scholarly journals Quantifying charge state heterogeneity for proteins with multiple ionizable residues

2021 ◽  
Author(s):  
Martin J Fossat ◽  
Ammon E Posey ◽  
Rohit V Pappu
Keyword(s):  
Charge State ◽  
Protein Solutions ◽  
Solution Ph ◽  
Net Charge ◽  
Pka Values ◽  
Intrinsically Disordered ◽  
Conformational Preferences ◽  
Charge States ◽  
Ionizable Residues ◽  
Analyze Data

For proteins with multiple ionizable residues, the canonical assumption is that ionization states of residues are fixed by their intrinsic pKa values. However, several studies have shown that protonation / deprotonation of acidic vs. basic sidechains is realizable even when the solution pH is kept fixed at values that are far away from the intrinsic pKa values. Indeed, protein solutions are best described as ensembles of charge microstates, with each member of the ensemble being a distinct charge microstate defined by differences in charge states for ionizable residues. Accordingly, for a given set of solution conditions, the true partition function is sum over all charge microstates and all the Boltzmann weights of all conformations associated with each of the charge microstates. Here, we leverage the advantages afforded by potentiometric titrations to measure global net charge as a function of pH, independent of considerations of conformational preferences. The systems studied are fragments of proteins with repetitive patterns of Lys and Glu. We analyze the potentiometry data using the recently introduced formalism of the q-canonical ensemble. In this ensemble, charge microstates can be grouped into mesostates. Each mesostate is a collection of microstates of the same net charge. We analyze data for global charge vs. pH to extract mesostate populations as a function of pH. Our findings reveal that the heterogeneity of charge states makes significant contributions to measured charge profiles. This has significant implications for the types of species that are present in solution, even for a fixed pH. Measurements of net charge, decoupled from measurements of conformational equilibria, and analyzed to extract the pH-dependent populations of different mesostates, will be significant for accurate understanding of how charge state heterogeneity contributes to conformational, binding, and phase equilibria of proteins, especially those that are intrinsically disordered.

scholarly journals The C Terminus of the Ribosomal-Associated Protein LrtA is an Intrinsically Disordered Oligomer

2018 ◽  
Vol 19 (12) ◽  
pp. 3902 ◽  
Author(s):  
José L. Neira ◽  
A. Marcela Giudici ◽  
Felipe Hornos ◽  
Arantxa Arbe ◽  
Bruno Rizzuti
Keyword(s):  
Computational Modelling ◽  
Terminal Region ◽  
Physiological Conditions ◽  
Intrinsically Disordered ◽  
Disordered Protein ◽  
C Terminus ◽  
Conformational Preferences ◽  
Self Association ◽  
Association Equilibrium ◽  
Post Stress

The 191-residue-long LrtA protein of Synechocystis sp. PCC 6803 is involved in post-stress survival and in stabilizing 70S ribosomal particles. It belongs to the hibernating promoting factor (HPF) family, intervening in protein synthesis. The protein consists of two domains: The N-terminal region (N-LrtA, residues 1101), which is common to all the members of the HPF, and seems to be well-folded; and the C-terminal region (C-LrtA, residues 102-191), which is hypothesized to be disordered. In this work, we studied the conformational preferences of isolated C-LrtA in solution. The protein was disordered, as shown by computational modelling, 1D-1H NMR, steady-state far- UV circular dichroism (CD) and chemical and thermal denaturations followed by fluorescence and far-UV CD. Moreover, at physiological conditions, as indicated by several biochemical and hydrodynamic techniques, isolated C-LrtA intervened in a self-association equilibrium, involving several oligomerization reactions. Thus, C-LrtA was an oligomeric disordered protein.

scholarly journals Arginine-enriched mixed-charge domains provide cohesion for nuclear speckle condensation

2019 ◽  
Author(s):  
Jamie A. Greig ◽  
Tu Anh Nguyen ◽  
Michelle Lee ◽  
Alex S. Holehouse ◽  
Ammon E. Posey ◽  
...  
Keyword(s):  
Low Complexity ◽  
Rna Recognition Motif ◽  
Rna Recognition ◽  
Nuclear Speckle ◽  
Net Charge ◽  
Intrinsically Disordered ◽  
Recognition Motif ◽  
Condensate Formation ◽  
Dynamic Material Properties

AbstractLow-complexity protein domains promote the formation of various biomolecular condensates. However, in many cases, the precise sequence features governing condensate formation and identity remain unclear. Here, we investigate the role of intrinsically disordered mixed-charge domains (MCDs) in nuclear speckle condensation. Proteins composed exclusively of arginine/aspartic-acid dipeptide repeats undergo length-dependent condensation and speckle incorporation. Substituting arginine with lysine in synthetic and natural speckle-associated MCDs abolishes these activities, identifying a key role for multivalent contacts through arginine’s guanidinium ion. MCDs can synergise with a speckle-associated RNA recognition motif to promote speckle specificity and residence. MCD behaviour is tuneable through net-charge: increasing negative charge abolishes condensation and speckle incorporation. By contrast, increasing positive charge through arginine leads to enhanced condensation, speckle enlargement, decreased splicing factor mobility, and defective mRNA export. Together, these results identify key sequence determinants of MCD-promoted speckle condensation, and link the speckle’s dynamic material properties with function in mRNA processing.

Structural characterization of intrinsically disordered proteins by the combined use of NMR and SAXS

2012 ◽  
Vol 40 (5) ◽  
pp. 955-962 ◽  
Author(s):  
Nathalie Sibille ◽  
Pau Bernadó
Keyword(s):  
Intrinsically Disordered Proteins ◽  
Dynamic Models ◽  
Dimensional Space ◽  
Three Dimensional ◽  
Main Tool ◽  
Structural Features ◽  
Disordered Proteins ◽  
Dimensional Structure ◽  
Intrinsically Disordered ◽  
Conformational Preferences

In recent years, IDPs (intrinsically disordered proteins) have emerged as pivotal actors in biology. Despite IDPs being present in all kingdoms of life, they are more abundant in eukaryotes where they are involved in the vast majority of regulation and signalling processes. The realization that, in some cases, functional states of proteins were partly or fully disordered was in contradiction to the traditional view where a well defined three-dimensional structure was required for activity. Several experimental evidences indicate, however, that structural features in IDPs such as transient secondary-structural elements and overall dimensions are crucial to their function. NMR has been the main tool to study IDP structure by probing conformational preferences at residue level. Additionally, SAXS (small-angle X-ray scattering) has the capacity to report on the three-dimensional space sampled by disordered states and therefore complements the local information provided by NMR. The present review describes how the synergy between NMR and SAXS can be exploited to obtain more detailed structural and dynamic models of IDPs in solution. These combined strategies, embedded into computational approaches, promise the elucidation of the structure–function properties of this important, but elusive, family of biomolecules.

scholarly journals DispHred: A Server to Predict pH-Dependent Order–Disorder Transitions in Intrinsically Disordered Proteins

2020 ◽  
Vol 21 (16) ◽  
pp. 5814 ◽  
Author(s):  
Jaime Santos ◽  
Valentín Iglesias ◽  
Carlos Pintado ◽  
Juan Santos-Suárez ◽  
Salvador Ventura
Keyword(s):  
Intrinsically Disordered Proteins ◽  
State Of The Art ◽  
Disordered Proteins ◽  
Protein Disorder ◽  
Net Charge ◽  
Intrinsically Disordered ◽  
Linear Boundary Condition ◽  
Natively Unfolded ◽  
Ph Dependent ◽  
Very High

The natively unfolded nature of intrinsically disordered proteins (IDPs) relies on several physicochemical principles, of which the balance between a low sequence hydrophobicity and a high net charge appears to be critical. Under this premise, it is well-known that disordered proteins populate a defined region of the charge–hydropathy (C–H) space and that a linear boundary condition is sufficient to distinguish between folded and disordered proteins, an approach widely applied for the prediction of protein disorder. Nevertheless, it is evident that the C–H relation of a protein is not unalterable but can be modulated by factors extrinsic to its sequence. Here, we applied a C–H-based analysis to develop a computational approach that evaluates sequence disorder as a function of pH, assuming that both protein net charge and hydrophobicity are dependent on pH solution. On that basis, we developed DispHred, the first pH-dependent predictor of protein disorder. Despite its simplicity, DispHred displays very high accuracy in identifying pH-induced order/disorder protein transitions. DispHred might be useful for diverse applications, from the analysis of conditionally disordered segments to the synthetic design of disorder tags for biotechnological applications. Importantly, since many disorder predictors use hydrophobicity as an input, the here developed framework can be implemented in other state-of-the-art algorithms.

Formation of different charge states and the charge state fractions of sputtered Ti and Ni

1986 ◽  
Vol 166 (2-3) ◽  
pp. 458-479 ◽  
Author(s):  
H.J. Barth ◽  
E. Mühling ◽  
W. Eckstein
Keyword(s):  
Charge State ◽  
Charge States

Neutralization Dialysis. Problems and Outlook

1995 ◽  
Vol 60 (11) ◽  
pp. 1888-1904 ◽  
Author(s):  
Galina A. Tishchenko ◽  
Genadii A. Denisov ◽  
Larisa K. Shataeva ◽  
Miroslav Bleha
Keyword(s):  
Ph Value ◽  
Ion Pairs ◽  
Model Systems ◽  
Protein Solutions ◽  
Solution Ph ◽  
Natural Substances ◽  
Constant Ph ◽  
Initial Salt ◽  
The Difference ◽  
Acidic Region

Neutralization dialysis (ND) of model systems containing 0.1-0.5 M NaCl and various high-molecular-weight synthetic or natural substances have been investigated. Desalination experiments were carried out in a three-compartment spiral module equipped with heterogeneous Ralex or homogeneous Neosepta membranes. Practically complete desalination proceeds due to the neutralization reaction in the desalination compartment. The time of desalination depends only on the membrane permeability and on the initial salt concentration in solution. An important feature hindering larger application of this method is sharp pH changes occurring in the desalination solution. This phenomenon predicted theoretically is mainly caused by the difference in diffusivities of H+/Na+ and OH-/Cl- ion pairs. Independently of the type of membrane and desalinated solution, pH value jumps into acidic region at the beginning of the ND. In the course of the process the pH value increases and the ND proceeds mostly at pH < 7. On the basis of theoretical prediction, an experimental method consisting in adjustment of acid concentration is proposed, which makes it possible to maintain constant pH in the desalination compartment. The convenience of this method was demonstrated by the ND of protein solutions.

scholarly journals Ion production by lasers using high–power densities in a near infrared region

1996 ◽  
Vol 14 (3) ◽  
pp. 335-345 ◽  
Author(s):  
K. Rohlena ◽  
B. Králiková ◽  
J. Krása ◽  
L. Láska ◽  
K. Mašek ◽  
...  
Keyword(s):  
Charge State ◽  
Near Infrared ◽  
Short Pulse ◽  
Target Surface ◽  
Infrared Region ◽  
Theoretical Explanation ◽  
Ion Production ◽  
Charge States ◽  
Focal Power ◽  
Power Densities

Results are presented of experiments on ion production from Ta targets using a short pulse (350–600 ps in focus) illumination with focal power densities exceeding 1014 Wcm-2 at the wavelength of an iodine photodissociation laser (1.315 μm) and its harmonics. Strong evidence of the existence of tantalum ions with the charge state +45 near the target surface was obtained by X-ray spectroscopy methods. The particle diagnostics point to the existence of frozen high charge states (<53+) of Ta ions in the far expansion zone at about 2 m from the target. The measured charge state-ion energy distribution indicates the highest energy (>4 MeV) for the highest observed charge states. A tentative theoretical explanation of the observed anomalous charge state freezing phenomenon in the expanding plasma produced by a subnanosecond laser pulse is given.

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