Rohit V. Pappu

Classification of Intrinsically Disordered Regions and Proteins.

1.1. Uncharacterized Protein Segments Are a Source of Functional Novelty Over the past decade, we have observed a massive increase in the amount of information describing protein sequences from a variety of organisms.1,2 While this may reflect the diversity in sequence space, and possibly also in function space,3 a large proportion of the sequences lacks any useful function annotation.4,5 Often these sequences are annotated as putative or hypothetical proteins, and for the majority their functions still remain unknown.6,7 Suggestions about potential protein function, primarily molecular function, often come from computational analysis of their sequences. For instance, homology detection allows for the transfer of information from well-characterized protein segments to those with similar sequences that lack annotation of molecular function.8−10 Other aspects of function, such as the biological processes proteins participate in, may come from genetic- and disease-association studies, expression and interaction network data, and comparative genomics approaches that investigate genomic context.11−17 Characterization of unannotated and uncharacterized protein segments is expected to lead to the discovery of novel functions as well as provide important insights into existing biological processes. In addition, it is likely to shed new light on molecular mechanisms of diseases that are not yet fully understood. Thus, uncharacterized protein segments are likely to be a large source of functional novelty relevant for discovering new biology.

Net charge per residue modulates conformational ensembles of intrinsically disordered proteins

Intrinsically disordered proteins (IDPs) adopt heterogeneous ensembles of conformations under physiological conditions. Understanding the relationship between amino acid sequence and conformational ensembles of IDPs can help clarify the role of disorder in physiological function. Recent studies revealed that polar IDPs favor collapsed ensembles in water despite the absence of hydrophobic groups—a result that holds for polypeptide backbones as well. By studying highly charged polypeptides, a different archetype of IDPs, we assess how charge content modulates the intrinsic preference of polypeptide backbones for collapsed structures. We characterized conformational ensembles for a set of protamines in aqueous milieus using molecular simulations and fluorescence measurements. Protamines are arginine-rich IDPs involved in the condensation of chromatin during spermatogenesis. Simulations based on the ABSINTH implicit solvation model predict the existence of a globule-to-coil transition, with net charge per residue serving as the discriminating order parameter. The transition is supported by quantitative agreement between simulation and experiment. Local conformational preferences partially explain the observed trends of polymeric properties. Our results lead to the proposal of a schematic protein phase diagram that should enable prediction of polymeric attributes for IDP conformational ensembles using easily calculated physicochemical properties of amino acid sequences. Although sequence composition allows the prediction of polymeric properties, interresidue contact preferences of protamines with similar polymeric attributes suggest that certain details of conformational ensembles depend on the sequence. This provides a plausible mechanism for specificity in the functions of IDPs.

Amyloid seeds formed by cellular uptake, concentration, and aggregation of the amyloid-beta peptide

One of the neuropathological hallmarks of Alzheimers disease (AD) is the amyloid plaque, primarily composed of aggregated amyloid-beta (Aβ) peptide. In vitro, Aβ1–42, the major alloform of Aβ found in plaques, self-assembles into fibrils at micromolar concentrations and acidic pH. Such conditions do not exist in the extracellular fluid of the brain where the pH is neutral and Aβ concentrations are in the nanomolar range. Here, we show that extracellular soluble Aβ (sAβ) at concentrations as low as 1 nM was taken up by murine cortical neurons and neuroblastoma (SHSY5Y) cells but not by human embryonic kidney (HEK293) cells. Following uptake, Aβ accumulated in Lysotracker-positive acidic vesicles (likely late endosomes or lysosomes) where effective concentrations (>2.5 μM) were greater than two orders of magnitude higher than that in the extracellular fluid (25 nM), as quantified by fluorescence intensity using laser scanning confocal microscopy. Furthermore, SHSY5Y cells incubated with 1 μM Aβ1–42 for several days demonstrated a time-dependent increase in intracellular high molecular weight (HMW) (>200 kDa) aggregates, which were absent in cells grown in the presence of Aβ1–40. Homogenates from these Aβ1–42-loaded cells were capable of seeding amyloid fibril growth. These results demonstrate that Aβ can be taken up by certain cells at low physiologically relevant concentrations of extracellular Aβ, and then concentrated into endosomes/lysosomes. At high concentrations, vesicular Aβ aggregates to form HMW species which are capable of seeding amyloid fibril growth. We speculate that extrusion of these aggregates may seed extracellular amyloid plaque formation during AD pathogenesis.

Conformations of intrinsically disordered proteins are influenced by linear sequence distributions of oppositely charged residues

The functions of intrinsically disordered proteins (IDPs) are governed by relationships between information encoded in their amino acid sequences and the ensembles of conformations that they sample as autonomous units. Most IDPs are polyampholytes, with sequences that include both positively and negatively charged residues. Accordingly, we focus here on the sequence–ensemble relationships of polyampholytic IDPs. The fraction of charged residues discriminates between weak and strong polyampholytes. Using atomistic simulations, we show that weak polyampholytes form globules, whereas the conformational preferences of strong polyampholytes are determined by a combination of fraction of charged residues values and the linear sequence distributions of oppositely charged residues. We quantify the latter using a patterning parameter κ that lies between zero and one. The value of κ is low for well-mixed sequences, and in these sequences, intrachain electrostatic repulsions and attractions are counterbalanced, leading to the unmasking of preferences for conformations that resemble either self-avoiding random walks or generic Flory random coils. Segregation of oppositely charged residues within linear sequences leads to high κ-values and preferences for hairpin-like conformations caused by long-range electrostatic attractions induced by conformational fluctuations. We propose a scaling theory to explain the sequence-encoded conformational properties of strong polyampholytes. We show that naturally occurring strong polyampholytes have low κ-values, and this feature implies a selection for random coil ensembles. The design of sequences with different κ-values demonstrably alters the conformational preferences of polyampholytic IDPs, and this ability could become a useful tool for enabling direct inquiries into connections between sequence–ensemble relationships and functions of IDPs.

Fluorescence correlation spectroscopy shows that monomeric polyglutamine molecules form collapsed structures in aqueous solutions

We have used fluorescence correlation spectroscopy measurements to quantify the hydrodynamic sizes of monomeric polyglutamine as a function of chain length (N) by measuring the scaling of translational diffusion times (τD) for the peptide series (Gly)-(Gln)N-Cys-Lys2 in aqueous solution. We find that τD scales with N as τoNν and therefore ln(τD) = ln(τo) + νln(N). The values for ν and ln(τo) are 0.32 ± 0.02 and 3.04 ± 0.08, respectively. Based on these observations, we conclude that water is a polymeric poor solvent for polyglutamine. Previous studies have shown that monomeric polyglutamine is intrinsically disordered. These observations combined with our fluorescence correlation spectroscopy data suggest that the ensemble for monomeric polyglutamine is made up of a heterogeneous collection of collapsed structures. This result is striking because the preference for collapsed structures arises despite the absence of residues deemed to be hydrophobic in the sequence constructs studied. Working under the assumption that the driving forces for collapse are similar to those for aggregation, we discuss the implications of our results for the thermodynamics and kinetics of polyglutamine aggregation, a process that has been implicated in the molecular mechanism of Huntingtons disease.

Role of Backbone−Solvent Interactions in Determining Conformational Equilibria of Intrinsically Disordered Proteins

Intrinsically disordered proteins (IDPs) are functional proteins that do not fold into well-defined three-dimensional structures under physiological conditions. IDP sequences have low hydrophobicity, and hence, recent experiments have focused on quantitative studies of conformational ensembles of archetypal IDP sequences such as polyglutamine and glycine-serine block copolypeptides. Results from these experiments show that, despite the absence of hydrophobic residues, polar IDPs prefer ensembles of collapsed structures in aqueous milieus. Do these preferences originate in interactions that are unique to polar sidechains? The current study addresses this issue by analyzing conformational equilibria for polyglycine and a glycine-serine block copolypeptide in two environments, namely, water and 8 M urea. Polyglycine, a poly secondary-amide, has no sidechains and is a useful model system for generic polypeptide backbones. Results based on large-scale molecular dynamics simulations show that polyglycine forms compact, albeit disordered, globules in water and swollen, disordered coils in 8 M urea. There is minimal overlap between conformational ensembles in the two environments. Analysis of order parameters derived from theories for flexible polymers show that water at ambient temperatures is a poor solvent for generic polypeptide backbones. Therefore, the experimentally observed preferences for polyglutamine and glycine-serine block copolypeptides must originate, at least partially, in polypeptide backbones. A preliminary analysis of the driving forces that lead to distinct conformational preferences for polyglycine in two different environments is presented. Implications for describing conformational ensembles of generic IDP sequences are also discussed.

ABSINTH: a new continuum solvation model for simulations of polypeptides in aqueous solutions.

A new implicit solvation model for use in Monte Carlo simulations of polypeptides is introduced. The model is termed ABSINTH for self‐Assembly of Biomolecules Studied by an Implicit, Novel, and Tunable Hamiltonian. It is designed primarily for simulating conformational equilibria and oligomerization reactions of intrinsically disordered proteins in aqueous solutions. The paradigm for ABSINTH is conceptually similar to the EEF1 model of Lazaridis and Karplus (Proteins 1999, 35, 133). In ABSINTH, the transfer of a polypeptide solute from the gas phase into a continuum solvent is the sum of a direct mean field interaction (DMFI), and a term to model the screening of polar interactions. Polypeptide solutes are decomposed into a set of distinct solvation groups. The DMFI is a sum of contributions from each of the solvation groups, which are analogs of model compounds. Continuum‐mediated screening of electrostatic interactions is achieved using a framework similar to the one used for the DMFI. Promising results are shown for a set of test cases. These include the calculation of NMR coupling constants for short peptides, the assessment of the thermal stability of two small proteins, reversible folding of both an α‐helix and a β‐hairpin forming peptide, and the polymeric properties of intrinsically disordered polyglutamine peptides of varying lengths. The tests reveal that the computational expense for simulations with the ABSINTH implicit solvation model increase by a factor that is in the range of 2.5–5.0 with respect to gas‐phase calculations.

Ebola Virus VP24 Targets a Unique NLS Binding Site on Karyopherin Alpha 5 to Selectively Compete with Nuclear Import of Phosphorylated STAT1.

During antiviral defense, interferon (IFN) signaling triggers nuclear transport of tyrosine-phosphorylated STAT1 (PY-STAT1), which occurs via a subset of karyopherin alpha (KPNA) nuclear transporters. Many viruses, including Ebola virus, actively antagonize STAT1 signaling to counteract the antiviral effects of IFN. Ebola virus VP24 protein (eVP24) binds KPNA to inhibit PY-STAT1 nuclear transport and render cells refractory to IFNs. We describe the structure of human KPNA5 C terminus in complex with eVP24. In the complex, eVP24 recognizes a unique nonclassical nuclear localization signal (NLS) binding site on KPNA5 that is necessary for efficient PY-STAT1 nuclear transport. eVP24 binds KPNA5 with very high affinity to effectively compete with and inhibit PY-STAT1 nuclear transport. In contrast, eVP24 binding does not affect the transport of classical NLS cargo. Thus, eVP24 counters cell-intrinsic innate immunity by selectively targeting PY-STAT1 nuclear import while leaving the transport of other cargo that may be required for viral replication unaffected.

A simple model for polyproline II structure in unfolded states of alanine‐based peptides

The striking similarity between observed circular dichroism spectra of nonprolyl homopolymers and that of regular left‐handed polyproline II (PII) helices prompted Tiffany and Krimm to propose in 1968 that unordered peptides and unfolded proteins are built of PII segments linked by sharp bends. A large body of experimental evidence, accumulated over the past three decades, provides compelling evidence in support of the original hypothesis of Tiffany and Krimm. Of particular interest are the recent experiments of Shi et al. who find significant PII structure in a short unfolded alanine‐based peptide. What is the physical basis for PII helices in peptide and protein unfolded states? The widely accepted view is that favorable chain‐solvent hydrogen bonds lead to a preference for dynamical fluctuations about noncooperative PII helices in water. Is this preference simply a consequence of hydrogen bonding or is it a manifestation of a more general trend for unfolded states which are appropriately viewed as chains in a good solvent? The prevalence of closely packed interiors in folded proteins suggests that under conditions that favor folding, water—which is a better solvent for itself than for any polypeptide chain—expels the chain from its midst, thereby maximizing chain packing. Implicit in this view is a complementary idea: under conditions that favor unfolding, chain‐solvent interactions are preferred and in a so‐called good solvent, chain packing density is minimized. In this work we show that minimization of chain packing density leads to preferred fluctuations for short polyalanyl chains around canonical, noncooperative PII‐like conformations. Minimization of chain packing is modeled using a purely repulsive soft‐core potential between polypeptide atoms. Details of chain‐solvent interactions are ignored. Remarkably, the simple model captures the essential physics behind the preference of short unfolded alanine‐based peptides for PII helices. Our results are based on a detailed analysis of the potential energy landscape which determines the systems structural and thermodynamic preferences. We use the inherent structure formalism of Stillinger and Weber, according to which the energy landscape is partitioned into basins of attraction around local minima. We find that the landscape for the experimentally studied seven‐residue alanine‐based peptide is dominated by fluctuations about two noncooperative structures: the left‐handed polyproline II helix and its symmetry mate.

Modulation of Polyglutamine Conformations and Dimer Formation by the N-Terminus of Huntingtin

Polyglutamine expansions within different proteins are associated with nine different neurodegenerative diseases. There is growing interest in understanding the roles of flanking sequences from disease-relevant proteins in the intrinsic conformational and aggregation properties of polyglutamine. We report results from atomistic simulations and circular dichroism experiments that quantify the effect of the N-terminal 17-residue (Nt17) segment of the huntingtin protein on polyglutamine conformations and intermolecular interactions. We show that the Nt17 segment and polyglutamine domains become increasingly disordered as polyglutamine length (N) increases in Nt17-Q(N) constructs. Hydrophobic groups within Nt17 become sequestered in intramolecular interdomain interfaces. We also show that the Nt17 segment suppresses the intrinsic propensity of polyglutamine aggregation. This inhibition arises from the incipient micellar structures adopted by monomeric forms of the peptides with Nt17 segments. The degree of intermolecular association increases with increasing polyglutamine length and is governed mainly by associations between polyglutamine domains. Comparative analysis of intermolecular associations for different polyglutamine-containing constructs leads to clearer interpretations of recently published experimental data. Our results suggest a framework for fibril formation and identify roles for flanking sequences in the modulation of polyglutamine aggregation.