Analysis of electrostatic interactions in the denatured state ensemble of the N-terminal domain of L9 under native conditions

2011 ◽  
Vol 79 (12) ◽  
pp. 3500-3510 ◽  
Author(s):  
Wenli Meng ◽  
Daniel P. Raleigh
Keyword(s):  
Electrostatic Interactions ◽  
Denatured State ◽  
Terminal Domain ◽  
State Ensemble

scholarly journals Electrostatic interactions in the denatured state ensemble: Their effect upon protein folding and protein stability

2008 ◽  
Vol 469 (1) ◽  
pp. 20-28 ◽  
Author(s):  
Jae-Hyun Cho ◽  
Satoshi Sato ◽  
Jia-Cherng Horng ◽  
Burcu Anil ◽  
Daniel P. Raleigh
Keyword(s):  
Protein Folding ◽  
Protein Stability ◽  
Electrostatic Interactions ◽  
Denatured State ◽  
State Ensemble

scholarly journals Role of a tryptophan anchor in human topoisomerase I structure, function and inhibition

2008 ◽  
Vol 411 (3) ◽  
pp. 523-530 ◽  
Author(s):  
Gary S. Laco ◽  
Yves Pommier
Keyword(s):  
Amino Acids ◽  
Topoisomerase I ◽  
Electrostatic Interactions ◽  
Functional Domain ◽  
Site Mutation ◽  
Supercoiled Dna ◽  
Terminal Domain ◽  
Tryptophan Residues ◽  
N Terminus

Human Top1 (topoisomerase I) relaxes supercoiled DNA during cell division and transcription. Top1 is composed of 765 amino acids and contains an unstructured N-terminal domain of 200 amino acids, and a structured functional domain of 565 amino acids that binds and relaxes supercoiled DNA. In the present study we examined the region spanning the junction of the N-terminal domain and functional domain (junction region). Analysis of several published Top1 structures revealed that three tryptophan residues formed a network of aromatic stacking interactions and electrostatic interactions that anchored the N-terminus of the functional domain to sub-domains containing the nose cone and active site. Mutation of the three tryptophan residues (Trp203/Trp205/Trp206) to an alanine residue, either individually or together, in silico revealed that the individual tryptophan residue's contribution to the tryptophan ‘anchor’ was additive. When the three tryptophan residues were mutated to alanine in vitro, the resulting mutant Top1 differed from wild-type Top1 in that it lacked processivity, exhibited resistance to camptothecin and was inactivated by urea. The results indicated that the tryptophan anchor stabilized the N-terminus of the functional domain and prevented the loss of Top1 structure and function.

scholarly journals Lipid bilayer induces contraction of the denatured state ensemble of a helical-bundle membrane protein

2021 ◽  
Vol 119 (1) ◽  
pp. e2109169119
Author(s):  
Kristen A. Gaffney ◽  
Ruiqiong Guo ◽  
Michael D. Bridges ◽  
Shaima Muhammednazaar ◽  
Daoyang Chen ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Lipid Bilayer ◽  
Lipid Bilayers ◽  
Limited Proteolysis ◽  
Water Soluble ◽  
Disordered Proteins ◽  
Resonance Spectroscopy ◽  
Denatured State ◽  
E Coli ◽  
State Ensemble

Defining the denatured state ensemble (DSE) and disordered proteins is essential to understanding folding, chaperone action, degradation, and translocation. As compared with water-soluble proteins, the DSE of membrane proteins is much less characterized. Here, we measure the DSE of the helical membrane protein GlpG of Escherichia coli (E. coli) in native-like lipid bilayers. The DSE was obtained using our steric trapping method, which couples denaturation of doubly biotinylated GlpG to binding of two streptavidin molecules. The helices and loops are probed using limited proteolysis and mass spectrometry, while the dimensions are determined using our paramagnetic biotin derivative and double electron–electron resonance spectroscopy. These data, along with our Upside simulations, identify the DSE as being highly dynamic, involving the topology changes and unfolding of some of the transmembrane (TM) helices. The DSE is expanded relative to the native state but only to 15 to 75% of the fully expanded condition. The degree of expansion depends on the local protein packing and the lipid composition. E. coli’s lipid bilayer promotes the association of TM helices in the DSE and, probably in general, facilitates interhelical interactions. This tendency may be the outcome of a general lipophobic effect of proteins within the cell membranes.

Structure of the N-terminal domain of Euprosthenops australis dragline silk suggests that conversion of spidroin dope to spider silk involves a conserved asymmetric dimer intermediate

2019 ◽  
Vol 75 (7) ◽  
pp. 618-627 ◽  
Author(s):  
Wangshu Jiang ◽  
Glareh Askarieh ◽  
Alexander Shkumatov ◽  
My Hedhammar ◽  
Stefan D. Knight
Keyword(s):  
Electrostatic Interactions ◽  
Spider Silk ◽  
Quaternary Structure ◽  
Nephila Clavipes ◽  
Trigger Mechanism ◽  
Dragline Silk ◽  
Terminal Domain ◽  
Structure Changes ◽  
Molecular Events ◽  
Asymmetric Dimer

Spider silk is a biomaterial with exceptional mechanical toughness, and there is great interest in developing biomimetic methods to produce engineered spider silk-based materials. However, the mechanisms that regulate the conversion of spider silk proteins (spidroins) from highly soluble dope into silk are not completely understood. The N-terminal domain (NT) of Euprosthenops australis dragline silk protein undergoes conformational and quaternary-structure changes from a monomer at a pH above 7 to a homodimer at lower pH values. Conversion from the monomer to the dimer requires the protonation of three conserved glutamic acid residues, resulting in a low-pH `locked' dimer stabilized by symmetric electrostatic interactions at the poles of the dimer. The detailed molecular events during this transition are still unresolved. Here, a 2.1 Å resolution crystal structure of an NT T61A mutant in an alternative, asymmetric, dimer form in which the electrostatic interactions at one of the poles are dramatically different from those in symmetrical dimers is presented. A similar asymmetric dimer structure from dragline silk of Nephila clavipes has previously been described. It is suggested that asymmetric dimers represent a conserved intermediate state in spider silk formation, and a revised `lock-and-trigger' mechanism for spider silk formation is presented.

Folding mechanism of the C‐terminal domain of nucleophosmin: residual structure in the denatured state and its pathophysiological significance

2009 ◽  
Vol 23 (8) ◽  
pp. 2360-2365 ◽  
Author(s):  
Flavio Scaloni ◽  
Stefano Gianni ◽  
Luca Federici ◽  
Brunangelo Falini ◽  
Maurizio Brunori
Keyword(s):  
Denatured State ◽  
Residual Structure ◽  
Folding Mechanism ◽  
Terminal Domain

scholarly journals NMR characterization of the interaction between the C-terminal domain of interferon-γ and heparin-derived oligosaccharides

2004 ◽  
Vol 384 (1) ◽  
pp. 93-99 ◽  
Author(s):  
Cécile VANHAVERBEKE ◽  
Jean-Pierre SIMORRE ◽  
Rabia SADIR ◽  
Pierre GANS ◽  
Hugues LORTAT-JACOB
Keyword(s):  
Electrostatic Interactions ◽  
Chemical Shifts ◽  
Heparan Sulphate ◽  
Interferon Γ ◽  
Activated T Cells ◽  
Terminal Domain ◽  
Physiological Processes ◽  
Nmr Characterization ◽  
Wide Range ◽  
Proliferation And Apoptosis

Interferons are cytokines that play a complex role in the resistance of mammalian hosts to pathogens. IFNγ (interferon-γ) is secreted by activated T-cells and natural killer cells. IFNγ is involved in a wide range of physiological processes, including antiviral activity, immune response, cell proliferation and apoptosis, as well as the stimulation and repression of a variety of genes. IFNγ activity is modulated by the binding of its C-terminal domain to HS (heparan sulphate), a glycosaminoglycan found in the extracellular matrix and at the cell surface. In the present study, we analysed the interaction of isolated heparin-derived oligosaccharides with the C-terminal peptide of IFNγ by NMR, in aqueous solution. We observed marked changes in the chemical shifts of both peptide and oligosaccharide compared with the free state. Our results provide evidence of a binding through electrostatic interactions between the charged side chains of the protein and the sulphate groups of heparin that does not induce specific conformation of the C-terminal part of IFNγ. Our data also indicate that an oligosaccharide size of at least eight residues displays the most efficient binding.

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