scholarly journals Site-Specific Modification and Segmental Isotope Labelling of HMGN1 Reveals Long-Range Conformational Perturbations Caused by Posttranslational Modifications

2020 ◽  
Author(s):  
Gerhard Niederacher ◽  
Debra Urwin ◽  
David J. Tremethick ◽  
K. Johan Rosengren ◽  
Christian F. W. Becker ◽  
...  
Keyword(s):  
Posttranslational Modifications ◽  
High Mobility ◽  
Intrinsically Disordered Protein ◽  
Site Specific ◽  
Isotope Labelling ◽  
Specific Modification ◽  
Intrinsically Disordered ◽  
Cellular Localisation ◽  
And Function ◽  
Nucleosome Binding

Interactions between histones, which package DNA in eukaryotes, and nuclear proteins such as the high mobility group nucleosome-binding protein HMGN1 are important for regulating access to DNA. HMGN1 is a highly charged and intrinsically disordered protein (IDP) that is modified at several sites by posttranslational modifications (PTMs) - acetylation, phosphorylation and ADP-ribosylation. These PTMs are thought to affect cellular localisation of HMGN1 and its ability to bind nucleosomes; however, little is known about how these PTMs regulate the structure and function of HMGN1 at a molecular level. Here, we combine the chemical biology tools of protein semi-synthesis and site-specific modification to generate a series of unique HMGN1 variants bearing precise PTMs at their N- and C-termini with segmental isotope labelling for NMR spectroscopy. This study demonstrates the power of combining protein semi-synthesis for introduction of site-specific PTMs with segmental isotope labelling for structural biology, allowing us to understand the roles of PTMs with atomic precision, from both structural and functional perspectives.<br>

scholarly journals Site-Specific Modification and Segmental Isotope Labelling of HMGN1 Reveals Long-Range Conformational Perturbations Caused by Posttranslational Modifications

2020 ◽  
Author(s):  
Gerhard Niederacher ◽  
Debra Urwin ◽  
David J. Tremethick ◽  
K. Johan Rosengren ◽  
Christian F. W. Becker ◽  
...  
Keyword(s):  
Posttranslational Modifications ◽  
High Mobility ◽  
Intrinsically Disordered Protein ◽  
Site Specific ◽  
Isotope Labelling ◽  
Specific Modification ◽  
Intrinsically Disordered ◽  
Cellular Localisation ◽  
And Function ◽  
Nucleosome Binding

Interactions between histones, which package DNA in eukaryotes, and nuclear proteins such as the high mobility group nucleosome-binding protein HMGN1 are important for regulating access to DNA. HMGN1 is a highly charged and intrinsically disordered protein (IDP) that is modified at several sites by posttranslational modifications (PTMs) - acetylation, phosphorylation and ADP-ribosylation. These PTMs are thought to affect cellular localisation of HMGN1 and its ability to bind nucleosomes; however, little is known about how these PTMs regulate the structure and function of HMGN1 at a molecular level. Here, we combine the chemical biology tools of protein semi-synthesis and site-specific modification to generate a series of unique HMGN1 variants bearing precise PTMs at their N- and C-termini with segmental isotope labelling for NMR spectroscopy. This study demonstrates the power of combining protein semi-synthesis for introduction of site-specific PTMs with segmental isotope labelling for structural biology, allowing us to understand the roles of PTMs with atomic precision, from both structural and functional perspectives.<br>

scholarly journals Site-specific modification and segmental isotope labelling of HMGN1 reveals long-range conformational perturbations caused by posttranslational modifications

2021 ◽  
Author(s):  
Gerhard Niederacher ◽  
Debra Urwin ◽  
Yasmin Dijkwel ◽  
David J. Tremethick ◽  
K. Johan Rosengren ◽  
...  
Keyword(s):  
Long Range ◽  
Posttranslational Modifications ◽  
Binding Protein ◽  
Site Specific ◽  
Isotope Labelling ◽  
Specific Modification ◽  
Nucleosome Binding

Using protein semi-synthesis, segmentally isotope-labelled variants of nucleosome-binding protein HMGN1 were generated with site-specific posttranslational modifications to explore their structural and functional effects.

scholarly journals Intrinsic Disorder in Tetratricopeptide Repeat Proteins

2020 ◽  
Vol 21 (10) ◽  
pp. 3709 ◽  
Author(s):  
Nathan W. Van Bibber ◽  
Cornelia Haerle ◽  
Roy Khalife ◽  
Bin Xue ◽  
Vladimir N. Uversky
Keyword(s):  
Protein Interactions ◽  
Posttranslational Modifications ◽  
Tandem Repeats ◽  
Intrinsically Disordered Protein ◽  
Intrinsic Disorder ◽  
Protein Protein Interactions ◽  
Systematic Analysis ◽  
Tetratricopeptide Repeats ◽  
Intrinsically Disordered ◽  
Tetratricopeptide Repeat Proteins

Among the realm of repeat containing proteins that commonly serve as “scaffolds” promoting protein-protein interactions, there is a family of proteins containing between 2 and 20 tetratricopeptide repeats (TPRs), which are functional motifs consisting of 34 amino acids. The most distinguishing feature of TPR domains is their ability to stack continuously one upon the other, with these stacked repeats being able to affect interaction with binding partners either sequentially or in combination. It is known that many repeat-containing proteins are characterized by high levels of intrinsic disorder, and that many protein tandem repeats can be intrinsically disordered. Furthermore, it seems that TPR-containing proteins share many characteristics with hybrid proteins containing ordered domains and intrinsically disordered protein regions. However, there has not been a systematic analysis of the intrinsic disorder status of TPR proteins. To fill this gap, we analyzed 166 human TPR proteins to determine the degree to which proteins containing TPR motifs are affected by intrinsic disorder. Our analysis revealed that these proteins are characterized by different levels of intrinsic disorder and contain functional disordered regions that are utilized for protein-protein interactions and often serve as targets of various posttranslational modifications.

scholarly journals Structural Proteomics Methods to Interrogate the Conformations and Dynamics of Intrinsically Disordered Proteins

2021 ◽  
Vol 9 ◽  
Author(s):  
Rebecca Beveridge ◽  
Antonio N. Calabrese
Keyword(s):  
Posttranslational Modifications ◽  
Intrinsically Disordered Proteins ◽  
Structure And Function ◽  
Disordered Proteins ◽  
Structural Proteomics ◽  
Intrinsically Disordered ◽  
Folded Proteins ◽  
Structural Mass Spectrometry ◽  
And Function ◽  
Structural Mass

Intrinsically disordered proteins (IDPs) and regions of intrinsic disorder (IDRs) are abundant in proteomes and are essential for many biological processes. Thus, they are often implicated in disease mechanisms, including neurodegeneration and cancer. The flexible nature of IDPs and IDRs provides many advantages, including (but not limited to) overcoming steric restrictions in binding, facilitating posttranslational modifications, and achieving high binding specificity with low affinity. IDPs adopt a heterogeneous structural ensemble, in contrast to typical folded proteins, making it challenging to interrogate their structure using conventional tools. Structural mass spectrometry (MS) methods are playing an increasingly important role in characterizing the structure and function of IDPs and IDRs, enabled by advances in the design of instrumentation and the development of new workflows, including in native MS, ion mobility MS, top-down MS, hydrogen-deuterium exchange MS, crosslinking MS, and covalent labeling. Here, we describe the advantages of these methods that make them ideal to study IDPs and highlight recent applications where these tools have underpinned new insights into IDP structure and function that would be difficult to elucidate using other methods.

scholarly journals Segmental and site specific isotope labelling strategies for structural analysis of posttranslationally modified proteins

2021 ◽  
Author(s):  
Dominik P Vogl ◽  
Anne C. Conibear ◽  
Christian F.W. Becker
Keyword(s):  
Nuclear Magnetic Resonance ◽  
Raman Spectroscopy ◽  
Structural Analysis ◽  
Posttranslational Modifications ◽  
Protein Structures ◽  
Spectroscopic Techniques ◽  
Site Specific ◽  
Isotope Labelling ◽  
Infrared And Raman Spectroscopy ◽  
Modified Proteins

Posttranslational modifications can alter protein structures, functions and localisation, and are important cellular regulatory and signalling mechanisms. Spectroscopic techniques such as nuclear magnetic resonance, infrared, and Raman spectroscopy, as well...

scholarly journals Combinatorial phosphorylation modulates the structure and function of the G protein γ subunit in yeast

2021 ◽  
Vol 14 (688) ◽  
pp. eabd2464
Author(s):  
Zahra Nassiri Toosi ◽  
Xinya Su ◽  
Ruth Austin ◽  
Shilpa Choudhury ◽  
Wei Li ◽  
...  
Keyword(s):  
G Protein ◽  
Posttranslational Modifications ◽  
Intrinsically Disordered Proteins ◽  
Structure And Function ◽  
Disordered Proteins ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Gpcr Activation ◽  
Essential Components ◽  
And Function

Intrinsically disordered regions (IDRs) in proteins are often targets of combinatorial posttranslational modifications, which serve to regulate protein structure and function. Emerging evidence suggests that the N-terminal tails of G protein γ subunits, which are essential components of heterotrimeric G proteins, are intrinsically disordered, phosphorylation-dependent determinants of G protein signaling. Here, we found that the yeast Gγ subunit Ste18 underwent combinatorial, multisite phosphorylation events within its N-terminal IDR. G protein–coupled receptor (GPCR) activation and osmotic stress induced phosphorylation at Ser7, whereas glucose and acid stress induced phosphorylation at Ser3, which was a quantitative indicator of intracellular pH. Each site was phosphorylated by a distinct set of kinases, and phosphorylation of one site affected phosphorylation of the other, as determined through exposure to serial stimuli and through phosphosite mutagenesis. Last, we showed that phosphorylation resulted in changes in IDR structure and that different combinations of phosphorylation events modulated the activation rate and amplitude of the downstream mitogen-activated protein kinase Fus3. These data place Gγ subunits among intrinsically disordered proteins that undergo combinatorial posttranslational modifications that govern signaling pathway output.

scholarly journals Conserved Glycines Control Disorder and Function in the Cold-Regulated Protein, COR15A

Biomolecules ◽  
2019 ◽  
Vol 9 (3) ◽  
pp. 84 ◽  
Author(s):  
Oluwakemi Sowemimo ◽  
Patrick Knox-Brown ◽  
Wade Borcherds ◽  
Tobias Rindfleisch ◽  
Anja Thalhammer ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Intrinsically Disordered Protein ◽  
Helical Structure ◽  
Cd Spectroscopy ◽  
Protective Function ◽  
Intrinsically Disordered ◽  
Thaw Cycle ◽  
Cellular Dehydration ◽  
And Function ◽  
The Impact

Cold-regulated (COR) 15A is an intrinsically disordered protein (IDP) from Arabidopsis thaliana important for freezing tolerance. During freezing-induced cellular dehydration, COR15A transitions from a disordered to mostly α-helical structure. We tested whether mutations that increase the helicity of COR15A also increase its protective function. Conserved glycine residues were identified and mutated to alanine. Nuclear magnetic resonance (NMR) spectroscopy was used to identify residue-specific changes in helicity for wildtype (WT) COR15A and the mutants. Circular dichroism (CD) spectroscopy was used to monitor the coil–helix transition in response to increasing concentrations of trifluoroethanol (TFE) and ethylene glycol. The impact of the COR15A mutants on the stability of model membranes during a freeze–thaw cycle was investigated by fluorescence spectroscopy. The results of these experiments showed the mutants had a higher content of α-helical structure and the increased α-helicity improved membrane stabilization during freezing. Comparison of the TFE- and ethylene glycol-induced coil–helix transitions support our conclusion that increasing the transient helicity of COR15A in aqueous solution increases its ability to stabilize membranes during freezing. Altogether, our results suggest the conserved glycine residues are important for maintaining the disordered structure of COR15A but are also compatible with the formation of α-helical structure during freezing induced dehydration.

scholarly journals Design of genetically encoded sensors to detect nucleosome ubiquitination in live cells

2021 ◽  
Vol 220 (4) ◽  
Author(s):  
Carolina dos Santos Passos ◽  
Yun-Seok Choi ◽  
Christopher D. Snow ◽  
Tingting Yao ◽  
Robert E. Cohen
Keyword(s):  
Posttranslational Modifications ◽  
Fluorescent Proteins ◽  
Live Cells ◽  
Transcriptional Elongation ◽  
Binding Domains ◽  
Kd Values ◽  
Binding Peptides ◽  
Genetically Encoded Sensors ◽  
And Function ◽  
Nucleosome Binding

Histone posttranslational modifications (PTMs) are dynamic, context-dependent signals that modulate chromatin structure and function. Ubiquitin (Ub) conjugation to different lysines of histones H2A and H2B is used to regulate diverse processes such as gene silencing, transcriptional elongation, and DNA repair. Despite considerable progress made to elucidate the players and mechanisms involved in histone ubiquitination, there remains a lack of tools to monitor these PTMs, especially in live cells. To address this, we combined an avidity-based strategy with in silico approaches to design sensors for specifically ubiquitinated nucleosomes. By linking Ub-binding domains to nucleosome-binding peptides, we engineered proteins that target H2AK13/15Ub and H2BK120Ub with Kd values from 10−8 to 10−6 M; when fused to fluorescent proteins, they work as PTM sensors in cells. The H2AK13/15Ub-specific sensor, employed to monitor signaling from endogenous DNA damage through the cell cycle, identified and differentiated roles for 53BP1 and BARD1 as mediators of this histone PTM.

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