scholarly journals Folding PDZ2 domain using the Molecular Transfer Model

2016 ◽  
Author(s):  
Zhenxing Liu ◽  
Govardhan Reddy ◽  
Dave Thirumalai
Keyword(s):  
Pdz Domain ◽  
High Energy ◽  
Coarse Grained ◽  
Radius Of Gyration ◽  
Transfer Model ◽  
Self Organized ◽  
Energy Profiles ◽  
X Ray Scattering ◽  
Molecular Transfer

A major challenge in molecular simulations is to describe denaturant-dependent folding of proteins order to make direct comparisons within vitroexperiments. We use the molecular transfer model (MTM), which is currently the only method that accomplishes this goal albeit phenomenologically, to quantitatively describe urea-dependent folding of PDZ domain, which plays a significant role in molecular recognition and signaling. Experiments show that urea-dependent unfolding rates of the PDZ2 domain exhibit a downward curvature at high urea concentrations ([C]s), which has been interpreted by invoking the presence of a sparsely populated high energy intermediate. Simulations using the MTM and a coarse-grained Self-Organized Polymer (SOP) representation of PDZ2 are used to show that the intermediate (IEQ), which has some native-like character, is present in equilibrium both in the presence and absence of urea. The free energy profiles as a function of the structural overlap order parameter show that there are two barriers separating the folded and unfolded states. Structures of the transition state ensembles, (TSE1 separating the unfolded and (IEQ) andTSE2 separatingIEQand the native state), determined using thePfoldmethod, show thatTSE1 is greatly expanded whileTSE2 is compact and native-like. Folding trajectories reveal that PDZ2 folds by parallel routes. In one pathway folding occurs exclusively throughI1, which resemblesIEQ. In a fraction of trajectories, constituting the second pathway, folding occurs through a combination ofI1and a kinetic intermediate. We establish that the radius of gyration (RUg) of the unfolded state is more compact (by ∼9%) under native conditions. Theory and simulations show that the decrease inRUgoccurs on the time scale on the order of utmost ∼20μs. The modest decrease inRUgand the rapid collapse suggest that high spatial and temporal resolution, currently beyond the scope of most small angle X-ray scattering experiments, are needed to detect compaction in finite-sized proteins. The present work further establishes that MTM is efficacious in producing nearly quantitative predictions for folding of proteins under conditions used to carry out experiments.

scholarly journals Denaturants Alter the Flux through Multiple Pathways in the Folding of PDZ Domain

2017 ◽  
Author(s):  
Zhenxing Liu ◽  
D. Thirumalai
Keyword(s):  
Pdz Domain ◽  
Transfer Model ◽  
Guanidinium Chloride ◽  
Self Organized ◽  
Small Proteins ◽  
Molecular Transfer ◽  
Unfolded State ◽  
The Stability ◽  
Folding Thermodynamics ◽  
Good Agreement

AbstractAlthough we understand many aspects of how small proteins (number of residues less than about hundred) fold, it is a major challenge to understand how large proteins self-assemble. To partially overcome this challenge, we performed simulations using the Self-Organized Polymer model with Side Chains (SOP-SC) in guanidinium chloride (GdmCl), using the Molecular Transfer Model (MTM), to describe the folding of the 110-residue PDZ3 domain. The simulations reproduce the folding thermodynamics accurately including the melting temperature (Tm), the stability of the folded state with respect to the unfolded state. We show that the calculated dependence of ln kobs (kobs is the relaxation rate) has the characteristic Chevron shape. The slopes of the Chevron plots are in good agreement with experiments. We show that PDZ3 folds by four major pathways populating two metastable intermediates, in accord with the kinetic partitioning mechanism. The structure of one of the intermediates, populated after polypeptide chain collapse, is structurally similar to an equilibrium intermediate. Surprisingly, the connectivities between the intermediates and hence, the fluxes through the pathways depend on the concentration of GdmCl. The results are used to predict possible outcomes for unfolding of PDZ domain subject to mechanical forces. Our study demonstrates that, irrespective of the size or topology, simulations based on MTM and SOP-SC offer a framework for describing the folding of proteins, mimicking precisely the conditions used in experiments.

scholarly journals Molecular Transfer Model for pH effects on Intrinsically Disordered Proteins: Theory and Applications

2020 ◽  
Author(s):  
Mauro L. Mugnai ◽  
D. Thirumalai
Keyword(s):  
Free Energy ◽  
Intrinsically Disordered Proteins ◽  
Conformational Space ◽  
Coarse Grained ◽  
Disordered Proteins ◽  
Transfer Model ◽  
Ph Effects ◽  
Transfer Free Energy ◽  
Intrinsically Disordered ◽  
Molecular Transfer

AbstractWe present a theoretical method to study how changes in pH shape the heterogeneous conformational ensemble explored by intrinsically disordered proteins (IDPs). The theory is developed in the context of coarse-grained models, which enable a fast, accurate, and extensive exploration of conformational space at a given protonation state. In order to account for pH effects, we generalize the Molecular Transfer Model (MTM), in which conformations are re-weighted using the transfer free energy, which is the free energy necessary for bringing to equilibrium in a new environment a “frozen” conformation of the system. Using the semi-grand ensemble, we derive an exact expression of the transfer free energy, which amounts to the appropriate summation over all the protonation states. Because the exact result is computationally too demanding to be useful for large polyelectrolytes or IDPs, we introduce a mean-field (MF) approximation of the transfer free energy. Using a lattice model, we compare the exact and MF results for the transfer free energy and a variety of observables associated with the model IDP. We find that the precise location of the charged groups (the sequence), and not merely the net charge, determines the structural properties. We demonstrate that some of the limitations previously noted for MF theory in the context of globular proteins are mitigated when disordered polymers are studied. The excellent agreement between the exact and MF results poises us to use the method presented here as a computational tool to study the properties of IDPs and other biological systems as a function of pH.

scholarly journals Collapse Precedes Folding in Denaturant-Dependent Assembly of Ubiquitin

2016 ◽  
Author(s):  
Govardhan Reddy ◽  
D. Thirumalai
Keyword(s):  
Single Molecule ◽  
Quantitative Agreement ◽  
Coarse Grained ◽  
Radius Of Gyration ◽  
Structural Elements ◽  
Neutral Ph ◽  
X Ray Scattering ◽  
Unfolded State ◽  
Polypeptide Chains ◽  
Kinetics Of

AbstractDespite the small size the folding of Ubiquitin (Ub), which plays an indispensable role in targeting proteins for degradation and DNA damage response, is complex. A number of experiments on Ub folding have reached differing conclusions regarding the relation between collapse and folding, and whether intermediates are populated. In order to resolve these vexing issues, we elucidate the denaturant-dependent thermodynamics and kinetics of Ub folding in low and neutral pH as a function of Guanidinium chloride and Urea using coarse-grained molecular simulations. The changes in the fraction of the folded Ub, and the radius of gyration (Rg) as a function of the denaturant concentration, [C], are in quantitative agreement with experiments. Under conditions used in experiments,Rgof the unfolded state at neutral pH changes only by ≈ 17% as the [GdmCl] decreases from 6 M to 0 M. We predict that the extent of compaction of the unfolded state increases as temperature decreases. A two-dimensional folding landscape as a function ofRgand a measure of similarity to the folded state reveals unambiguously that the native state assembly is preceded by collapse, as discovered in fast mixing experiments on several proteins. Analyses of the folding trajectories, under mildly denaturing conditions ([GdmCl]=1.0M or [Urea]=1.0M), shows that Ub folds by collision between preformed secondary structural elements involving kinetic intermediates that are primarily stabilized by long-range contacts. Our work explains the results of Small Angle X-Ray Scattering (SAXS) experiments on Ub quantitatively, and establishes that evolved globular proteins are poised to collapse. In the process, we explain the discrepancy between SAXS and single molecule fluorescent resonant energy transfer (smFRET) experiments, which have arrived at a contradicting conclusion concerning the collapse of polypeptide chains.

scholarly journals Interplay of folded domains and the disordered low-complexity domain in mediating hnRNPA1 phase separation

2020 ◽  
Author(s):  
Erik W. Martin ◽  
F. Emil Thomasen ◽  
Nicole M. Milkovic ◽  
Matthew J. Cuneo ◽  
Christy R. Grace ◽  
...  
Keyword(s):  
Phase Separation ◽  
Phase Behavior ◽  
Rna Binding ◽  
Mechanistic Explanation ◽  
Md Simulations ◽  
Low Complexity ◽  
Coarse Grained ◽  
Intrinsically Disordered ◽  
X Ray Scattering

AbstractLiquid-liquid phase separation underlies the membrane-less compartmentalization of cells. Intrinsically disordered low-complexity domains (LCDs) often mediate phase separation, but how their phase behavior is modulated by folded domains is incompletely understood. Here, we interrogate the interplay between folded and disordered domains of the RNA-binding protein hnRNPA1. The LCD of hnRNPA1 is sufficient for mediating phase separation in vitro. However, we show that the folded RRM domains and a folded solubility-tag modify the phase behavior, even in the absence of RNA. Notably, the presence of the folded domains reverses the salt dependence of the driving force for phase separation relative to the LCD alone. Small-angle X-ray scattering experiments and coarse-grained MD simulations show that the LCD interacts transiently with the RRMs and/or the solubility-tag in a salt-sensitive manner, providing a mechanistic explanation for the observed salt-dependent phase separation. These data point to two effects from the folded domains: (1) electrostatically mediated interactions that compact hnRNPA1 and contribute to phase separation, and (2) increased solubility at higher ionic strengths mediated by the folded domains. The interplay between disordered and folded domains can modify the dependence of phase behavior on solution conditions and can obscure signatures of physicochemical interactions underlying phase separation.Graphical abstracthnRNPA1 phase separation is highly salt sensitive.Phase separation of the low-complexity domain (LCD) of hnRNPA1 increases with NaCl. In contrast, phase separation of full-length hnRNPA1 is saltsensitive. At low NaCl concentrations, electrostatic RRM-LCD interactions occur and can contribute positively to phase separation, but they are screened at high NaCl concentrations. The folded domains solubilize hnRNPA1 under these conditions and prevent phase separation.

scholarly journals Interplay of folded domains and the disordered low-complexity domain in mediating hnRNPA1 phase separation

2021 ◽  
Author(s):  
Erik W Martin ◽  
F Emil Thomasen ◽  
Nicole M Milkovic ◽  
Matthew J Cuneo ◽  
Christy R Grace ◽  
...  
Keyword(s):  
Phase Separation ◽  
Phase Behavior ◽  
Rna Binding ◽  
Mechanistic Explanation ◽  
Md Simulations ◽  
Low Complexity ◽  
Coarse Grained ◽  
Intrinsically Disordered ◽  
X Ray Scattering

Abstract Liquid–liquid phase separation underlies the membrane-less compartmentalization of cells. Intrinsically disordered low-complexity domains (LCDs) often mediate phase separation, but how their phase behavior is modulated by folded domains is incompletely understood. Here, we interrogate the interplay between folded and disordered domains of the RNA-binding protein hnRNPA1. The LCD of hnRNPA1 is sufficient for mediating phase separation in vitro. However, we show that the folded RRM domains and a folded solubility-tag modify the phase behavior, even in the absence of RNA. Notably, the presence of the folded domains reverses the salt dependence of the driving force for phase separation relative to the LCD alone. Small-angle X-ray scattering experiments and coarse-grained MD simulations show that the LCD interacts transiently with the RRMs and/or the solubility-tag in a salt-sensitive manner, providing a mechanistic explanation for the observed salt-dependent phase separation. These data point to two effects from the folded domains: (i) electrostatically-mediated interactions that compact hnRNPA1 and contribute to phase separation and (ii) increased solubility at higher ionic strengths mediated by the folded domains. The interplay between disordered and folded domains can modify the dependence of phase behavior on solution conditions and can obscure signatures of physicochemical interactions underlying phase separation.

Time-Resolved Cryo-Electron Microscopy of Oscillating Microtubules

1990 ◽  
Vol 48 (1) ◽  
pp. 502-503
Author(s):  
Eva-Maria Mandelkow ◽  
Ron Milligan
Keyword(s):  
Electron Microscopy ◽  
Dynamic Instability ◽  
Entire Solution ◽  
Reaction Scheme ◽  
Time Resolved ◽  
X Ray ◽  
X Ray Scattering ◽  
A Chain ◽  
Ray Scattering

Microtubules form part of the cytoskeleton of eukaryotic cells. They are hollow libers of about 25 nm diameter made up of 13 protofilaments, each of which consists of a chain of heterodimers of α-and β-tubulin. Microtubules can be assembled in vitro at 37°C in the presence of GTP which is hydrolyzed during the reaction, and they are disassembled at 4°C. In contrast to most other polymers microtubules show the behavior of “dynamic instability”, i.e. they can switch between phases of growth and phases of shrinkage, even at an overall steady state [1]. In certain conditions an entire solution can be synchronized, leading to autonomous oscillations in the degree of assembly which can be observed by X-ray scattering (Fig. 1), light scattering, or electron microscopy [2-5]. In addition such solutions are capable of generating spontaneous spatial patterns [6].In an earlier study we have analyzed the structure of microtubules and their cold-induced disassembly by cryo-EM [7]. One result was that disassembly takes place by loss of protofilament fragments (tubulin oligomers) which fray apart at the microtubule ends. We also looked at microtubule oscillations by time-resolved X-ray scattering and proposed a reaction scheme [4] which involves a cyclic interconversion of tubulin, microtubules, and oligomers (Fig. 2). The present study was undertaken to answer two questions: (a) What is the nature of the oscillations as seen by time-resolved cryo-EM? (b) Do microtubules disassemble by fraying protofilament fragments during oscillations at 37°C?

dra (down regulated in adenema) binds in vitro to the second PDZ domain of NHERF and E3KARP

2001 ◽  
Vol 120 (5) ◽  
pp. A526-A526
Author(s):  
G LAMPRECHT ◽  
A HEIL ◽  
E WU ◽  
C YUN ◽  
M GREGOR ◽  
...  
Keyword(s):  
Pdz Domain

scholarly journals Albumin in patients with liver disease shows an altered conformation

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Margret Paar ◽  
Vera H. Fengler ◽  
Daniel J. Rosenberg ◽  
Angelika Krebs ◽  
Rudolf E. Stauber ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Liver Disease ◽  
Chronic Liver Disease ◽  
Chronic Liver ◽  
X Ray Scattering ◽  
Pathological Conditions ◽  
Healthy Donors ◽  
Relevant Variables ◽  
End Stage

AbstractHuman serum albumin (HSA) constitutes the primary transporter of fatty acids, bilirubin, and other plasma compounds. The binding, transport, and release of its cargos strongly depend on albumin conformation, which is affected by bound ligands induced by physiological and pathological conditions. HSA is both highly oxidized and heavily loaded with fatty acids and bilirubin in chronic liver disease. By employing small-angle X-ray scattering we show that HSA from the plasma of chronic liver disease patients undergoes a distinct opening compared to healthy donors. The extent of HSA opening correlates with clinically relevant variables, such as the model of end-stage liver disease score, bilirubin, and fatty acid levels. Although the mild oxidation of HSA in vitro does not alter overall structure, the alteration of patients’ HSA correlates with its redox state. This study connects clinical data with structural visualization of albumin dynamicity in solution and underlines the functional importance of albumin’s inherent flexibility.

Mirror to measure small angle x-ray scattering signal in high energy density experiments

2020 ◽  
Vol 91 (12) ◽  
pp. 123501
Author(s):  
M. Šmíd ◽  
C. Baehtz ◽  
A. Pelka ◽  
A. Laso García ◽  
S. Göde ◽  
...  
Keyword(s):  
Energy Density ◽  
Small Angle ◽  
High Energy ◽  
High Energy Density ◽  
X Ray ◽  
X Ray Scattering ◽  
Scattering Signal ◽  
Ray Scattering
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