scholarly journals Hsp70 biases the folding pathways of client proteins

2016 ◽  
Vol 113 (20) ◽  
pp. E2794-E2801 ◽  
Author(s):  
Ashok Sekhar ◽  
Rina Rosenzweig ◽  
Guillaume Bouvignies ◽  
Lewis E. Kay
Keyword(s):  
Long Range ◽  
Structural Information ◽  
Structural Features ◽  
Telomere Repeat ◽  
Hsp70 Family ◽  
Binding Factor ◽  
Long Range Interactions ◽  
Unfolded State ◽  
Folding Pathways ◽  
Client Proteins

The 70-kDa heat shock protein (Hsp70) family of chaperones bind cognate substrates to perform a variety of different processes that are integral to cellular homeostasis. Although detailed structural information is available on the chaperone, the structural features of folding competent substrates in the bound form have not been well characterized. Here we use paramagnetic relaxation enhancement (PRE) NMR spectroscopy to probe the existence of long-range interactions in one such folding competent substrate, human telomere repeat binding factor (hTRF1), which is bound to DnaK in a globally unfolded conformation. We show that DnaK binding modifies the energy landscape of the substrate by removing long-range interactions that are otherwise present in the unbound, unfolded conformation of hTRF1. Because the unfolded state of hTRF1 is only marginally populated and transiently formed, it is inaccessible to standard NMR approaches. We therefore developed a 1H-based CEST experiment that allows measurement of PREs in sparse states, reporting on transiently sampled conformations. Our results suggest that DnaK binding can significantly bias the folding pathway of client substrates such that secondary structure forms first, followed by the development of longer-range contacts between more distal parts of the protein.

scholarly journals The role of CTCF in regulating nuclear organization

2008 ◽  
Vol 205 (4) ◽  
pp. 747-750 ◽  
Author(s):  
Adam Williams ◽  
Richard A. Flavell
Keyword(s):  
Long Range ◽  
Dna Sequences ◽  
Spatial Organization ◽  
Zinc Finger Protein ◽  
Nuclear Organization ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Binding Factor ◽  
Long Range Interactions ◽  
Regulatory Functions

The spatial organization of the genome is thought to play an important part in the coordination of gene regulation. New techniques have been used to identify specific long-range interactions between distal DNA sequences, revealing an ever-increasing complexity to nuclear organization. CCCTC-binding factor (CTCF) is a versatile zinc finger protein with diverse regulatory functions. New data now help define how CTCF mediates both long-range intrachromosomal and interchromosomal interactions, and highlight CTCF as an important factor in determining the three-dimensional structure of the genome.

scholarly journals Mapping the conformation of a client protein through the Hsp70 functional cycle

2015 ◽  
Vol 112 (33) ◽  
pp. 10395-10400 ◽  
Author(s):  
Ashok Sekhar ◽  
Rina Rosenzweig ◽  
Guillaume Bouvignies ◽  
Lewis E. Kay
Keyword(s):  
Structural Changes ◽  
Bound State ◽  
Structural Features ◽  
Client Protein ◽  
Telomere Repeat ◽  
Conformational Ensembles ◽  
Cellular Processes ◽  
Substrate Protein ◽  
Binding Factor ◽  
Atp Binding And Hydrolysis

The 70 kDa heat shock protein (Hsp70) chaperone system is ubiquitous, highly conserved, and involved in a myriad of diverse cellular processes. Its function relies on nucleotide-dependent interactions with client proteins, yet the structural features of folding-competent substrates in their Hsp70-bound state remain poorly understood. Here we use NMR spectroscopy to study the human telomere repeat binding factor 1 (hTRF1) in complex with Escherichia coli Hsp70 (DnaK). In the complex, hTRF1 is globally unfolded with up to 40% helical secondary structure in regions distal to the binding site. Very similar conformational ensembles are observed for hTRF1 bound to ATP-, ADP- and nucleotide-free DnaK. The patterns in substrate helicity mirror those found in the unfolded state in the absence of denaturants except near the site of chaperone binding, demonstrating that DnaK-bound hTRF1 retains its intrinsic structural preferences. To our knowledge, our study presents the first atomic resolution structural characterization of a client protein bound to each of the three nucleotide states of DnaK and establishes that the large structural changes in DnaK and the associated energy that accompanies ATP binding and hydrolysis do not affect the overall conformation of the bound substrate protein.

scholarly journals Double Mutant Cycles as a Tool to Address Folding, Binding, and Allostery

2021 ◽  
Vol 22 (2) ◽  
pp. 828
Author(s):  
Livia Pagano ◽  
Angelo Toto ◽  
Francesca Malagrinò ◽  
Lorenzo Visconti ◽  
Per Jemth ◽  
...  
Keyword(s):  
Long Range ◽  
Double Mutant ◽  
Quantitative Measurement ◽  
System A ◽  
Long Range Interactions ◽  
Folding Pathways ◽  
Protein Folding Pathways ◽  
Basic Equations ◽  
Allosteric Mechanisms ◽  
Double Mutant Cycle

Quantitative measurement of intramolecular and intermolecular interactions in protein structure is an elusive task, not easy to address experimentally. The phenomenon denoted ‘energetic coupling’ describes short- and long-range interactions between two residues in a protein system. A powerful method to identify and quantitatively characterize long-range interactions and allosteric networks in proteins or protein–ligand complexes is called double-mutant cycles analysis. In this review we describe the thermodynamic principles and basic equations that underlie the double mutant cycle methodology, its fields of application and latest employments, and caveats and pitfalls that the experimentalists must consider. In particular, we show how double mutant cycles can be a powerful tool to investigate allosteric mechanisms in protein binding reactions as well as elusive states in protein folding pathways.

Measurement and Reduction of Radiation Damage in Frozen Hydrated Crystalline Specimens

1978 ◽  
Vol 36 (3) ◽  
pp. 70-77 ◽  
Author(s):  
R.M. Glaeser ◽  
S.B. Hayward
Keyword(s):  
Diffraction Pattern ◽  
Structural Information ◽  
Structural Disorder ◽  
Structural Features ◽  
Biological Macromolecules ◽  
Diffraction Intensity ◽  
Object Structure ◽  
Specimen Material ◽  
Unit Cells ◽  
Identical Unit

Highly ordered or crystalline biological macromolecules become severely damaged and structurally disordered after a brief electron exposure. Evidence that damage and structural disorder are occurring is clearly given by the fading and eventual disappearance of the specimen's electron diffraction pattern. The fading and disappearance of sharp diffraction spots implies a corresponding disappearance of periodic structural features in the specimen. By the same token, there is a oneto- one correspondence between the disappearance of the crystalline diffraction pattern and the disappearance of reproducible structural information that can be observed in the images of identical unit cells of the object structure. The electron exposures that result in a significant decrease in the diffraction intensity will depend somewhat upon the resolution (Bragg spacing) involved, and can vary considerably with the chemical makeup and composition of the specimen material.

HAZARD: An Expert System for Risk Assessment of Environmental Chemicals

1987 ◽  
Vol 26 (01) ◽  
pp. 13-23 ◽  
Author(s):  
H. W. Gottinger
Keyword(s):  
Expert System ◽  
Structural Information ◽  
Chemical Carcinogenesis ◽  
Structural Features ◽  
Environmental Chemicals ◽  
Chemical Carcinogens ◽  
Carcinogenic Activity ◽  
Current State ◽  
Structure Activity ◽  
Carcinogenic Potential

AbstractThe purpose of this paper is to report on an expert system in design that screens for potential hazards from environmental chemicals on the basis of structure-activity relationships in the study of chemical carcinogenesis, particularly with respect to analyzing the current state of known structural information about chemical carcinogens and predicting the possible carcinogenicity of untested chemicals. The structure-activity tree serves as an index of known chemical structure features associated with carcinogenic activity. The basic units of the tree are the principal recognized classes of chemical carcinogens that are subdivided into subclasses known as nodes according to specific structural features that may reflect differences in carcinogenic potential among chemicals in the class. An analysis of a computerized data base of known carcinogens (knowledge base) is proposed using the structure-activity tree in order to test the validity of the tree as a classification scheme (inference engine).

Rapid Online Buffer Exchange: A Method for Screening of Proteins, Protein Complexes, and Cell Lysates by Native Mass Spectrometry

2019 ◽  
Author(s):  
Zachary VanAernum ◽  
Florian Busch ◽  
Benjamin J. Jones ◽  
Mengxuan Jia ◽  
Zibo Chen ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Speed ◽  
Structural Information ◽  
Protein Complexes ◽  
High Sensitivity ◽  
Native Mass Spectrometry ◽  
Structural Features ◽  
Consumer Products ◽  
Cell Lysates ◽  
Protein Expression And Purification

It is important to assess the identity and purity of proteins and protein complexes during and after protein purification to ensure that samples are of sufficient quality for further biochemical and structural characterization, as well as for use in consumer products, chemical processes, and therapeutics. Native mass spectrometry (nMS) has become an important tool in protein analysis due to its ability to retain non-covalent interactions during measurements, making it possible to obtain protein structural information with high sensitivity and at high speed. Interferences from the presence of non-volatiles are typically alleviated by offline buffer exchange, which is timeconsuming and difficult to automate. We provide a protocol for rapid online buffer exchange (OBE) nMS to directly screen structural features of pre-purified proteins, protein complexes, or clarified cell lysates. Information obtained by OBE nMS can be used for fast (<5 min) quality control and can further guide protein expression and purification optimization.

Protein Interaction Domains and Post-Translational Modifications: Structural Features and Drug Discovery Applications

2020 ◽  
Vol 27 (37) ◽  
pp. 6306-6355 ◽  
Author(s):  
Marian Vincenzi ◽  
Flavia Anna Mercurio ◽  
Marilisa Leone
Keyword(s):  
Drug Discovery ◽  
Protein Interaction ◽  
Protein Interactions ◽  
Structural Information ◽  
Protein Complexes ◽  
Structural Features ◽  
Protein Protein Interactions ◽  
Modular Architecture ◽  
Post Translational Modifications ◽  
Interaction Domains

Background:: Many pathways regarding healthy cells and/or linked to diseases onset and progression depend on large assemblies including multi-protein complexes. Protein-protein interactions may occur through a vast array of modules known as protein interaction domains (PIDs). Objective:: This review concerns with PIDs recognizing post-translationally modified peptide sequences and intends to provide the scientific community with state of art knowledge on their 3D structures, binding topologies and potential applications in the drug discovery field. Method:: Several databases, such as the Pfam (Protein family), the SMART (Simple Modular Architecture Research Tool) and the PDB (Protein Data Bank), were searched to look for different domain families and gain structural information on protein complexes in which particular PIDs are involved. Recent literature on PIDs and related drug discovery campaigns was retrieved through Pubmed and analyzed. Results and Conclusion:: PIDs are rather versatile as concerning their binding preferences. Many of them recognize specifically only determined amino acid stretches with post-translational modifications, a few others are able to interact with several post-translationally modified sequences or with unmodified ones. Many PIDs can be linked to different diseases including cancer. The tremendous amount of available structural data led to the structure-based design of several molecules targeting protein-protein interactions mediated by PIDs, including peptides, peptidomimetics and small compounds. More studies are needed to fully role out, among different families, PIDs that can be considered reliable therapeutic targets, however, attacking PIDs rather than catalytic domains of a particular protein may represent a route to obtain selective inhibitors.

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