scholarly journals The solution structures of higher-order human telomere G-quadruplex multimers

2021 ◽  
Vol 49 (3) ◽  
pp. 1749-1768
Author(s):  
Robert C Monsen ◽  
Srinivas Chakravarthy ◽  
William L Dean ◽  
Jonathan B Chaires ◽  
John O Trent
Keyword(s):  
Analytical Ultracentrifugation ◽  
Persistence Length ◽  
Md Simulations ◽  
Size Exclusion ◽  
Scattering Data ◽  
Solution Structures ◽  
X Ray Scattering ◽  
G Quadruplex ◽  
Dynamics Simulations ◽  
Global Fitting

Abstract Human telomeres contain the repeat DNA sequence 5′-d(TTAGGG), with duplex regions that are several kilobases long terminating in a 3′ single-stranded overhang. The structure of the single-stranded overhang is not known with certainty, with disparate models proposed in the literature. We report here the results of an integrated structural biology approach that combines small-angle X-ray scattering, circular dichroism (CD), analytical ultracentrifugation, size-exclusion column chromatography and molecular dynamics simulations that provide the most detailed characterization to date of the structure of the telomeric overhang. We find that the single-stranded sequences 5′-d(TTAGGG)n, with n = 8, 12 and 16, fold into multimeric structures containing the maximal number (2, 3 and 4, respectively) of contiguous G4 units with no long gaps between units. The G4 units are a mixture of hybrid-1 and hybrid-2 conformers. In the multimeric structures, G4 units interact, at least transiently, at the interfaces between units to produce distinctive CD signatures. Global fitting of our hydrodynamic and scattering data to a worm-like chain (WLC) model indicates that these multimeric G4 structures are semi-flexible, with a persistence length of ∼34 Å. Investigations of its flexibility using MD simulations reveal stacking, unstacking, and coiling movements, which yield unique sites for drug targeting.

scholarly journals The solution structures of higher-order human telomere G-quadruplex multimers

2020 ◽  
Author(s):  
Robert C. Monsen ◽  
Srinivas Chakravarthy ◽  
William L. Dean ◽  
Jonathan B. Chaires ◽  
John O. Trent
Keyword(s):  
Analytical Ultracentrifugation ◽  
Persistence Length ◽  
Md Simulations ◽  
Size Exclusion ◽  
Scattering Data ◽  
Solution Structures ◽  
X Ray Scattering ◽  
G Quadruplex ◽  
Dynamics Simulations ◽  
Global Fitting

ABSTRACTHuman telomeres contain the repeat DNA sequence 5’(TTAGGG), with duplex regions that are several kilobases long terminating in a 3’ single-stranded overhang. The structure of the single-stranded overhang is not known with certainty, with disparate modes proposed in the literature. We report here the results of an integrated structural biology approach that combines small-angle X-ray scattering, circular dichroism (CD), analytical ultracentrifugation, size-exclusion column chromatography and molecular dynamics simulations that provide the most detailed characterization to date of the structure of the telomeric overhang. We find that the single-stranded sequences 5’(TTAGGG)n, with n=8, 12, and 16, fold into multimeric structures containing the maximal number (2, 3, and 4, respectively) of contiguous G4 units with no long gaps between units. The G4 units are a mixture of hybrid-1 and hybrid-2 conformers. In the multimeric structures, G4 units interact, at least transiently, at the interfaces between units to produce distinctive CD signatures. Global fitting of our hydrodynamic and scattering data to a worm-like chain (WLC) model indicates that these multimeric G4 structures are semi-flexible, with a persistence length of about 34 Å. Investigations of its flexibility using MD simulations reveal stacking, unstacking, and coiling movements, which yield unique sites for drug targeting.

scholarly journals Sorafenib as an Inhibitor of RUVBL2

Biomolecules ◽  
2020 ◽  
Vol 10 (4) ◽  
pp. 605 ◽  
Author(s):  
Nardin Nano ◽  
Francisca Ugwu ◽  
Thiago V. Seraphim ◽  
Tangzhi Li ◽  
Gina Azer ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Competitive Inhibitor ◽  
Telomerase Activity ◽  
Size Exclusion ◽  
Scattering Data ◽  
Cellular Processes ◽  
Dna Damage Signaling ◽  
X Ray Scattering ◽  
Exclusion Chromatography ◽  
Aaa Atpases

RUVBL1 and RUVBL2 are highly conserved ATPases that belong to the AAA+ (ATPases Associated with various cellular Activities) superfamily and are involved in various complexes and cellular processes, several of which are closely linked to oncogenesis. The proteins were implicated in DNA damage signaling and repair, chromatin remodeling, telomerase activity, and in modulating the transcriptional activities of proto-oncogenes such as c-Myc and β-catenin. Moreover, both proteins were found to be overexpressed in several different types of cancers such as breast, lung, kidney, bladder, and leukemia. Given their various roles and strong involvement in carcinogenesis, the RUVBL proteins are considered to be novel targets for the discovery and development of therapeutic cancer drugs. Here, we describe the identification of sorafenib as a novel inhibitor of the ATPase activity of human RUVBL2. Enzyme kinetics and surface plasmon resonance experiments revealed that sorafenib is a weak, mixed non-competitive inhibitor of the protein’s ATPase activity. Size exclusion chromatography and small angle X-ray scattering data indicated that the interaction of sorafenib with RUVBL2 does not cause a significant effect on the solution conformation of the protein; however, the data suggested that the effect of sorafenib on RUVBL2 activity is mediated by the insertion domain in the protein. Sorafenib also inhibited the ATPase activity of the RUVBL1/2 complex. Hence, we propose that sorafenib could be further optimized to be a potent inhibitor of the RUVBL proteins.

scholarly journals Solution structures of long-acting insulin analogues and their complexes with albumin

2019 ◽  
Vol 75 (3) ◽  
pp. 272-282 ◽  
Author(s):  
Line A. Ryberg ◽  
Pernille Sønderby ◽  
Fabian Barrientos ◽  
Jens T. Bukrinski ◽  
Günther H. J. Peters ◽  
...  
Keyword(s):  
Solution Structure ◽  
Insulin Analogues ◽  
Size Exclusion ◽  
Life Extension ◽  
Once Daily ◽  
Solution Structures ◽  
X Ray Scattering ◽  
Exclusion Chromatography ◽  
Self Association ◽  
Long Acting

The lipidation of peptide drugs is one strategy to obtain extended half-lives, enabling once-daily or even less frequent injections for patients. The half-life extension results from a combination of self-association and association with human serum albumin (albumin). The self-association and association with albumin of two insulin analogues, insulin detemir and insulin degludec, were investigated by small-angle X-ray scattering (SAXS) and dynamic light scattering (DLS) in phenolic buffers. Detemir shows concentration-dependent self-association, with an equilibrium between hexamer, dihexamer, trihexamer and larger species, while degludec appears as a dihexamer independent of concentration. The solution structure of the detemir trihexamer has a bent shape. The stoichiometry of the association with albumin was studied using DLS. For albumin–detemir the molar stoichiometry was determined to be 1:6 (albumin:detemir ratio) and for albumin–degludec it was between 1:6 and 1:12 (albumin:degludec ratio). Batch SAXS measurements of a 1:6 albumin:detemir concentration series revealed a concentration dependence of complex formation. The data allowed the modelling of a complex between albumin and a detemir hexamer and a complex consisting of two albumins binding to opposite ends of a detemir dihexamer. Measurements of size-exclusion chromatography coupled to SAXS revealed a complex between a degludec dihexamer and albumin. Based on the results, equilibria for the albumin–detemir and albumin–degludec mixtures are proposed.

scholarly journals Non-linearity of the collagen triple helix in solution and implications for collagen function

2017 ◽  
Vol 474 (13) ◽  
pp. 2203-2217 ◽  
Author(s):  
Kenneth T. Walker ◽  
Ruodan Nan ◽  
David W. Wright ◽  
Jayesh Gor ◽  
Anthony C. Bishop ◽  
...  
Keyword(s):  
Analytical Ultracentrifugation ◽  
Linear Structure ◽  
Small Degree ◽  
Scattering Data ◽  
Helical Conformation ◽  
End Effects ◽  
Helical Peptides ◽  
Atomistic Modelling ◽  
Triple Helices ◽  
Dynamics Simulations

Collagen adopts a characteristic supercoiled triple helical conformation which requires a repeating (Xaa-Yaa-Gly)n sequence. Despite the abundance of collagen, a combined experimental and atomistic modelling approach has not so far quantitated the degree of flexibility seen experimentally in the solution structures of collagen triple helices. To address this question, we report an experimental study on the flexibility of varying lengths of collagen triple helical peptides, composed of six, eight, ten and twelve repeats of the most stable Pro-Hyp-Gly (POG) units. In addition, one unblocked peptide, (POG)10unblocked, was compared with the blocked (POG)10 as a control for the significance of end effects. Complementary analytical ultracentrifugation and synchrotron small angle X-ray scattering data showed that the conformations of the longer triple helical peptides were not well explained by a linear structure derived from crystallography. To interpret these data, molecular dynamics simulations were used to generate 50 000 physically realistic collagen structures for each of the helices. These structures were fitted against their respective scattering data to reveal the best fitting structures from this large ensemble of possible helix structures. This curve fitting confirmed a small degree of non-linearity to exist in these best fit triple helices, with the degree of bending approximated as 4–17° from linearity. Our results open the way for further studies of other collagen triple helices with different sequences and stabilities in order to clarify the role of molecular rigidity and flexibility in collagen extracellular and immune function and disease.

scholarly journals Size-exclusion chromatography small-angle X-ray scattering of water soluble proteins on a laboratory instrument

2018 ◽  
Vol 51 (6) ◽  
pp. 1623-1632 ◽  
Author(s):  
Saskia Bucciarelli ◽  
Søren Roi Midtgaard ◽  
Martin Nors Pedersen ◽  
Søren Skou ◽  
Lise Arleth ◽  
...  
Keyword(s):  
Size Exclusion Chromatography ◽  
Small Angle ◽  
Size Exclusion ◽  
Scattering Data ◽  
X Ray ◽  
Laboratory Instrument ◽  
X Ray Scattering ◽  
Home Institution ◽  
Exclusion Chromatography ◽  
Ray Scattering

Coupling of size-exclusion chromatography with biological solution small-angle X-ray scattering (SEC-SAXS) on dedicated synchrotron beamlines enables structural analysis of challenging samples such as labile proteins and low-affinity complexes. For this reason, the approach has gained increased popularity during the past decade. Transportation of perishable samples to synchrotrons might, however, compromise the experiments, and the limited availability of synchrotron beamtime renders iterative sample optimization tedious and lengthy. Here, the successful setup of laboratory-based SEC-SAXS is described in a proof-of-concept study. It is demonstrated that sufficient quality data can be obtained on a laboratory instrument with small sample consumption, comparable to typical synchrotron SEC-SAXS demands. UV/vis measurements directly on the SAXS exposure cell ensure accurate concentration determination, crucial for direct molecular weight determination from the scattering data. The absence of radiation damage implies that the sample can be fractionated and subjected to complementary analysis available at the home institution after SEC-SAXS. Laboratory-based SEC-SAXS opens the field for analysis of biological samples at the home institution, thus increasing productivity of biostructural research. It may further ensure that synchrotron beamtime is used primarily for the most suitable and optimized samples.

scholarly journals Solution scattering at the Life Science X-ray Scattering (LiX) beamline

2020 ◽  
Vol 27 (3) ◽  
pp. 804-812 ◽  
Author(s):  
Lin Yang ◽  
Stephen Antonelli ◽  
Shirish Chodankar ◽  
James Byrnes ◽  
Edwin Lazo ◽  
...  
Keyword(s):  
Data Analysis ◽  
Data Storage ◽  
User Interfaces ◽  
Life Science ◽  
Size Exclusion ◽  
Scattering Data ◽  
Batch Mode ◽  
X Ray ◽  
X Ray Scattering ◽  
Ray Scattering

This work reports the instrumentation and software implementation at the Life Science X-ray Scattering (LiX) beamline at NSLS-II in support of biomolecular solution scattering. For automated static measurements, samples are stored in PCR tubes and grouped in 18-position sample holders. Unattended operations are enabled using a six-axis robot that exchanges sample holders between a storage box and a sample handler, transporting samples from the PCR tubes to the X-ray beam for scattering measurements. The storage box has a capacity of 20 sample holders. At full capacity, the measurements on all samples last for ∼9 h. For in-line size-exclusion chromatography, the beamline-control software coordinates with a commercial high-performance liquid chromatography (HPLC) system to measure multiple samples in batch mode. The beamline can switch between static and HPLC measurements instantaneously. In all measurements, the scattering data span a wide q-range of typically 0.006–3.2 Å−1. Functionalities in the Python package py4xs have been developed to support automated data processing, including azimuthal averaging, merging data from multiple detectors, buffer scattering subtraction, data storage in HDF5 format and exporting the final data in a three-column text format that is acceptable by most data analysis tools. These functionalities have been integrated into graphical user interfaces that run in Jupyter notebooks, with hooks for external data analysis software.

scholarly journals The solution structures of native and patient monomeric human IgA1 reveal asymmetric extended structures: implications for function and IgAN disease

2015 ◽  
Vol 471 (2) ◽  
pp. 167-185 ◽  
Author(s):  
Gar Kay Hui ◽  
David W. Wright ◽  
Owen L. Vennard ◽  
Lucy E. Rayner ◽  
Melisa Pang ◽  
...  
Keyword(s):  
Neutron Scattering ◽  
Receptor Binding ◽  
Analytical Ultracentrifugation ◽  
Scattering Data ◽  
X Ray ◽  
Solution Structures ◽  
Binding Function ◽  
Atomistic Modelling ◽  
Modelling Approach

Detailed analytical ultracentrifugation and X-ray/neutron scattering data and a new atomistic modelling approach revealed asymmetric extended solution structures for human IgA1 that account for its receptor-binding function. IgA1 with different hinge O-galactosylation patterns showed similar structures.

scholarly journals Structural and functional characterization of TesB fromYersinia pestisreveals a unique octameric arrangement of hotdog domains

2015 ◽  
Vol 71 (4) ◽  
pp. 986-995 ◽  
Author(s):  
C. M. D. Swarbrick ◽  
M. A. Perugini ◽  
N. Cowieson ◽  
J. K. Forwood
Keyword(s):  
Analytical Ultracentrifugation ◽  
Quaternary Structure ◽  
Functional Characterization ◽  
Fatty Acyl ◽  
Size Exclusion ◽  
X Ray Scattering ◽  
Wide Range ◽  
Exclusion Chromatography ◽  
Enzyme Families

Acyl-CoA thioesterases catalyse the hydrolysis of the thioester bonds present within a wide range of acyl-CoA substrates, releasing free CoASH and the corresponding fatty-acyl conjugate. The TesB-type thioesterases are members of the TE4 thioesterase family, one of 25 thioesterase enzyme families characterized to date, and contain two fused hotdog domains in both prokaryote and eukaryote homologues. Only two structures have been elucidated within this enzyme family, and much of the current understanding of the TesB thioesterases has been based on theEscherichia colistructure.Yersinia pestis, a highly virulent bacterium, encodes only one TesB-type thioesterase in its genome; here, the structural and functional characterization of this enzyme are reported, revealing unique elements both within the protomer and quaternary arrangements of the hotdog domains which have not been reported previously in any thioesterase family. The quaternary structure, confirmed using a range of structural and biophysical techniques including crystallography, small-angle X-ray scattering, analytical ultracentrifugation and size-exclusion chromatography, exhibits a unique octameric arrangement of hotdog domains. Interestingly, the same biological unit appears to be present in both TesB structures solved to date, and is likely to be a conserved and distinguishing feature of TesB-type thioesterases. Analysis of theY. pestisTesB thioesterase activity revealed a strong preference for octanoyl-CoA and this is supported by structural analysis of the active site. Overall, the results provide novel insights into the structure of TesB thioesterases which are likely to be conserved and distinguishing features of the TE4 thioesterase family.

scholarly journals A Mechanically Weak Extracellular Membrane-Adjacent Domain Induces Dimerization of Protocadherin-15

2018 ◽  
Author(s):  
P. De-la-Torre ◽  
D. Choudhary ◽  
R. Araya-Secchi ◽  
Y. Narui ◽  
M. Sotomayor
Keyword(s):  
Protein Interactions ◽  
Analytical Ultracentrifugation ◽  
Size Exclusion ◽  
Molecular Architecture ◽  
Protein Protein Interactions ◽  
Tip Link ◽  
Exclusion Chromatography ◽  
Protocadherin 15 ◽  
Cadherin 23 ◽  
Dynamics Simulations

ABSTRACTThe cadherin superfamily of proteins is defined by the presence of extracellular cadherin (EC) repeats that engage in protein-protein interactions to mediate cell-cell adhesion, cell signaling, and mechanotransduction. The extracellular domains of non-classical cadherins often have a large number of EC repeats along with other subdomains of various folds. Protocadherin-15 (PCDH15), a protein component of the inner-ear tip link filament essential for mechanotransduction, has eleven EC repeats and a membrane adjacent domain (MAD12) of atypical fold. Here we report the crystal structure of a pig PCDH15 fragment including EC10, EC11, and MAD12 in a parallel dimeric arrangement. MAD12 has a unique molecular architecture and folds as a ferredoxin-like domain similar to that found in the nucleoporin protein Nup54. Analytical ultracentrifugation experiments along with size exclusion chromatography coupled to multi-angle laser light scattering and small-angle X-ray scattering corroborate the crystallographic dimer and show that MAD12 induces parallel dimerization of PCDH15 near its membrane insertion point. In addition, steered molecular dynamics simulations suggest that MAD12 is mechanically weak and may unfold before tip-link rupture. Sequence analyses and structural modeling predict the existence of similar domains in cadherin-23, protocadherin-24, and the “giant” FAT and CELSR cadherins, indicating that some of them may also exhibit MAD-induced parallel dimerization.

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