of a balanced protein homeostasis. Here, we investigate how protein kinases are recruited and loaded to the Hsp90-Cdc37 complex, the ...first step during Hsp90-mediated chaperoning that leads to enhanced client kinase stability and activation. We show that conformational dynamics of all partners is a critical feature of the underlying loading mechanism. The kinome co-chaperone Cdc37 exists primarily in a dynamic extended conformation but samples a low-populated, well-defined compact structure. Exchange between these two states is maintained in an assembled Hsp90-Cdc37 complex and is necessary for substrate loading. Breathing motions at the N-lobe of a free kinase domain partially expose the kinase segment trapped in the Hsp90 dimer downstream in the cycle. Thus, client dynamics poise for chaperone dependence. Hsp90 is not directly involved during loading, and Cdc37 is assigned the task of sensing clients by stabilizing the preexisting partially unfolded client state.
Unique to targeting the C-terminal domain of Hsp90 (C-Hsp90) is the ability to uncouple the cytotoxic and cytoprotective outcomes of Hsp90 modulation. After the identification of novobiocin as a ...C-Hsp90 interacting ligand a diverse gamut of novologues emerged, from which KU-32 and KU-596 exhibited strong neuroprotective activity. However, further development of these ligands is hampered by the difficulty to obtain structural information on their complexes with Hsp90. Using saturation transfer difference (STD) NMR spectroscopy, we found that the primary binding epitopes of KU-32 and KU596 map at the ring systems of the ligands and specifically the coumarin and biphenyl structures, respectively. Based on both relative and absolute STD effects, we identified KU-596 sites that can be explored to design novel third-generation novologues. In addition, chemical shift perturbations obtained by methyl-TROSY reveal that novologues bind at the cryptic, C-Hsp90 ATP-binding pocket and produce global, long-range structural rearrangements to dimeric Hsp90.
Abstract Introduction We present the protocol of a large population-based case-control study of 5 common tumors in Spain (MCC-Spain) that evaluates environmental exposures and genetic factors. ...Methods Between 2008-2013, 10,183 persons aged 20-85 years were enrolled in 23 hospitals and primary care centres in 12 Spanish provinces including 1,115 cases of a new diagnosis of prostate cancer, 1,750 of breast cancer, 2,171 of colorectal cancer, 492 of gastro-oesophageal cancer, 554 cases of chronic lymphocytic leukaemia (CLL) and 4,101 population-based controls matched by frequency to cases by age, sex and region of residence. Participation rates ranged from 57% (stomach cancer) to 87% (CLL cases) and from 30% to 77% in controls. Participants completed a face-to-face computerized interview on sociodemographic factors, environmental exposures, occupation, medication, lifestyle, and personal and family medical history. In addition, participants completed a self-administered food-frequency questionnaire and telephone interviews. Blood samples were collected from 76% of participants while saliva samples were collected in CLL cases and participants refusing blood extractions. Clinical information was recorded for cases and paraffin blocks and/or fresh tumor samples are available in most collaborating hospitals. Genotyping was done through an exome array enriched with genetic markers in specific pathways. Multiple analyses are planned to assess the association of environmental, personal and genetic risk factors for each tumor and to identify pleiotropic effects. Discussion This study, conducted within the Spanish Consortium for Biomedical Research in Epidemiology & Public Health (CIBERESP), is a unique initiative to evaluate etiological factors for common cancers and will promote cancer research and prevention in Spain.
Sorption of microamounts of Cs from highly saline solutions (bottom residues from nuclear power plants) on various types of ferrocyanide sorbents was studied. The dependences of the distribution ...coefficient (
K
d
) of
137
Cs on pH of solution were determined under static conditions. They strongly depend on the type of the sorbent used. FS-2 sorbent prepared by joint precipitation of silicic acid and copper ferrocyanide exhibits the best sorption characteristics toward Cs. On this sorbent,
K
d
of
137
Cs in a model solution with pH < 11 is (5–6) × 10
5
cm
3
g
−1
. In alkaline solutions at pH > 11, the Cs sorption drastically decreases because of dissolution of the ferrocyanide component of the sorbent. Data on Cs sorption under dynamic conditions on various types of ferrocyanide sorbents from the bottom residue with pH 8–11 are presented. The volume of the solution passed up to 1%
137
Cs breakthrough was determined. FS-2 sorbent exhibits the best dynamic characteristics. Its use allows decontamination of 1000–4500 column volumes of the bottom residue depending on pH.
The solidification of partially evaporated bottoms of RBMK and VVER with salt concentration 500–650 g/liter by compositional binders consisting of Portland cement and silicic additives – aerosil, ...microsilica, opoka, silicic acid, liquid glass, and diatomite is examined. The additions were used to obtain matrices that satisfy the requirements of safe storage of cemented radwastes. The partition coefficients of
137
Cs in partially evaporated bottoms are determined for all additives studied. The most effective additive for solidification of partially evaporated bottoms of VVER is diatomite. Matrices with diatomite have strength 50–81 kg/cm
2
, the rate of leaching of
137
Cs ~ 10
–3
–10
–4
g/(cm
2
·day) and the fill with respect to salts reaches 20.9 wt.%. On the solidification of partially evaporated RBMK bottoms the most effective hardening additives are aerosil and microsilica and the most effective sorbing additives are bentonite, opoka, and diatomite. The matrices so obtained have strength 59–93 kg/cm
2
,
137
Cs leach rate ~ 10
–3
–10
–4
g/(cm
2
·day) and contain to 25.1 wt.% salts.
We discuss the physics of large impact parameter interactions at the LHC: ultraperipheral collisions (UPCs). The dominant processes in UPCs are photon–nucleon (nucleus) interactions. The current LHC ...detector configurations can explore hard phenomena at small
x
with nuclei and nucleons at photon–nucleon center-of-mass energies above 1 TeV, extending the
x
range of HERA by a factor of ten. In particular, it will be possible to probe diffractive and inclusive parton densities in nuclei using several processes. The interaction of small dipoles with protons and nuclei can be investigated in elastic and quasi-elastic
J
/
ψ
and
Υ
production as well as in high
t
ρ
0
production accompanied by a rapidity gap. Several of these phenomena provide clean signatures of the onset of the new high gluon density QCD regime. The LHC is in the kinematic range where nonlinear effects are several times larger than those at HERA. Two-photon processes in UPCs are also studied. In addition, while UPCs play a role in limiting the maximum beam luminosity, they can also be used as a luminosity monitor by measuring mutual electromagnetic dissociation of the beam nuclei. We also review similar studies at HERA and RHIC as well as describe the potential use of the LHC detectors for UPC measurements.
Expanded low-carbon baseload power production through the use of nuclear fission can be enabled by recycling long-lived actinide isotopes within the nuclear fuel cycle. This approach provides the ...benefits of (a) more completely utilizing the energy potential of mined uranium, (b) reducing the footprint of nuclear geological repositories, and (c) reducing the time required for the radiotoxicity of the disposed waste to decrease to the level of uranium ore from one hundred thousand years to a few hundred years. A key step in achieving this goal is the separation of long-lived isotopes of americium (Am) and curium (Cm) for recycle into fast reactors. To achieve this goal, a novel process was successfully demonstrated on a laboratory scale using a bank of 1.25-cm centrifugal contactors, fabricated by additive manufacturing, and a simulant containing the major fission product elements. Americium and Cm were separated from the lanthanides with over 99.9% completion. The sum of the impurities of the Am/Cm product stream using the simulated raffinate was found to be 3.2 × 10
g/L. The process performance was validated using a genuine high burnup used nuclear fuel raffinate in a batch regime. Separation factors of nearly 100 for
Eu over
Am were achieved. All these results indicate the process scalability to an engineering scale.
The characteristics of various sorbents based on tetraoctyldiglycolamide (TODGA) in sorption of REE(III), Th(IV), and U(VI) ions from nitric acid solutions were studied. The static capacity of the ...sorbents increases as the TODGA content of the sorbent and HNO
3
concentration in the initial solution are increased. Europium can be desorbed with dilute HNO
3
(0.01 M) and complexone (DTPA) solutions. The sorption characteristics of the sorbents in repeated sorption–desorption cycles remain stable. The sorbents prepared by joint copolymerization (solid extactants, SEs) exhibit better sorption and kinetic characteristics compared to the sorbents prepared by impregnation of the ready polymeric matrix.
Unique to targeting the C-terminal domain of Hsp90 (C-Hsp90) is the ability to uncouple the cytotoxic and cytoprotective outcomes of Hsp90 modulation. After the identification of novobiocin as a ...C-Hsp90 interacting ligand a diverse gamut of novologues emerged, from which KU-32 and KU-596 exhibited strong neuroprotective activity. However, further development of these ligands is hampered by the difficulty to obtain structural information on their complexes with Hsp90. Using saturation transfer difference (STD) NMR spectroscopy, we found that the primary binding epitopes of KU-32 and KU596 map at the ring systems of the ligands and specifically the coumarin and biphenyl structures, respectively. Based on both relative and absolute STD effects, we identified KU-596 sites that can be explored to design novel third-generation novologues. In addition, chemical shift perturbations obtained by methyl-TROSY reveal that novologues bind at the cryptic, C-Hsp90 ATP-binding pocket and produce global, long-range structural rearrangements to dimeric Hsp90.
Novologues KU-32 and KU-596 bind at the C-terminal domain of Hsp90 and exploit a long-range allosteric network to modulate the chaperone cycle.
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