scholarly journals Michler’s hydrol blue elucidates structural differences in prion strains

2020 ◽  
Vol 117 (47) ◽  
pp. 29677-29683
Author(s):  
Yiling Xiao ◽  
Sandra Rocha ◽  
Catherine C. Kitts ◽  
Anna Reymer ◽  
Tamás Beke-Somfai ◽  
...  
Keyword(s):  
Binding Site ◽  
Conformational Changes ◽  
Density Functional ◽  
Amyloid Fibrils ◽  
Site Specific ◽  
Yeast Prions ◽  
Structural Details ◽  
Structural Differences ◽  
Environmentally Sensitive ◽  
A Site

Yeast prions provide self-templating protein-based mechanisms of inheritance whose conformational changes lead to the acquisition of diverse new phenotypes. The best studied of these is the prion domain (NM) of Sup35, which forms an amyloid that can adopt several distinct conformations (strains) that confer distinct phenotypes when introduced into cells that do not carry the prion. Classic dyes, such as thioflavin T and Congo red, exhibit large increases in fluorescence when bound to amyloids, but these dyes are not sensitive to local structural differences that distinguish amyloid strains. Here we describe the use of Michler’s hydrol blue (MHB) to investigate fibrils formed by the weak and strong prion fibrils of Sup35NM and find that MHB differentiates between these two polymorphs. Quantum mechanical time-dependent density functional theory (TDDFT) calculations indicate that the fluorescence properties of amyloid-bound MHB can be correlated to the change of binding site polarity and that a tyrosine to phenylalanine substitution at a binding site could be detected. Through the use of site-specific mutants, we demonstrate that MHB is a site-specific environmentally sensitive probe that can provide structural details about amyloid fibrils and their polymorphs.

scholarly journals Structural and functional studies of pyruvate carboxylase regulation by cyclic di-AMP in lactic acid bacteria

2017 ◽  
Vol 114 (35) ◽  
pp. E7226-E7235 ◽  
Author(s):  
Philip H. Choi ◽  
Thu Minh Ngoc Vu ◽  
Huong Thi Pham ◽  
Joshua J. Woodward ◽  
Mark S. Turner ◽  
...  
Keyword(s):  
Binding Site ◽  
Conformational Changes ◽  
Pyruvate Carboxylase ◽  
Adenosine Monophosphate ◽  
Binding Mode ◽  
Functional Studies ◽  
Cellular Processes ◽  
Wide Range ◽  
A Site ◽  
Amp Inhibition

Cyclic di-3′,5′-adenosine monophosphate (c-di-AMP) is a broadly conserved bacterial second messenger that has been implicated in a wide range of cellular processes. Our earlier studies showed that c-di-AMP regulates central metabolism inListeria monocytogenesby inhibiting its pyruvate carboxylase (LmPC), a biotin-dependent enzyme with biotin carboxylase (BC) and carboxyltransferase (CT) activities. We report here structural, biochemical, and functional studies on the inhibition ofLactococcus lactisPC (LlPC) by c-di-AMP. The compound is bound at the dimer interface of the CT domain, at a site equivalent to that in LmPC, although it has a distinct binding mode in the LlPC complex. This binding site is not well conserved among PCs, and only a subset of these bacterial enzymes are sensitive to c-di-AMP. Conformational changes in the CT dimer induced by c-di-AMP binding may be the molecular mechanism for its inhibitory activity. Mutations of residues in the binding site can abolish c-di-AMP inhibition. InL. lactis, LlPC is required for efficient milk acidification through its essential role in aspartate biosynthesis. The aspartate pool inL. lactisis negatively regulated by c-di-AMP, and high aspartate levels can be restored by expression of a c-di-AMP–insensitive LlPC. LlPC has high intrinsic catalytic activity and is not sensitive to acetyl-CoA activation, in contrast to other PC enzymes.

scholarly journals Structural basis of adenylyl cyclase 9 activation

2021 ◽  
Author(s):  
Chao Qi ◽  
Pia Lavriha ◽  
Ved Mehta ◽  
Basavraj Khanppnavar ◽  
Inayathulla Mohammed ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Binding Site ◽  
Adenylyl Cyclase ◽  
Conformational Changes ◽  
Structural Basis ◽  
Structural Studies ◽  
Nucleotide Analogue ◽  
C Terminus ◽  
Membrane Bound ◽  
A Site

Adenylyl cyclase 9 (AC9) is a membrane-bound enzyme that converts ATP into cAMP. The enzyme is weakly activated by forskolin, fully activated by the G protein Gαs subunit and is autoinhibited by the AC9 C-terminus. Although our recent structural studies of the AC9-Gαs complex provided the framework for understanding AC9 autoinhibition, the conformational changes that AC9 undergoes in response to activator binding remains poorly understood. Here, we present the cryo-EM structures of AC9 in several distinct states: (i) AC9 bound to a nucleotide inhibitor MANT-GTP, (ii) bound to an artificial activator (DARPin C4) and MANT-GTP, (iii) bound to DARPin C4 and a nucleotide analogue ATPαS, (iv) bound to Gαs and MANT-GTP. The artificial activator DARPin C4 partially activates AC9 by binding at a site that overlaps with the Gαs binding site. Together with the previously observed occluded and forskolin-bound conformations, structural comparisons of AC9 in the four new conformations show that secondary structure rearrangements in the region surrounding the forskolin binding site are essential for AC9 activation.

Human Cdc5, a regulator of mitotic entry, can act as a site-specific DNA binding protein

2000 ◽  
Vol 113 (24) ◽  
pp. 4523-4531
Author(s):  
X.H. Lei ◽  
X. Shen ◽  
X.Q. Xu ◽  
H.S. Bernstein
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Transcriptional Activation ◽  
Mammalian Cells ◽  
Family Members ◽  
Site Specific ◽  
Coordinated Expression ◽  
Preferential Binding ◽  
Dna Protein Interaction ◽  
A Site

G(2)/M progression requires coordinated expression of many gene products, but little is known about the transcriptional regulators involved. We recently identified human Cdc5, a positive regulator of G(2)/M in mammalian cells. We also demonstrated the presence of a latent activation domain in its carboxyl terminus, suggesting that human Cdc5 regulates G(2)/M through transcriptional activation. Despite the presence of a DNA binding domain, studies by others have failed to identify a preferential binding site for Cdc5 family members. In addition, Cdc5 recently has been associated with the splicesome in several organisms, suggesting that it may not act through DNA binding. We now report the identification of a 12 bp sequence to which human Cdc5 binds specifically and with high affinity through its amino terminus. We show that this DNA-protein interaction is capable of activating transcription. We also used a selection system in yeast to identify human genomic fragments that interact with human Cdc5. Several of these contained sequences similar to the binding site. We demonstrate that these bind human Cdc5 with similar specificity and affinity. These experiments provide the first evidence that Cdc5 family members can act as site-specific DNA binding proteins, and that human Cdc5 may interact with specific, low abundance sequences in the human genome. This raises the possibility that Cdc5 proteins may participate in more than one process necessary for regulated cell division.

A Density Functional Theory Study of the Iron-Binding Site of Human Serum Transferrin

2004 ◽  
Vol 57 (12) ◽  
pp. 1219 ◽  
Author(s):  
David Rinaldo ◽  
Martin J. Field
Keyword(s):  
Human Serum ◽  
Binding Site ◽  
Conformational Changes ◽  
Metal Binding ◽  
Density Functional ◽  
Release Mechanism ◽  
Serum Transferrin ◽  
Iron Binding ◽  
Iron Release ◽  
Human Serum Transferrin

Human serum transferrin binds ferric ions with high affinity in the blood stream and releases them into cells by a process involving receptor-mediated endocytosis and a decrease in pH. The iron-release mechanism is unclear but protonation events and conformational changes are known to be important. In this study, we investigate properties of the iron-binding site theoretically. Our results suggest that an equatorial histidine could be in its histidinate form when bound to iron at neutral and high pH and that protonation of an axial tyrosine is a key event in iron release. Support for this mechanism from other metal-binding enzymes is also presented.

scholarly journals Structural characterization of tau in fuzzy tau:tubulin complexes

2019 ◽  
Author(s):  
Ho Yee Joyce Fung ◽  
Kristen McKibben ◽  
Jennifer Ramirez ◽  
Kushol Gupta ◽  
Elizabeth Rhoades
Keyword(s):  
Structural Characterization ◽  
Neurodegenerative Disorders ◽  
Intrinsic Disorder ◽  
Structural Features ◽  
Dynamic Nature ◽  
Site Specific ◽  
Significant Interest ◽  
Environmentally Sensitive ◽  
A Site

SummaryTau is a neuronal microtubule (MT) associated protein of significant interest due to its association with several neurodegenerative disorders. Tau’s intrinsic disorder and the dynamic nature of its interactions with tubulin and MTs make its structural characterization challenging. Here we use an environmentally sensitive fluorophore as a site-specific probe of tau bound to soluble tubulin. Comparison of our results with recently published tau:MT cryo-EM model reveals structural similarities between tubulin- and MT-bound tau. Analysis of residues across the repeat regions reveal a hierarchy in tubulin occupancy, which may be relevant to tau’s ability to differentiate between tubulin and MTs. As binding to soluble tubulin is a critical first step in MT polymerization, our characterization of the structural features of tau in dynamic, fuzzy tau:tubulin assemblies advances our understanding of how tau functions in the cell and how function may be disrupted in disease.

scholarly journals Selected peptide-based fluorescent probes for biological applications

2020 ◽  
Vol 16 ◽  
pp. 2971-2982
Author(s):  
Debabrata Maity
Keyword(s):  
Fluorescent Probes ◽  
Direct Detection ◽  
Aqueous Media ◽  
Live Cells ◽  
Biological Applications ◽  
Site Specific ◽  
Specific Manner ◽  
Environmentally Sensitive ◽  
Living Organisms ◽  
A Site

To understand the molecular interactions, present in living organisms and their environments, chemists are trying to create novel chemical tools. In this regard, peptide-based fluorescence techniques have attracted immense interest. Synthetic peptide-based fluorescent probes are advantageous over protein-based sensors, since they are synthetically accessible, more stable, and can be easily modified in a site-specific manner for selective biological applications. Peptide receptors labeled with environmentally sensitive/FRET fluorophores have allowed direct detection/monitoring of biomolecules in aqueous media and in live cells. In this review, key peptide-based approaches for different biological applications are presented.

scholarly journals Molecular Modelling of the Ni(II)-Responsive Synechocystis PCC 6803 Transcriptional Regulator InrS in the Metal Bound Form

Inorganics ◽  
2019 ◽  
Vol 7 (6) ◽  
pp. 76 ◽  
Author(s):  
Elia Barchi ◽  
Francesco Musiani
Keyword(s):  
Binding Site ◽  
Conformational Changes ◽  
Metal Binding ◽  
Computational Study ◽  
Transcriptional Regulator ◽  
Planar Geometry ◽  
Synechocystis Pcc 6803 ◽  
Square Planar ◽  
Structural Details ◽  
Pcc 6803

InrS (internal nickel-responsive sensor) is a transcriptional regulator found in cyanobacteria that represses the transcription of the nickel exporter NrsD in the apo form and de-represses expression of the exporter upon Ni(II) binding. Although a crystal structure of apo-InrS from Synechocystis PCC 6803 has been reported, no structure of the protein with metal ions bound is available. Here we report the results of a computational study aimed to reconstruct the metal binding site by taking advantage of recent X-ray absorption spectroscopy (XAS) data and to envisage the structural rearrangements occurring upon Ni(II) binding. The modelled Ni(II) binding site shows a square planar geometry consistent with experimental data. The structural details of the conformational changes occurring upon metal binding are also discussed in the framework of trying to rationalize the different affinity of the apo- and holo-forms of the protein for DNA.

scholarly journals Monitoring site-specific conformational changes in real-time reveals a misfolding mechanism of the prion protein

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Ishita Sengupta ◽  
Jayant Udgaonkar
Keyword(s):  
Prion Protein ◽  
Conformational Changes ◽  
Amyloid Fibrils ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Monitoring Site ◽  
Site Specific ◽  
Experimental Strategy ◽  
Β Sheets ◽  
Kinetics Of

During pathological aggregation, proteins undergo remarkable conformational re-arrangements to anomalously assemble into a heterogeneous collection of misfolded multimers, ranging from soluble oligomers to insoluble amyloid fibrils. Inspired by fluorescence resonance energy transfer (FRET) measurements of protein folding, an experimental strategy to study site-specific misfolding kinetics during aggregation, by effectively suppressing contributions from inter-molecular FRET, is described. Specifically, the kinetics of conformational changes across different secondary and tertiary structural segments of the mouse prion protein (moPrP) were monitored independently, after the monomeric units transformed into large oligomers OL, which subsequently disaggregated reversibly into small oligomers OS at pH 4. The sequence segments spanning helices α2 and α3 underwent a compaction during the formation of OL and elongation into β-sheets during the formation of OS. The β1-α1-β2 and α2-α3 subdomains were separated, and the helix α1 was unfolded to varying extents in both OL and OS.

scholarly journals Unusual binding-site-specific photophysical properties of a benzothiazole-based optical probe in amyloid beta fibrils

2018 ◽  
Vol 20 (31) ◽  
pp. 20334-20339 ◽  
Author(s):  
N. Arul Murugan ◽  
Robert Zaleśny ◽  
Hans Ågren
Keyword(s):  
Electronic Structure ◽  
Binding Site ◽  
Amyloid Beta ◽  
Binding Sites ◽  
Amyloid Fibrils ◽  
Photophysical Properties ◽  
Optical Probe ◽  
Site Specific

Varying electronic structure of BTA-3 probe in different binding sites in amyloid fibrils is the key mechanism behind its site-specific photophysical properties.

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