RNA Ensembles from Solvent Accessibility Data: Application to the SAM-I Riboswitch Aptamer Domain

2021 ◽  
Author(s):  
Jingru Xie ◽  
Aaron T. Frank
Keyword(s):  
Solvent Accessibility ◽  
Data Application ◽  
Aptamer Domain

scholarly journals Structural Ensembles of Ribonucleic Acids From Solvent Accessibility Data: Application to the S-Adenosylmethionine (SAM)-Responsive Riboswitch

2020 ◽  
Author(s):  
Jingru Xie ◽  
Aaron T. Frank
Keyword(s):  
Solvent Accessibility ◽  
Binding Pocket ◽  
Accessible Surface Area ◽  
Solvent Accessible Surface Area ◽  
Structural Examination ◽  
Ribonucleic Acids ◽  
Data Application ◽  
Accessible Surface ◽  
Aptamer Domain ◽  
Solvent Accessible Surface

ABSTRACTRiboswitches are regulatory ribonucleic acid (RNA) elements that act as ligand-dependent conformational switches. In the apo form, the aptamer domain, the region of a riboswitch that binds to its cognate ligand, is dynamic, thus requiring an ensemble-representation of its structure. Analysis of such ensembles can provide molecular insights into the sensing mechanism and capabilities of riboswitches. Here, as a proof-of-concept, we constructed a pair of atomistic ensembles of the well-studied S-adenosylmethionine (SAM)-responsive riboswitch in the absence (-SAM) and presence (+SAM) of SAM. To achieve this, we first generated a large conformational pool and then reweighted conformers in the pool using solvent accessible surface area (SASA) data derived from recently reported light-activated structural examination of RNA (LASER) reactivities, measured in the −SAM and +SAM states of the riboswitch. The differences in the resulting −SAM and +SAM ensembles are consistent with a SAM-dependent reshaping of the free landscape of the riboswitch. Interestingly, within the −SAM ensemble, we identified a conformer that harbors a hidden binding pocket, which was discovered using ensemble docking. The method we have applied to the SAM riboswitch is general, and could, therefore, be used to construct atomistic ensembles for other riboswitches, and more broadly, other classes of structured RNAs.

scholarly journals A 300-fold enhancement of imino nucleic acid resonances by hyperpolarized water provides a new window for probing RNA refolding by 1D and 2D NMR

2020 ◽  
Vol 117 (5) ◽  
pp. 2449-2455 ◽  
Author(s):  
Mihajlo Novakovic ◽  
Gregory L. Olsen ◽  
György Pintér ◽  
Daniel Hymon ◽  
Boris Fürtig ◽  
...  
Keyword(s):  
Nmr Spectra ◽  
Chemical Exchange ◽  
Solvent Accessibility ◽  
2D Nmr ◽  
Rapid Acquisition ◽  
H Nmr ◽  
Rapid Monitoring ◽  
Nmr Sensitivity ◽  
A Site ◽  
Aptamer Domain

NMR sensitivity-enhancement methods involving hyperpolarized water could be of importance for solution-state biophysical investigations. Hyperpolarized water (HyperW) can enhance the 1H NMR signals of exchangeable sites by orders of magnitude over their thermal counterparts, while providing insight into chemical exchange and solvent accessibility at a site-resolved level. As HyperW’s enhancements are achieved by exploiting fast solvent exchanges associated with minimal interscan delays, possibilities for the rapid monitoring of chemical reactions and biomolecular (re)folding are opened. HyperW NMR can also accommodate heteronuclear transfers, facilitating the rapid acquisition of 2-dimensional (2D) 15N-1H NMR correlations, and thereby combining an enhanced spectral resolution with speed and sensitivity. This work demonstrates how these qualities can come together for the study of nucleic acids. HyperW injections were used to target the guanine-sensing riboswitch aptamer domain (GSRapt) of the xpt-pbuX operon in Bacillus subtilis. Unlike what had been observed in proteins, where residues benefited of HyperW NMR only if/when sufficiently exposed to water, these enhancements applied to every imino resonance throughout the RNA. The >300-fold enhancements observed in the resulting 1H NMR spectra allowed us to monitor in real time the changes that GSRapt undergoes upon binding hypoxanthine, a high-affinity interaction leading to conformational refolding on a ∼1-s timescale at 36 °C. Structural responses could be identified for several nucleotides by 1-dimensional (1D) imino 1H NMR as well as by 2D HyperW NMR spectra acquired upon simultaneous injection of hyperpolarized water and hypoxanthine. The folding landscape revealed by this HyperW strategy for GSRapt, is briefly discussed.

Short-Form Application of the Screening Test of Spanish Grammar and the Northwestern Syntax Screening Test to Latino Children

1978 ◽  
Vol 9 (4) ◽  
pp. 220-235
Author(s):  
David L. Ratusnik ◽  
Carol Melnick Ratusnik ◽  
Karen Sattinger
Keyword(s):  
New York ◽  
Case Studies ◽  
Metropolitan Area ◽  
Screening Test ◽  
Normative Data ◽  
Short Form ◽  
Latino Children ◽  
Data Application ◽  
York Metropolitan Area ◽  
Multiple Regression Technique

Short-form versions of the Screening Test of Spanish Grammar (Toronto, 1973) and the Northwestern Syntax Screening Test (Lee, 1971) were devised for use with bilingual Latino children while preserving the original normative data. Application of a multiple regression technique to data collected on 60 lower social status Latino children (four years and six months to seven years and one month) from Spanish Harlem and Yonkers, New York, yielded a small but powerful set of predictor items from the Spanish and English tests. Clinicians may make rapid and accurate predictions of STSG or NSST total screening scores from administration of substantially shortened versions of the instruments. Case studies of Latino children from Chicago and Miami serve to cross-validate the procedure outside the New York metropolitan area.

Expression and Characterization of Clustered Charge-to-Alanine Mutants of Low Mr Single-Chain Urokinase-Type Plasminogen Activator

1994 ◽  
Vol 71 (01) ◽  
pp. 134-140 ◽  
Author(s):  
S Ueshima ◽  
P Holvoet ◽  
H R Lijnen ◽  
L Nelles ◽  
V Seghers ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Solvent Accessibility ◽  
Site Directed Mutagenesis ◽  
Clot Lysis ◽  
Wild Type ◽  
Single Chain ◽  
Charged Amino Acids ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Pharmacokinetic Properties

SummaryIn an effort to modify the fibrinolytic and/or pharmacokinetic properties of recombinant low M r single-chain urokinase-type plasminogen activator (rscu-PA-32k), mutants were prepared by site-directed mutagenesis of clusters of charged amino acids with the highest solvent accessibility. The following mutants of rscu-PA-32k were prepared: LUK-2 (Lys 212, Glu 213 and Asp 214 to Ala), LUK-3 (Lys 243 and Asp 244 to Ala), LUK-4 (Arg 262, Lys 264, Glu 265 and Arg 267 to Ala), LUK-5 (Lys 300, Glu 301 and Asp 305 to Ala) and LUK-6 (Arg 400, Lys 404, Glu 405 and Glu 406 to Ala).The rscu-PA 32k moictic3 were expressed in High Five Ttichoplasiani cells, and purified to humugciicily from the conditioned cell culture medium, with recoveries of 0.8 to 3.7 mg/1. The specific fibrinolytic activities (220,000 to 300,000 IU/mg), the rates of plasminogen activation by the single-chain moieties and the rates of conversion In lwo chain moieties by plasmin were comparable for mutant and wild-type rscu PA 32k moieties, with the exception of LUK-5 which was virtually inactive. Equi-effective lysis (50% in 2 h) of 60 pi 125I-fibrin labeled plasma clots submerged in 0.5 ml normal human plasma was obtained with 0.7 to 0.8 μg/ml of wild-type or mutant rscu-PA-3?.k, except with LUK-5 (no significant lysis with 16 pg/ml). Following bolus injection in hamsters, all rscu-PA-32k moieties had a comparably rapid plasma clearance (1.3 to 2.7 ml/min), as a result of a short initial half-life (1.4 to 2.5 min). In hamsters with pulmonary embolism, continuous intravenous infusion over 60 min at a dose of 1 mg/kg, resulted in 53 to 72% clot lysis with the mutants, but only 23% with LUK-5, as compared to 36% for wild-type rscu-PA-32k.These data indicate that clustered charge-to-alanine mutants of rscu-PA-32k, designed to eliminate charged regions with the highest solvent accessibility, do not have significantly improved functional, fibrinolytic or pharmacokinetic properties.

Development of Synchrotron Footprinting at NSLS and NSLS-II

2019 ◽  
Vol 26 (1) ◽  
pp. 55-60 ◽  
Author(s):  
Jen Bohon
Keyword(s):  
Solvent Accessibility ◽  
Biological Systems ◽  
In Vivo Studies ◽  
Aqueous Samples ◽  
Biological Mechanisms ◽  
Macromolecular Assembly ◽  
Structure And Dynamics ◽  
Synchrotron Light ◽  
Ideal Technique

Background: First developed in the 1990’s at the National Synchrotron Light Source, xray synchrotron footprinting is an ideal technique for the analysis of solution-state structure and dynamics of macromolecules. Hydroxyl radicals generated in aqueous samples by intense x-ray beams serve as fine probes of solvent accessibility, rapidly and irreversibly reacting with solvent exposed residues to provide a “snapshot” of the sample state at the time of exposure. Over the last few decades, improvements in instrumentation to expand the technology have continuously pushed the boundaries of biological systems that can be studied using the technique. Conclusion: Dedicated synchrotron beamlines provide important resources for examining fundamental biological mechanisms of folding, ligand binding, catalysis, transcription, translation, and macromolecular assembly. The legacy of synchrotron footprinting at NSLS has led to significant improvement in our understanding of many biological systems, from identifying key structural components in enzymes and transporters to in vivo studies of ribosome assembly. This work continues at the XFP (17-BM) beamline at NSLS-II and facilities at ALS, which are currently accepting proposals for use.

Current Trends in Biotherapeutic Higher Order Structure Characterization by Irreversible Covalent Footprinting Mass Spectrometry

2019 ◽  
Vol 26 (1) ◽  
pp. 35-43 ◽  
Author(s):  
Natalie K. Garcia ◽  
Galahad Deperalta ◽  
Aaron T. Wecksler
Keyword(s):  
Mass Spectrometry ◽  
Protein Structure ◽  
Protein Interactions ◽  
Quaternary Structure ◽  
Solvent Accessibility ◽  
Higher Order ◽  
Structure Characterization ◽  
Order Structure ◽  
Protein Footprinting ◽  
Wide Range

Background: Biotherapeutics, particularly monoclonal antibodies (mAbs), are a maturing class of drugs capable of treating a wide range of diseases. Therapeutic function and solutionstability are linked to the proper three-dimensional organization of the primary sequence into Higher Order Structure (HOS) as well as the timescales of protein motions (dynamics). Methods that directly monitor protein HOS and dynamics are important for mapping therapeutically relevant protein-protein interactions and assessing properly folded structures. Irreversible covalent protein footprinting Mass Spectrometry (MS) tools, such as site-specific amino acid labeling and hydroxyl radical footprinting are analytical techniques capable of monitoring the side chain solvent accessibility influenced by tertiary and quaternary structure. Here we discuss the methodology, examples of biotherapeutic applications, and the future directions of irreversible covalent protein footprinting MS in biotherapeutic research and development. Conclusion: Bottom-up mass spectrometry using irreversible labeling techniques provide valuable information for characterizing solution-phase protein structure. Examples range from epitope mapping and protein-ligand interactions, to probing challenging structures of membrane proteins. By paring these techniques with hydrogen-deuterium exchange, spectroscopic analysis, or static-phase structural data such as crystallography or electron microscopy, a comprehensive understanding of protein structure can be obtained.

Assessing the Impact of Secondary Structure and Solvent Accessibility on Protein Evolution

Genetics ◽  
1998 ◽  
Vol 149 (1) ◽  
pp. 445-458 ◽  
Author(s):  
Nick Goldman ◽  
Jeffrey L Thorne ◽  
David T Jones
Keyword(s):  
Amino Acid ◽  
Secondary Structure ◽  
Protein Evolution ◽  
Solvent Accessibility ◽  
Strong Association ◽  
Length Distribution ◽  
Parametric Bootstrap ◽  
Amino Acid Replacement ◽  
Physical Constraints ◽  
The Impact

Abstract Empirically derived models of amino acid replacement are employed to study the association between various physical features of proteins and evolution. The strengths of these associations are statistically evaluated by applying the models of protein evolution to 11 diverse sets of protein sequences. Parametric bootstrap tests indicate that the solvent accessibility status of a site has a particularly strong association with the process of amino acid replacement that it experiences. Significant association between secondary structure environment and the amino acid replacement process is also observed. Careful description of the length distribution of secondary structure elements and of the organization of secondary structure and solvent accessibility along a protein did not always significantly improve the fit of the evolutionary models to the data sets that were analyzed. As indicated by the strength of the association of both solvent accessibility and secondary structure with amino acid replacement, the process of protein evolution—both above and below the species level—will not be well understood until the physical constraints that affect protein evolution are identified and characterized.

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