scholarly journals The Picornavirus Precursor 3CD Has Different Conformational Dynamics Compared to 3Cpro and 3Dpol in Functionally Relevant Regions

Viruses ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 442
Author(s):  
Dennis S. Winston ◽  
David D. Boehr
Keyword(s):  
Active Sites ◽  
Conformational Dynamics ◽  
Genetic Material ◽  
Lipid Binding ◽  
Relaxation Dispersion ◽  
Chemical Exchange Saturation Transfer ◽  
Resonance Spectroscopy ◽  
Conformational Ensemble ◽  
Multiple Quantum ◽  
Functional Differences

Viruses have evolved numerous strategies to maximize the use of their limited genetic material, including proteolytic cleavage of polyproteins to yield products with different functions. The poliovirus polyprotein 3CD is involved in important protein-protein, protein-RNA and protein-lipid interactions in viral replication and infection. It is a precursor to the 3C protease and 3D RNA-dependent RNA polymerase, but has different protease specificity, is not an active polymerase, and participates in other interactions differently than its processed products. These functional differences are poorly explained by the known X-ray crystal structures. It has been proposed that functional differences might be due to differences in conformational dynamics between 3C, 3D and 3CD. To address this possibility, we conducted nuclear magnetic resonance spectroscopy experiments, including multiple quantum relaxation dispersion, chemical exchange saturation transfer and methyl spin-spin relaxation, to probe conformational dynamics across multiple timescales. Indeed, these studies identified differences in conformational dynamics in functionally important regions, including enzyme active sites, and RNA and lipid binding sites. Expansion of the conformational ensemble available to 3CD may allow it to perform additional functions not observed in 3C and 3D alone despite having nearly identical lowest-energy structures.

scholarly journals Binding kinetics and substrate selectivity in HIV-1 protease−Gag interactions probed at atomic resolution by chemical exchange NMR

2017 ◽  
Vol 114 (46) ◽  
pp. E9855-E9862 ◽  
Author(s):  
Lalit Deshmukh ◽  
Vitali Tugarinov ◽  
John M. Louis ◽  
G. Marius Clore
Keyword(s):  
Chemical Exchange ◽  
Conformational Dynamics ◽  
Specific Antigen ◽  
Relaxation Dispersion ◽  
Chemical Exchange Saturation Transfer ◽  
Cleavage Sites ◽  
Gag Polyprotein ◽  
Intrinsically Disordered ◽  
Catalytic Cleft ◽  
Hiv 1

The conversion of immature noninfectious HIV-1 particles to infectious virions is dependent upon the sequential cleavage of the precursor group-specific antigen (Gag) polyprotein by HIV-1 protease. The precise mechanism whereby protease recognizes distinct Gag cleavage sites, located in the intrinsically disordered linkers connecting the globular domains of Gag, remains unclear. Here, we probe the dynamics of the interaction of large fragments of Gag and various variants of protease (including a drug resistant construct) using Carr−Purcell−Meiboom−Gill relaxation dispersion and chemical exchange saturation transfer NMR experiments. We show that the conformational dynamics within the flaps of HIV-1 protease that form the lid over the catalytic cleft play a significant role in substrate specificity and ordered Gag processing. Rapid interconversion between closed and open protease flap conformations facilitates the formation of a transient, sparsely populated productive complex between protease and Gag substrates. Flap closure traps the Gag cleavage sites within the catalytic cleft of protease. Modulation of flap opening through protease−Gag interactions fine-tunes the lifetime of the productive complex and hence the likelihood of Gag proteolysis. A productive complex can also be formed in the presence of a noncognate substrate but is short-lived owing to lack of optimal complementarity between the active site cleft of protease and the substrate, resulting in rapid flap opening and substrate release, thereby allowing protease to differentiate between cognate and noncognate substrates.

scholarly journals Ligand Entry into Fatty Acid Binding Protein via Local Unfolding instead of Gap Widening

2019 ◽  
Author(s):  
T Xiao ◽  
Y Lu ◽  
J Fan ◽  
D Yang
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Binding Proteins ◽  
Lipid Binding ◽  
Relaxation Dispersion ◽  
Chemical Exchange Saturation Transfer ◽  
Fatty Acid Binding Proteins ◽  
Fatty Acid Binding ◽  
Protein Cavity ◽  
Local Unfolding

AbstractFatty acid binding proteins (FABPs) play an important role in transportation of fatty acids. Despite intensive studies, how fatty acids enter the protein cavity for binding is still controversial. Here, a gap-closed variant of human intestinal FABP was generated by mutagenesis, in which the gap is locked by a disulfide bridge. According to its structure determined here by NMR, this variant has no obvious openings as the ligand entrance and the gap cannot be widened by internal dynamics. Nevertheless, it still uptakes fatty acids and other ligands. NMR relaxation dispersion, chemical exchange saturation transfer and hydrogen-deuterium exchange experiments show that the variant exists in a major native state, two minor native-like state, and two locally unfolded states in aqueous solution. Local unfolding of either βB–βD or helix 2 can generate an opening large enough for ligands to enter the protein cavity, but only the fast local unfolding of helix 2 is relevant to the ligand entry process.Statement of SignificanceFatty acid binding proteins transport fatty acids to specific organelles in the cell. To enable the transport, fatty acids must enter and leave the protein cavity. In spite of many studies, how fatty acids enter the protein cavity remains controversial. Using mutagenesis and biophysical techniques, we have resolved the disagreement and further showed that local unfolding of the second helix can generate a transient opening to allow ligands to enter the protein cavity. Since lipid binding proteins are highly conserved in 3D structures and ligand binding, all of them may use the same local unfolding mechanism for ligand uptake and release.

scholarly journals Lactate Chemical Exchange Saturation Transfer (LATEST) Imaging in vivo: A Biomarker for LDH Activity

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Catherine DeBrosse ◽  
Ravi Prakash Reddy Nanga ◽  
Puneet Bagga ◽  
Kavindra Nath ◽  
Mohammad Haris ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Magnetic Resonance ◽  
Musculoskeletal Diseases ◽  
Chemical Exchange ◽  
Radioactive Isotopes ◽  
Chemical Exchange Saturation Transfer ◽  
Resonance Spectroscopy ◽  
Multiple Quantum ◽  
Saturation Transfer ◽  
Wide Range

Abstract Non-invasive imaging of lactate is of enormous significance in cancer and metabolic disorders where glycolysis dominates. Here, for the first time, we describe a chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI) method (LATEST), based on the exchange between lactate hydroxyl proton and bulk water protons to image lactate with high spatial resolution. We demonstrate the feasibility of imaging lactate with LATEST in lactate phantoms under physiological conditions, in a mouse model of lymphoma tumors and in skeletal muscle of healthy human subjects pre- and post-exercise. The method is validated by measuring LATEST changes in lymphoma tumors pre- and post-infusion of pyruvate and correlating them with lactate determined from multiple quantum filtered proton magnetic resonance spectroscopy (SEL-MQC 1H-MRS). Similarly, dynamic LATEST changes in exercising human skeletal muscle are correlated with lactate determined from SEL-MQC 1H-MRS. The LATEST method does not involve injection of radioactive isotopes or labeled metabolites. It has over two orders of magnitude higher sensitivity compared to conventional 1H-MRS. It is anticipated that this technique will have a wide range of applications including diagnosis and evaluation of therapeutic response of cancer, diabetes, cardiac and musculoskeletal diseases. The advantages of LATEST over existing methods and its potential challenges are discussed.

The A39G FF domain folds on a volcano-shaped free energy surface via separate pathways

2021 ◽  
Vol 118 (46) ◽  
pp. e2115113118
Author(s):  
Ved P. Tiwari ◽  
Yuki Toyama ◽  
Debajyoti De ◽  
Lewis E. Kay ◽  
Pramodh Vallurupalli
Keyword(s):  
Free Energy ◽  
Chemical Exchange ◽  
Exchange Process ◽  
Conformational Dynamics ◽  
Chemical Exchange Saturation Transfer ◽  
Folding Kinetics ◽  
Free Energy Surface ◽  
Wide Range ◽  
Order Of Magnitude ◽  
Energy Surfaces

Conformational dynamics play critical roles in protein folding, misfolding, function, misfunction, and aggregation. While detecting and studying the different conformational states populated by protein molecules on their free energy surfaces (FESs) remain a challenge, NMR spectroscopy has emerged as an invaluable experimental tool to explore the FES of a protein, as conformational dynamics can be probed at atomic resolution over a wide range of timescales. Here, we use chemical exchange saturation transfer (CEST) to detect “invisible” minor states on the energy landscape of the A39G mutant FF domain that exhibited “two-state” folding kinetics in traditional experiments. Although CEST has mostly been limited to studies of processes with rates between ∼5 to 300 s−1 involving sparse states with populations as low as ∼1%, we show that the line broadening that is often associated with minor state dips in CEST profiles can be exploited to inform on additional conformers, with lifetimes an order of magnitude shorter and populations close to 10-fold smaller than what typically is characterized. Our analysis of CEST profiles that exploits the minor state linewidths of the 71-residue A39G FF domain establishes a folding mechanism that can be described in terms of a four-state exchange process between interconverting states spanning over two orders of magnitude in timescale from ∼100 to ∼15,000 μs. A similar folding scheme is established for the wild-type domain as well. The study shows that the folding of this small domain proceeds through a pair of sparse, partially structured intermediates via two discrete pathways on a volcano-shaped FES.

scholarly journals Interactions and Dissociation Constants of Galactomannan Rendered Cellulose Films with Concavalin A by SPR Spectroscopy

Polymers ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 3040
Author(s):  
Pilar Vilaró ◽  
Carina Sampl ◽  
Gundula Teichert ◽  
Werner Schlemmer ◽  
Mathias Hobisch ◽  
...  
Keyword(s):  
Active Sites ◽  
Dissociation Constants ◽  
Resonance Spectroscopy ◽  
Irreversible Adsorption ◽  
Force Microscopy ◽  
Cellulose Films ◽  
The Past ◽  
Physiological Processes ◽  
Atomic Force ◽  
Dissociation Constant Kd

Interactions of biomolecules at interfaces are important for a variety of physiological processes. Among these, interactions of lectins with monosaccharides have been investigated extensively in the past, while polysaccharide-lectin interactions have scarcely been investigated. Here, we explore the adsorption of galactomannans (GM) extracted from Prosopis affinis on cellulose thin films determined by a combination of multi-parameter surface plasmon resonance spectroscopy (MP-SPR) and atomic force microscopy (AFM). The galactomannan adsorbs spontaneously on the cellulose surfaces forming monolayer type coverage (0.60 ± 0.20 mg·m−2). The interaction of a lectin, Concavalin A (ConA), with these GM rendered cellulose surfaces using MP-SPR has been investigated and the dissociation constant KD (2.1 ± 0.8 × 10−8 M) was determined in a range from 3.4 to 27.3 nM. The experiments revealed that the galactose side chains as well as the mannose reducing end of the GM are weakly interacting with the active sites of the lectins, whereas these interactions are potentially amplified by hydrophobic effects between the non-ionic GM and the lectins, thereby leading to an irreversible adsorption.

scholarly journals Conformational propensities of intrinsically disordered proteins influence the mechanism of binding and folding

2015 ◽  
Vol 112 (31) ◽  
pp. 9614-9619 ◽  
Author(s):  
Munehito Arai ◽  
Kenji Sugase ◽  
H. Jane Dyson ◽  
Peter E. Wright
Keyword(s):  
Intrinsically Disordered Proteins ◽  
Terminal Region ◽  
Relaxation Dispersion ◽  
Disordered Proteins ◽  
Conformational Ensemble ◽  
Induced Fit ◽  
Conformational Selection ◽  
Intrinsically Disordered ◽  
Factor C ◽  
Binding Mechanisms

Intrinsically disordered proteins (IDPs) frequently function in protein interaction networks that regulate crucial cellular signaling pathways. Many IDPs undergo transitions from disordered conformational ensembles to folded structures upon binding to their cellular targets. Several possible binding mechanisms for coupled folding and binding have been identified: folding of the IDP after association with the target (“induced fit”), or binding of a prefolded state in the conformational ensemble of the IDP to the target protein (“conformational selection”), or some combination of these two extremes. The interaction of the intrinsically disordered phosphorylated kinase-inducible domain (pKID) of the cAMP-response element binding (CREB) protein with the KIX domain of a general transcriptional coactivator CREB-binding protein (CBP) provides an example of the induced-fit mechanism. Here we show by NMR relaxation dispersion experiments that a different intrinsically disordered ligand, the transactivation domain of the transcription factor c-Myb, interacts with KIX at the same site as pKID but via a different binding mechanism that involves elements of conformational selection and induced fit. In contrast to pKID, the c-Myb activation domain has a strong propensity for spontaneous helix formation in its N-terminal region, which binds to KIX in a predominantly folded conformation. The C-terminal region of c-Myb exhibits a much smaller helical propensity and likely folds via an induced-fit process after binding to KIX. We propose that the intrinsic secondary structure propensities of pKID and c-Myb determine their binding mechanisms, consistent with their functions as inducible and constitutive transcriptional activators.

Sign in / Sign up

Export Citation Format

Share Document