scholarly journals 1H, 13C and 15N resonance assignment of the SARS-CoV-2 full-length nsp1 protein and its mutants reveals its unique secondary structure features in solution

PLoS ONE ◽  
2021 ◽  
Vol 16 (12) ◽  
pp. e0251834
Author(s):  
Tatiana Agback ◽  
Francisco Dominguez ◽  
Ilya Frolov ◽  
Elena I. Frolova ◽  
Peter Agback
Keyword(s):  
Secondary Structure ◽  
Molecular Mechanisms ◽  
Full Length ◽  
Intramolecular Interactions ◽  
Nmr Studies ◽  
Intrinsically Disordered ◽  
Structure And Dynamics ◽  
High Resolution Nmr ◽  
In The Beginning

Structural characterization of the SARS-CoV-2 full length nsp1 protein will be an essential tool for developing new target-directed antiviral drugs against SARS-CoV-2 and for further understanding of intra- and intermolecular interactions of this protein. As a first step in the NMR studies of the protein, we report the 1H, 13C and 15N resonance backbone assignment as well as the Cβ of the apo form of the full-lengthSARS-CoV-2 nsp1 including the folded domain together with the flaking N- and C- terminal intrinsically disordered fragments. The 19.8 kD protein was characterized by high-resolution NMR. Validation of assignment have been done by using two different mutants, H81P and K129E/D48E as well as by amino acid specific experiments. According to the obtained assignment, the secondary structure of the folded domain in solution was almost identical to its previously published X-ray structure as well as another published secondary structure obtained by NMR, but some discrepancies have been detected. In the solution SARS-CoV-2 nsp1 exhibited disordered, flexible N- and C-termini with different dynamic characteristics. The short peptide in the beginning of the disordered C-terminal domain adopted two different conformations distinguishable on the NMR time scale. We propose that the disordered and folded nsp1 domains are not fully independent units but are rather involved in intramolecular interactions. Studies of the structure and dynamics of the SARS-CoV-2 mutant in solution are on-going and will provide important insights into the molecular mechanisms underlying these interactions.

scholarly journals Stability and flexibility of full-length human oligodendrocytic QKI6

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Arne Raasakka ◽  
Petri Kursula
Keyword(s):  
Rna Binding ◽  
Full Length ◽  
Rna Transport ◽  
Myelin Membrane ◽  
X Ray ◽  
Intrinsically Disordered ◽  
X Ray Scattering ◽  
The Central Nervous System ◽  
Sequential Unfolding

Abstract Objective Oligodendrocytes account for myelination in the central nervous system. During myelin compaction, key proteins are translated in the vicinity of the myelin membrane, requiring targeted mRNA transport. Quaking isoform 6 (QKI6) is a STAR domain-containing RNA transport protein, which binds a conserved motif in the 3′-UTR of certain mRNAs, affecting the translation of myelination-involved proteins. RNA binding has been earlier structurally characterized, but information about full-length QKI6 conformation is lacking. Based on known domains and structure predicitons, we expected full-length QKI6 to be flexible and carry disordered regions. Hence, we carried out biophysical and structural characterization of human QKI6. Results We expressed and purified full-length QKI6 and characterized it using mass spectrometry, light scattering, small-angle X-ray scattering, and circular dichroism spectroscopy. QKI6 was monodisperse, folded, and mostly dimeric, being oxidation-sensitive. The C-terminal tail was intrinsically disordered, as predicted. In the absence of RNA, the RNA-binding subdomain is likely to present major flexibility. In thermal stability assays, a double sequential unfolding behaviour was observed in the presence of phosphate, which may interact with the RNA-binding domain. The results confirm the flexibility and partial disorder of QKI6, which may be functionally relevant.

The gene expression profiling of sporadic pheochromocytoma and novel full-length cDNAs cloning.

2003 ◽  
pp. 621-627
Author(s):  
Y-S Yang ◽  
H-D Song ◽  
Y-D Peng ◽  
Q-H Huang ◽  
R-Y Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Adrenal Gland ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Molecular Mechanisms ◽  
Sequence Data ◽  
Full Length ◽  
Familial Pheochromocytoma ◽  
Sporadic Pheochromocytoma

Pheochromocytoma is a chromaffin cell neoplasm that typically causes symptoms and signs of episodic catecholamine release. Pheochromocytoma can be divided into two types: familial and sporadic. The molecular mechanisms involved in familial pheochromocytoma have been unraveled, but the detailed molecular mechanism of sporadic pheochromocytoma remains unknown. The present study thus aimed at characterization of gene expression profiling of sporadic pheochromocytoma using expressed sequence tags (ESTs), and established a preliminary catalog of genes expressed in the tumor. In total, 4115 ESTs were generated from the tumor library. The gene expression profilings of the pheochromocytoma and the normal adrenal gland were compared, and 341 genes were identified to be significantly expressed differently between the two libraries. Interestingly, 16 known genes participating in cell division or apoptosis were notably differently expressed between the tumor and the normal adrenal gland. Twenty-four novel full-length cDNAs were cloned from the tumor library and five of them were significantly up-regulated in the tumor. Some of them may be involved in the tumorigenesis of pheochromocytoma. The sequence data of ESTs and novel full-length cDNAs described in this paper have been submitted to the GeneBank library.

Intrinsically disordered proteins: lessons from colicins

2012 ◽  
Vol 40 (6) ◽  
pp. 1534-1538 ◽  
Author(s):  
Oliver Hecht ◽  
Colin Macdonald ◽  
Geoffrey R. Moore
Keyword(s):  
Intrinsically Disordered Proteins ◽  
Structural Features ◽  
Intramolecular Interactions ◽  
Disordered Proteins ◽  
Target Cells ◽  
Bacterial Cells ◽  
Intrinsically Disordered ◽  
Self Recognition ◽  
Protein Partners

Defining structural features of IDPs (intrinsically disordered proteins) and relating these to biological function requires characterization of their dynamical properties. In the present paper, we review what is known about the IDPs of colicins, protein antibiotics that use their IDPs to enter bacterial cells. The structurally characterized colicin IDPs we consider contain linear binding epitopes for proteins within their target cells that the colicin hijacks during entry. We show that these binding epitopes take part in intramolecular interactions in the absence of protein partners, i.e. self-recognition, and consider the structural origins of this and its functional implications. We suggest that self-recognition is common in other IDPs that contain similar types of binding epitopes.

NMR structures of biomolecules using field oriented media and residual dipolar couplings

2000 ◽  
Vol 33 (4) ◽  
pp. 371-424 ◽  
Author(s):  
J. H. Prestegard ◽  
H. M. Al-Hashimi ◽  
J. R. Tolman
Keyword(s):  
Magnetic Field ◽  
Simulated Annealing ◽  
Protein Structure ◽  
High Resolution ◽  
Residual Dipolar Couplings ◽  
Multiple Alignment ◽  
Dipolar Couplings ◽  
Structure And Dynamics ◽  
High Resolution Nmr

1. Introduction 3721.1 Residual dipolar couplings as a route to structure and dynamics 3721.2 A brief history of oriented phase high resolution NMR 3742. Theoretical treatment of dipolar interactions 3762.1 Anisotropic interactions as probes of macromolecular structure and dynamics 3762.1.1 The dipolar interaction 3762.1.2 Averaging in the solution state 3772.2 Ordering of a rigid body 3772.2.1 The Saupe order tensor 3782.2.2 Orientational probability distribution function 3802.2.3 The generalized degree of order 3802.3 Molecular structure and internal dynamics 3813. Inducing molecular order in high resolution NMR 3833.1 Tensorial interactions between the magnetic field and anisotropic magnetic susceptibilities 3833.2 Dilute liquid crystal media: a tunable source of order 3843.2.1 Bicelles : from membrane mimics to aligning media 3853.2.2 Filamentous phage 3873.2.3 Transfer of alignment from ordered media to macromolecules 3883.3 Magnetic field alignment 3893.3.1 Paramagnetic assisted alignment 3893.3.2 Advantages of using magnetic alignment 3894. The measurement of residual dipolar couplings 3914.1 Introduction 3914.2 Frequency based methods 3924.2.1 Coupling enhanced pulse schemes 3924.2.2 In phase anti-phase methods (IPAP): 1DNH couplings in proteins 3934.2.3 Exclusive correlated spectroscopy (E-COSY): 1DNH, 1DNC′ and 2DHNC′ 3954.2.4 Extraction of splitting values from the frequency domain 3964.3 Intensity based experiments 3974.3.1 J-Modulated experiments: the measurement of 1DCαHα in proteins 3974.3.2 Phase modulated methods 3994.3.3 Constant time COSY – the measurement of DHH couplings 3994.3.4 Systematic errors in intensity based experiments 4005. Interpretation of residual dipolar coupling data 4015.1 Structure determination protocols utilizing orientational constraints 4015.1.1 The simulated annealing approach 4015.1.2 Order matrix analysis of dipolar couplings 4025.1.3 A discussion of the two approaches 4025.2 Reducing orientational degeneracy 4035.2.1 Multiple alignment media in the simulated annealing approach 4045.2.2 Multiple alignment media in the order matrix approach 4055.3 Simplifying effects arising due to molecular symmetry 4065.4 Database approaches for determining protein structure 4076. Applications to the characterization of macromolecular systems 4086.1 Protein structure refinement 4086.2 Protein domain orientation 4096.3 Oligosaccharides 4136.4 Biomolecular complexes 4156.5 Exchanging systems 4167. Acknowledgements 4188. References 419Within its relatively short history, nuclear magnetic resonance (NMR) spectroscopy has managed to play an important role in the characterization of biomolecular structure. However, the methods on which most of this characterization has been based, Nuclear Overhauser Effect (NOE) measurements for short-range distance constraints and scalar couplings measurements for torsional constraints, have limitations (Wüthrich, 1986). For extended structures, such as DNA helices, for example, propagation of errors in the short distance constraints derived from NOEs leaves the relative orientation of remote parts of the structures poorly defined. Also, the low density of observable protons in contact regions of molecules held together by factors other than hydrophobic packing, leads to poorly defined structures. This is especially true in carbohydrate containing complexes where hydrogen bonds often mediate contacts, and in multi-domain proteins where the area involved in domain–domain contact can also be small. Moreover, most NMR based structural applications are concerned with the characterization of a single, rigid conformer for the final structure. This can leave out important mechanistic information that depends on dynamic aspects and, when motion is present, this can lead to incorrect structural representations. This review focuses on one approach to alleviating some of the existing limitations in NMR based structure determination: the use of constraints derived from the measurement of residual dipolar couplings (D).

scholarly journals High-resolution NMR studies of structure and dynamics of human ERp27 indicate extensive interdomain flexibility

2013 ◽  
Vol 450 (2) ◽  
pp. 321-332 ◽  
Author(s):  
Nader T. Amin ◽  
A. Katrine Wallis ◽  
Stephen A. Wells ◽  
Michelle L. Rowe ◽  
Richard A. Williamson ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
High Resolution ◽  
Nmr Relaxation ◽  
Full Length ◽  
15N Nmr ◽  
Nmr Studies ◽  
Average Order ◽  
X Ray ◽  
Nmr Data ◽  
High Resolution Nmr

ERp27 (endoplasmic reticulum protein 27.7 kDa) is a homologue of PDI (protein disulfide-isomerase) localized to the endoplasmic reticulum. ERp27 is predicted to consist of two thioredoxin-fold domains homologous with the non-catalytic b and b′ domains of PDI. The structure in solution of the N-terminal b-like domain of ERp27 was solved using high-resolution NMR data. The structure confirms that it has the thioredoxin fold and that ERp27 is a member of the PDI family. 15N-NMR relaxation data were obtained and ModelFree analysis highlighted limited exchange contributions and slow internal motions, and indicated that the domain has an average order parameter S2 of 0.79. Comparison of the single-domain structure determined in the present study with the equivalent domain within full-length ERp27, determined independently by X-ray diffraction, indicated very close agreement. The domain interface inferred from NMR data in solution was much more extensive than that observed in the X-ray structure, suggesting that the domains flex independently and that crystallization selects one specific interdomain orientation. This led us to apply a new rapid method to simulate the flexibility of the full-length protein, establishing that the domains show considerable freedom to flex (tilt and twist) about the interdomain linker, consistent with the NMR data.

Capturing dynamic conformational shifts in protein–ligand recognition using integrative structural biology in solution

2018 ◽  
Vol 2 (1) ◽  
pp. 107-119
Author(s):  
Hyun-Seo Kang ◽  
Michael Sattler
Keyword(s):  
Molecular Mechanisms ◽  
Protein Complexes ◽  
Conformational Dynamics ◽  
Integrated Approach ◽  
Conformational Ensemble ◽  
Biological Macromolecules ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Structure And Dynamics ◽  
Complementary Techniques

In recent years, a dynamic view of the structure and function of biological macromolecules is emerging, highlighting an essential role of dynamic conformational equilibria to understand molecular mechanisms of biological functions. The structure of a biomolecule, i.e. protein or nucleic acid in solution, is often best described as a dynamic ensemble of conformations, rather than a single structural state. Strikingly, the molecular interactions and functions of the biological macromolecule can then involve a shift between conformations that pre-exist in such an ensemble. Upon external cues, such population shifts of pre-existing conformations allow gradually relaying the signal to the downstream biological events. An inherent feature of this principle is conformational dynamics, where intrinsically disordered regions often play important roles to modulate the conformational ensemble. Unequivocally, solution-state NMR spectroscopy is a powerful technique to study the structure and dynamics of such biomolecules in solution. NMR is increasingly combined with complementary techniques, including fluorescence spectroscopy and small angle scattering. The combination of these techniques provides complementary information about the conformation and dynamics in solution and thus affords a comprehensive description of biomolecular functions and regulations. Here, we illustrate how an integrated approach combining complementary techniques can assess the structure and dynamics of proteins and protein complexes in solution.

Characterization of Tamm-Horsfall Urinary Glycoprotein

1973 ◽  
Vol 31 ◽  
pp. 420-421
Author(s):  
John P. Robinson ◽  
J. David Puett
Keyword(s):  
Electron Microscopy ◽  
Circular Dichroism ◽  
Secondary Structure ◽  
Quaternary Structure ◽  
Normal Adult ◽  
Morphological Properties ◽  
Adult Males ◽  
Circular Dichroïsm ◽  
Urinary Glycoprotein

Much work has been reported on the chemical, physical and morphological properties of urinary Tamm-Horsfall glycoprotein (THG). Although it was once reported that cystic fibrotic (CF) individuals had a defective THG, more recent data indicate that THG and CF-THG are similar if not identical.No studies on the conformational aspects have been reported on this glycoprotein using circular dichroism (CD). We examined the secondary structure of THG and derivatives under various conditions and have correlated these results with quaternary structure using electron microscopy.THG was prepared from normal adult males and CF-THG from a 16-year old CF female by the method of Tamm and Horsfall. CF female by the method of Tamm and Horsfall.

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