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Intrinsically disordered protein regions (IDRs) are well established as contributors to intermolecular interactions and the formation of biomolecular condensates. In particular, RNA-binding proteins (RBPs) often harbor IDRs in addition to folded RNA-binding domains that contribute to RBP function. To understand the dynamic interactions of an IDR-RNA complex, we characterized the RNA-binding features of a small (68 residues), positively charged IDR-containing protein, Small ERDK-Rich Factor (SERF). At high concentrations, SERF and RNA undergo charge-driven associative phase separation to form a protein- and RNA-rich dense phase. A key advantage of this model system is that this threshold for demixing is sufficiently high that we could use solution-state biophysical methods to interrogate the stoichiometric complexes of SERF with RNA in the one-phase regime. Herein, we describe our comprehensive characterization of SERF alone and in complex with a small fragment of the HIV-1 Trans-Activation Response (TAR) RNA with complementary biophysical methods and molecular simulations. We find that this binding event is not accompanied by the acquisition of structure by either molecule; however, we see evidence for a modest global compaction of the SERF ensemble when bound to RNA. This behavior likely reflects attenuated charge repulsion within SERF via binding to the polyanionic RNA and provides a rationale for the higher-order assembly of SERF in the context of RNA. We envision that the SERF-RNA system will lower the barrier to accessing the details that support IDR-RNA interactions and likewise deepen our understanding of the role of IDR-RNA contacts in complex formation and liquid-liquid phase separation., Competing Interests: Competing interests statement:The authors declare no competing interest.

Molecular descriptions of intrinsically disordered protein regions (IDRs) are fundamental to understanding their cellular functions and regulation. NMR spectroscopy has been a leading tool in characterizing IDRs at the atomic level. In this review, we highlight recent conceptual breakthroughs in the study of IDRs facilitated by NMR and discuss emerging NMR techniques that bridge molecular descriptions to cellular functions. First, we review the assemblies formed by IDRs at various scales, from one-to-one complexes to non-stoichiometric clusters and condensates, discussing how NMR characterizes their structural dynamics and molecular interactions. Next, we explore several unique interaction modes of IDRs that enable regulatory mechanisms such as selective transport and switch-like inhibition. Finally, we highlight recent progress in solid-state NMR and in-cell NMR on IDRs, discussing how these methods allow for atomic characterization of full-length IDR complexes in various phases and cellular environments. This review emphasizes recent conceptual and methodological advancements in IDR studies by NMR and offers future perspectives on bridging the gap between in vitro molecular descriptions and the cellular functions of IDRs., Competing Interests: The authors have no conflicts to disclose., (© 2024 Author(s).)

Acetylation of the histone H4 tail (H4Kac) has been established as a significant regulator of chromatin architecture and accessibility; however, the molecular mechanisms that underlie these observations remain elusive. Here, we characterize the ensemble features of the histone H4 tail and determine how they change following acetylation on specific sets of lysine residues. Our comprehensive account is enabled by a robust combination of experimental and computational biophysical methods that converge on molecular details including conformer size, intramolecular contacts, and secondary structure propensity. We find that acetylation significantly alters the chemical environment of basic patch residues (16-20) and leads to tail compaction that is partially mediated by transient intramolecular contacts established between the basic patch and N-terminal amino acids. Beyond acetylation, we identify that the protonation state of H18, which is affected by the acetylation state, is a critical regulator of ensemble characteristics, highlighting the potential for interplay between the sequence context and post-translational modifications to define the ensemble features of intrinsically disordered regions. This study elucidates molecular details that could link H4Kac with the regulation of chromatin architecture, illuminating a small piece of the complex network of molecular mechanisms underlying the histone code hypothesis.

Intrinsically disordered protein regions (IDRs) are well-established as contributors to intermolecular interactions and the formation of biomolecular condensates. In particular, RNA-binding proteins (RBPs) often harbor IDRs in addition to folded RNA-binding domains that contribute to RBP function. To understand the dynamic interactions of an IDR-RNA complex, we characterized the RNA-binding features of a small (68 residues), positively charged IDR-containing protein, SERF. At high concentrations, SERF and RNA undergo charge-driven associative phase separation to form a protein- and RNA-rich dense phase. A key advantage of this model system is that this threshold for demixing is sufficiently high that we could use solution-state biophysical methods to interrogate the stoichiometric complexes of SERF with RNA in the one-phase regime. Herein, we describe our comprehensive characterization of SERF alone and in complex with a small fragment of the HIV-1 TAR RNA (TAR) with complementary biophysical methods and molecular simulations. We find that this binding event is not accompanied by the acquisition of structure by either molecule; however, we see evidence for a modest global compaction of the SERF ensemble when bound to RNA. This behavior likely reflects attenuated charge repulsion within SERF via binding to the polyanionic RNA and provides a rationale for the higher-order assembly of SERF in the context of RNA. We envision that the SERF-RNA system will lower the barrier to accessing the details that support IDR-RNA interactions and likewise deepen our understanding of the role of IDR-RNA contacts in complex formation and liquid-liquid phase separation., Competing Interests: DECLARATION OF INTERESTS The authors declare that they have no competing financial interests.

Enzymes that regulate the degree of histone H3 lysine 4 (H3K4) methylation are crucial for proper cellular differentiation and are frequently mutated in cancer. The Mixed lineage leukemia (MLL) family of enzymes deposit H3K4 mono-, di-, or trimethylation at distinct genomic locations, requiring precise spatial and temporal control. Despite evidence that the degree of H3K4 methylation is controlled in part by a hierarchical assembly pathway with key subcomplex components, we previously found that the assembled state of the MLL1 core complex is not favored at physiological temperature. To better understand this paradox, we tested the hypothesis that increasing the concentration of subunits in a biomolecular condensate overcomes this thermodynamic barrier via mass action. Here, we demonstrate that MLL1 core complex phase separation stimulates enzymatic activity up to 60-fold but not primarily by concentrating subunits into droplets. Instead, we found that stimulated activity is largely due to the formation of an altered oligomeric scaffold that greatly reduces substrate K m . We posit that phase separation-induced scaffolding of the MLL1 core complex is a potential "switch-like" mechanism for spatiotemporal control of H3K4 methylation through the rapid formation or dissolution of biomolecular condensates within RNA Pol II transcription factories., Competing Interests: Conflicts of interest M. S. C. owns stock and serves on the Consultant Advisory Board for Kathera Bioscience Inc, the makers of antifungal technologies. M. S. C. also hold US patents (8133690), (8715678), and (10392423) for compounds and methods for inhibiting SET1/MLL family complexes. The other authors declare no competing financial interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Post-translational modifications (PTMs) are reversible chemical modifications that can modulate protein structure and function. Methylation and acetylation are two such PTMs with integral and well-characterized biological roles, including modulation of chromatin structure; and unknown or poorly understood roles, exemplified by the influence of these PTMs on transcription factor structure and function. The need for biological insights into the function of these PTMs motivates the development of a nondestructive and label-free method that enables pursuit of molecular mechanisms. Here, we present a protocol for implementing nuclear magnetic resonance (NMR) methods that allow for unambiguous detection of methylation and acetylation events and demonstrate their utility by observing these marks on histone H3 tail as a model system. We leverage strategic isotopic enrichment of cofactor and peptide for visualization by [ 1 H, 13 C]-HSQC and 13 C direct-detect NMR measurements. Finally, we present 13 C-labeling schemes that facilitate one-dimensional NMR experiments, which combine reduced measurement time relative to two-dimensional spectroscopy with robust filtering of background signals that would otherwise create spectral crowding or limit detection of low-abundance analytes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Intrinsically disordered proteins are frequent targets for functional regulation through post-translational modification due to their high accessibility to modifying enzymes and the strong influence of changes in primary structure on their chemical properties. While lysine N ε -acetylation was first observed as a common modification of histone tails, proteomic data suggest that lysine acetylation is ubiquitous among both nuclear and cytosolic proteins. However, compared with our biophysical understanding of the other common post-translational modifications, mechanistic studies to document how lysine N ε -acetyl marks are placed, utilized to transduce signals, and eliminated when signals need to be turned off, have not kept pace with proteomic discoveries. Herein we report a nuclear magnetic resonance method to monitor N ε -lysine acetylation through enzymatic installation of a 13 C-acetyl probe on a protein substrate, followed by detection through 13 C direct-detect spectroscopy. We demonstrate the ease and utility of this method using histone H3 tail acetylation as a model. The clearest advantage to this method is that it requires no exogenous tags that would otherwise add steric bulk, change the chemical properties of the modified lysine, or generally interfere with downstream biochemical processes. The non-perturbing nature of this tagging method is beneficial for application in any system where changes to local structure and chemical properties beyond those imparted by lysine modification are unacceptable, including intrinsically disordered proteins, bromodomain containing protein complexes, and lysine deacetylase enzyme assays., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Fraser, Dewing, Usher, George and Showalter.)

The pancreatic and duodenal homeobox 1 (PDX1) is a central regulator of glucose-dependent transcription of insulin in pancreatic β cells. PDX1 transcription factor activity is integral to the development and sustained health of the pancreas; accordingly, deciphering the complex network of cellular cues that lead to PDX1 activation or inactivation is an important step toward understanding the etiopathologies of pancreatic diseases and the development of novel therapeutics. Despite nearly 3 decades of research into PDX1 control of Insulin expression, the molecular mechanisms that dictate the function of PDX1 in response to glucose are still elusive. The transcriptional activation functions of PDX1 are regulated, in part, by its two intrinsically disordered regions, which pose a barrier to its structural and biophysical characterization. Indeed, many studies of PDX1 interactions, clinical mutations, and posttranslational modifications lack molecular level detail. Emerging methods for the quantitative study of intrinsically disordered regions and refined models for transactivation now enable us to validate and interrogate the biochemical and biophysical features of PDX1 that dictate its function. The goal of this review is to summarize existing PDX1 studies and, further, to generate a comprehensive resource for future studies of transcriptional control via PDX1., Competing Interests: Conflicts of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Post-translational modification (PTM) of proteins is of critical importance to the regulation of many cellular processes in eukaryotic organisms. One of the most well-studied protein PTMs is methylation, wherein an enzyme catalyzes the transfer of a methyl group from a cofactor to a lysine or arginine side chain. Lysine methylation is especially abundant in the histone tails and is an important marker for denoting active or repressed genes. Given their relevance to transcriptional regulation, the study of methyltransferase function through in vitro experiments is an important stepping stone toward understanding the complex mechanisms of regulated gene expression. To date, most methyltransferase characterization strategies rely on the use of radioactive cofactors, detection of a methyl transfer byproduct, or discontinuous-type assays. Although such methods are suitable for some applications, information about multiple methylation events and kinetic intermediates is often lost. Herein, we describe the use of two-dimensional NMR to monitor mono-, di-, and trimethylation in a single reaction tube. To do so, we incorporated 13 C into the donor methyl group of the enzyme cofactor S-adenosyl methionine. In this way, we may study enzymatic methylation by monitoring the appearance of distinct resonances corresponding to mono-, di-, or trimethyl lysine without the need to isotopically enrich the substrate. To demonstrate the capabilities of this method, we evaluated the activity of three lysine methyltransferases, Set7, MWRAD 2 (MLL1 complex), and PRDM9, toward the histone H3 tail. We monitored mono- or multimethylation of histone H3 tail at lysine 4 through sequential short two-dimensional heteronuclear single quantum coherence experiments and fit the resulting progress curves to first-order kinetic models. In summary, NMR detection of PTMs in one-pot, real-time reaction using facile cofactor isotopic enrichment shows promise as a method toward understanding the intricate mechanisms of methyltransferases and other enzymes., (Copyright © 2021 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Intrinsically disordered protein (IDP) sequences often contain a high proportion of charged residues in conjunction with their high degree of hydrophilicity and solvation. For high net charge IDPs, long-range electrostatic interactions are thought to play a role in modulating the strength or kinetics of protein-protein interactions. In this work, we examined intramolecular interactions mediated by charged regions of a model IDP, the C-terminal tail of the phosphatase Fcp1. Specifically, this work focuses on intermolecular interactions between acidic and basic patches in the primary structure of Fcp1 and their contributions to binding its predominantly basic partner, the winged helix domain of Rap74. We observe both intramolecular and intermolecular interactions through paramagnetic relaxation enhancement (PRE) consistent with oppositely charged regions associating with one another, both in unbound Fcp1 and in the Fcp1-Rap74 complex. Formation of this complex is strongly driven by hydrophobic interactions in the minimal binding motif. Here, we test the hypothesis that charged residues in Fcp1 that flank the binding helix also contribute to the strength of binding. Charge inversion mutations in Fcp1 generally support this hypothesis, while PRE data suggest substitution of observed transient interactions in the unbound ensemble for similarly transient interactions with Rap74 in the complex.

Speckle-type POZ protein (SPOP) is a ubiquitin ligase adaptor that binds substrate proteins and facilitates their proteasomal degradation. Most SPOP substrates present multiple SPOP-binding (SB) motifs and undergo liquid-liquid phase separation with SPOP. Pancreatic and duodenal homeobox 1 (Pdx1), an insulin transcription factor, is downregulated by interaction with SPOP. Unlike other substrates, only one SB motif has previously been reported within the Pdx1 C-terminal intrinsically disordered region (Pdx1-C). Given this difference, we aimed to determine the specific mode of interaction of Pdx1 with SPOP and how it is similar or different to that of other SPOP substrates. Here, we identify a second SB motif in Pdx1-C, but still find that the resulting moderate valency is insufficient to support phase separation with SPOP in cells. Although Pdx1 does not phase separate with SPOP, Pdx1 and SPOP interaction prompts SPOP relocalization from nuclear speckles to the diffuse nucleoplasm. Accordingly, we find that SPOP-mediated ubiquitination activity of Pdx1 occurs in the nucleoplasm and that highly efficient Pdx1 turnover requires both SB motifs. Our results suggest that the subnuclear localization of SPOP-substrate interactions and substrate ubiquitination may be directed by the properties of the substrate itself., Competing Interests: Conflicts of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Although conformational dynamics of RNA molecules are potentially important in microRNA (miRNA) processing, the role of the protein binding partners in facilitating the requisite structural changes is not well understood. In previous work, we and others have demonstrated that nonduplex structural elements and the conformational flexibility they support are necessary for efficient RNA binding and cleavage by the proteins associated with the two major stages of miRNA processing. However, recent studies showed that the protein DGCR8 binds primary miRNA and duplex RNA with similar affinities. Here, we study RNA binding by a small recombinant construct of the DGCR8 protein and the RNA conformation changes that result. This construct, the DGCR8 core, contains two double-stranded RNA-binding domains (dsRBDs) and a C-terminal tail. To assess conformational changes resulting from binding, we applied small-angle x-ray scattering with contrast variation to detect conformational changes of primary-miR-16-1 in complex with the DGCR8 core. This method reports only on the RNA conformation within the complex and suggests that the protein bends the RNA upon binding. Supporting work using smFRET to study the conformation of RNA duplexes bound to the core also shows bending. Together, these studies elucidate the role of DGCR8 in interacting with RNA during the early stages of miRNA processing., (Copyright © 2020 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

A key challenge in antibiotic stewardship is figuring out how to use antibiotics therapeutically without promoting the evolution of antibiotic resistance. Here, we demonstrate proof of concept for an adjunctive therapy that allows intravenous antibiotic treatment without driving the evolution and onward transmission of resistance. We repurposed the FDA-approved bile acid sequestrant cholestyramine, which we show binds the antibiotic daptomycin, as an 'anti-antibiotic' to disable systemically-administered daptomycin reaching the gut. We hypothesized that adjunctive cholestyramine could enable therapeutic daptomycin treatment in the bloodstream, while preventing transmissible resistance emergence in opportunistic pathogens colonizing the gastrointestinal tract. We tested this idea in a mouse model of Enterococcus faecium gastrointestinal tract colonization. In mice treated with daptomycin, adjunctive cholestyramine therapy reduced the fecal shedding of daptomycin-resistant E. faecium by up to 80-fold. These results provide proof of concept for an approach that could reduce the spread of antibiotic resistance for important hospital pathogens., Competing Interests: VM, CK, DS, SO, LJ, EH, GU, SS, MP, RW, AR No competing interests declared, (© 2020, Morley et al.)

The inability to spatiotemporally guide proteins in tissues and efficiently deliver them into cells remains a key barrier to realizing their full potential in precision medicine. Here, we report ultrasound-sensitive fluoro-protein nanoemulsions which can be acoustically tracked, guided, and activated for on-demand cytosolic delivery of proteins, including antibodies, using clinically relevant diagnostic ultrasound. This advance is accessed through the discovery of a family of fluorous tags, or FTags, that transiently mask proteins to mediate their efficient dispersion into ultrasound-sensitive liquid perfluorocarbons, a phenomenon akin to dissolving an egg in liquid Teflon. We identify the biochemical basis for protein fluorous masking and confirm FTag coatings are shed during delivery, without disrupting the protein structure or function. Harnessing the ultrasound sensitivity of fluorous emulsions, real-time imaging is used to simultaneously monitor and activate FTag-protein complexes to enable controlled cytosolic antibody delivery in vitro and in vivo . These findings may advance the development of image-guided, protein-based biosensing and therapeutic modalities.

Lanmodulin (LanM) is a high-affinity lanthanide (Ln)-binding protein recently identified in Methylobacterium extorquens, a bacterium that requires Lns for the function of at least two enzymes. LanM possesses four EF-hands, metal coordination motifs generally associated with Ca II binding, but it undergoes a metal-dependent conformational change with a 100 million-fold selectivity for Ln III s and Y III over Ca II . Here we present the nuclear magnetic resonance solution structure of LanM complexed with Y III . This structure reveals that LanM features an unusual fusion of adjacent EF-hands, resulting in a compact fold to the best of our knowledge unique among EF-hand-containing proteins. It also supports the importance of an additional carboxylate ligand in contributing to the protein's picomolar affinity for Ln III s, and it suggests a role of unusual N i+1 -H···N i hydrogen bonds, in which LanM's unique EF-hand proline residues are engaged, in selective Ln III recognition. This work sets the stage for a detailed mechanistic understanding of LanM's Ln selectivity, which may inspire new strategies for binding, detecting, and sequestering these technologically important metals.

The pancreatic and duodenal homeobox 1 (Pdx1) is an essential pancreatic transcription factor. The C-terminal intrinsically disordered domain of Pdx1 (Pdx1-C) has a heavily biased amino acid composition; most notably, 18 of 83 residues are proline, including a hexaproline cluster near the middle of the chain. For these reasons, Pdx1-C is an attractive target for structure characterization, given the availability of suitable methods. To determine the solution ensembles of disordered proteins, we have developed a suite of 13 C direct-detect NMR experiments that provide high spectral quality, even in the presence of strong proline enrichment. Here, we have extended our suite of NMR experiments to include four new pulse programs designed to record backbone residual dipolar couplings in a 13 C, 15 N-CON detection format. Using our NMR strategy, in combination with small-angle X-ray scattering measurements and Monte Carlo simulations, we have determined that Pdx1-C is extended in solution, with a radius of gyration and internal scaling similar to that of an excluded volume polymer, and a subtle tendency toward a collapsed structure to the N-terminal side of the hexaproline sequence. This structure leaves Pdx1-C exposed for interactions with trans-regulatory co-factors that contribute with Pdx1 to transcription control in the cell.

NMR spectroscopy remains the only experimental technique that provides (near) atomistic structural information for intrinsically disordered proteins (IDPs), but their sequence and structure characteristics still pose major challenges for high-resolution spectroscopy. Carbon-13 direct-detect NMR spectroscopy can overcome poor spectral dispersion and other difficulties associated with traditional 1 H-detected NMR of nonaggregating disordered proteins. This chapter presents spectroscopic protocols suitable for complete characterization of IDPs that rely exclusively on 13 C direct-detect experiments. The protocols described span initial characterization and chemical shift assignment; structure constraint through residual dipolar coupling and paramagnetic relaxation enhancement measurements; and assessment of intramolecular dynamics through 15 N spin relaxation. The experiments described empower investigators to establish molecular mechanisms and structure-function relationships for IDPs and other proteins characterized by high internal flexibility., (© 2018 Elsevier Inc. All rights reserved.)

Proline isomerization is ubiquitous in proteins and is important for regulating important processes such as folding, recognition, and enzymatic activity. In humans, peptidyl-prolyl isomerase cis-trans isomerase NIMA interacting 1 (Pin1) is responsible for mediating fast conversion between cis- and trans-conformations of serine/threonine-proline (S/T-P) motifs in a large number of cellular pathways, many of which are involved in normal development as well as progression of several cancers and diseases. One of the major processes that Pin1 regulates is phosphatase activity against the RNA polymerase II C-terminal domain (RNAPII CTD). However, molecular tools capable of distinguishing the effects of proline conformation on phosphatase function have been lacking. A key tool that allows us to understand isomeric specificity of proteins toward their substrates is the usage of proline mimicking isosteres that are locked to prevent cis/trans-proline conversion. These locked isosteres can be incorporated into standard peptide synthesis and then used in replacement of native substrates in various experimental techniques such as kinetic and thermodynamic assays as well as X-ray crystallography. We will describe the application of these chemical tools in detail using CTD phosphatases as an example. We will also discuss alternative methods for analyzing the effect of proline conformation such as 13 C NMR and the biological implications of proline isomeric specificity of proteins. The chemical and analytical tools presented in this chapter are widely applicable and should help elucidate many questions on the role of proline isomerization in biology., (© 2018 Elsevier Inc. All rights reserved.)

The positive transcription elongation factor b (P-TEFb) promotes transcription elongation through phosphorylation of the RNA polymerase II C-terminal domain. This process is not well understood, partly due to difficulties in determining the specificity of P-TEFb toward the various heptad repeat motifs within the C-terminal domain. A simple assay using mass spectrometry was developed to identify the substrate specificity of the Drosophila melanogaster P-TEFb (DmP-TEFb) in vitro. This assay demonstrated that DmP-TEFb preferentially phosphorylates Ser5 and, surprisingly, that pre-phosphorylation or conserved amino acid variation at the 7-position in the heptad can alter DmP-TEFb specificity, leading to the creation of distinct double-phosphorylation marks., (Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The prevalence of intrinsically disordered protein regions, particularly in eukaryotic proteins, and their clear functional advantages for signaling and gene regulation have created an imperative for high-resolution structural and mechanistic studies. NMR spectroscopy has played a central role in enhancing not only our understanding of the intrinsically disordered native state, but also how that state contributes to biological function. While pathological functions associated with protein aggregation are well established, it has recently become clear that disordered regions also mediate functionally advantageous assembly into high-order structures that promote the formation of membrane-less sub-cellular compartments and even hydrogels. Across the range of functional assembly states accessed by disordered regions, post-translational modifications and regulatory macromolecular interactions, which can also be investigated by NMR spectroscopy, feature prominently. Here we will explore the many ways in which NMR has advanced our understanding of the physical-chemical phase space occupied by disordered protein regions and provide prospectus for the future role of NMR in this emerging and exciting field., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Canonical processing of miRNA begins in the nucleus with the Microprocessor complex, which is minimally composed of the RNase III enzyme Drosha and two copies of its cofactor protein DGCR8. In structural analogy to most RNase III enzymes, Drosha possesses a modular domain with the double-stranded RNA binding domain (dsRBD) fold. Unlike the dsRBDs found in most members of the RNase III family, the Drosha-dsRBD does not display double-stranded RNA binding activity; perhaps related to this, the Drosha-dsRBD amino acid sequence does not conform well to the canonical patterns expected for a dsRBD. In this article, we investigate the impact on miRNA processing of engineering double-stranded RNA binding activity into Drosha's non-canonical dsRBD. Our findings corroborate previous studies that have demonstrated the Drosha-dsRBD is necessary for miRNA processing and suggest that the amino acid composition in the second α-helix of the domain is critical to support its evolved function.

RNA polymerase II contains a repetitive, intrinsically disordered, C-terminal domain (CTD) composed of heptads of the consensus sequence YSPTSPS. The CTD is heavily phosphorylated and serves as a scaffold, interacting with factors involved in transcription initiation, elongation and termination, RNA processing and chromatin modification. Despite being a nexus of eukaryotic gene regulation, the structure of the CTD and the structural implications of phosphorylation are poorly understood. Here we present a biophysical and biochemical interrogation of the structure of the full length CTD of Drosophila melanogaster, which we conclude is a compact random coil. Surprisingly, we find that the repetitive CTD is structurally heterogeneous. Phosphorylation causes increases in radius, protein accessibility and stiffness, without disrupting local structural heterogeneity. Additionally, we show the human CTD is also structurally heterogeneous and able to substitute for the D. melanogaster CTD in supporting fly development to adulthood. This finding implicates conserved structural organization, not a precise array of heptad motifs, as important to CTD function.

The carboxy-terminal domain (CTD) of the RNA polymerase II (Pol II) large subunit cycles through phosphorylation states that correlate with progression through the transcription cycle and regulate nascent mRNA processing. Structural analyses of yeast and mammalian CTD are hampered by their repetitive sequences. Here we identify a region of the Drosophila melanogaster CTD that is essential for Pol II function in vivo and capitalize on natural sequence variations within it to facilitate structural analysis. Mass spectrometry and NMR spectroscopy reveal that hyper-Ser5 phosphorylation transforms the local structure of this region via proline isomerization. The sequence context of this switch tunes the activity of the phosphatase Ssu72, leading to the preferential de-phosphorylation of specific heptads. Together, context-dependent conformational switches and biased dephosphorylation suggest a mechanism for the selective recruitment of cis-proline-specific regulatory factors and region-specific modulation of the CTD code that may augment gene regulation in developmentally complex organisms.

Intrinsically disordered protein regions (IDRs) partially or completely lack a cooperatively folded structure under native conditions, preventing their equilibrium state from being adequately described by a single structural model. As a direct consequence of their disorder, remarkably few experimental studies have quantified the ensembles IDRs adopt in solution. Here, we conduct unbiased computer simulations of the RAP74 interaction motif from the human phosphatase FCP1 in the unbound state, which provides an ensemble in quantitative agreement with both experimental NMR chemical shift information and small-angle X-ray scattering data. The partially α-helical short linear motif found in the C-terminus of FCP1 has been the subject of extensive biophysical characterization aimed at developing a molecular description for the mechanism of coupled folding and binding and establishing the functional relevance of partial order in the unbound state. The analysis presented here yields a remarkably consistent molecular picture enumerating the diversity of structures present in a "partially formed" helix. Specific interactions, including anticorrelations in backbone dihedral angle fluctuations as well as the transient formation of a helix-stabilizing salt bridge, stabilize the preformed structure in the unbound state. The general consequences of these findings for mechanistic analysis of protein-protein interactions are discussed.

Protein-nucleic acid interactions are central to a variety of biological processes, many of which involve large-scale conformational changes that lead to bending of the nucleic acid helix. Here, we focus on the nonsequence-specific protein TRBP, whose double-stranded RNA-binding domains (dsRBDs) interact with the A-form geometry of double-stranded RNA (dsRNA). Crystal structures of dsRBD-dsRNA interactions suggest that the dsRNA helix must bend in such a way that its major groove expands to conform to the dsRBD's binding surface. We show through isothermal titration calorimetry experiments that dsRBD2 of TRBP binds dsRNA with a temperature-independent observed binding affinity (KD ∼500 nM). Furthermore, a near-zero observed heat capacity change (ΔCp = 70 ± 40 cal·mol(-1)·K(-1)) suggests that large-scale conformational changes do not occur upon binding. This result is bolstered by molecular-dynamics simulations in which dsRBD-dsRNA interactions generate only modest bending of the RNA along its helical axis. Overall, these results suggest that this particular dsRBD-dsRNA interaction produces little to no change in the A-form geometry of dsRNA in solution. These results further support our previous hypothesis, based on extensive gel-shift assays, that TRBP preferentially binds to sites of nearly ideal A-form structure while being excluded from sites of local deformation in the RNA helical structure. The implications of this mechanism for efficient micro-RNA processing will be discussed., (Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Many labs have conventional calorimeters where denaturation and binding experiments are setup and run one at a time. While these systems are highly informative to biopolymer folding and ligand interaction, they require considerable manual intervention for cleaning and setup. As such, the throughput for such setups is limited typically to a few runs a day. With a large number of experimental parameters to explore including different buffers, macromolecule concentrations, temperatures, ligands, mutants, controls, replicates, and instrument tests, the need for high-throughput automated calorimeters is on the rise. Lower sample volume requirements and reduced user intervention time compared to the manual instruments have improved turnover of calorimetry experiments in a high-throughput format where 25 or more runs can be conducted per day. The cost and efforts to maintain high-throughput equipment typically demands that these instruments be housed in a multiuser core facility. We describe here the steps taken to successfully start and run an automated biological calorimetry facility at Pennsylvania State University. Scientists from various departments at Penn State including Chemistry, Biochemistry and Molecular Biology, Bioengineering, Biology, Food Science, and Chemical Engineering are benefiting from this core facility. Samples studied include proteins, nucleic acids, sugars, lipids, synthetic polymers, small molecules, natural products, and virus capsids. This facility has led to higher throughput of data, which has been leveraged into grant support, attracting new faculty hire and has led to some exciting publications., (© 2016 Elsevier Inc. All rights reserved.)

Broad interest in the thermodynamic driving forces of coupled macromolecular folding and binding is motivated by the prevalence of disorder-to-order transitions observed when intrinsically disordered proteins (IDPs) bind to their partners. Isothermal titration calorimetry (ITC) is one of the few methods available for completely evaluating the thermodynamic parameters describing a protein-ligand binding event. Significantly, when the effective ΔH° for the coupled folding and binding process is determined by ITC in a temperature series, the constant-pressure heat capacity change (ΔCp) associated with these coupled equilibria is experimentally accessible, offering a unique opportunity to investigate the driving forces behind them. Notably, each of these molecular-scale events is often accompanied by strongly temperature-dependent enthalpy changes, even over the narrow temperature range experimentally accessible for biomolecules, making single temperature determinations of ΔH° less informative than typically assumed. Here, we will document the procedures we have adopted in our laboratory for designing, executing, and globally analyzing temperature-dependent ITC studies of coupled folding and binding in IDP interactions. As a biologically significant example, our recent evaluation of temperature-dependent interactions between the disordered tail of FCP1 and the winged-helix domain from Rap74 will be presented. Emphasis will be placed on the use of publically available analysis programs written in MATLAB that facilitate quantification of the thermodynamic forces governing IDP interactions. Although motivated from the perspective of IDPs, the experimental design principles and data fitting procedures presented here are general to the study of most noncooperative ligand binding equilibria., (© 2016 Elsevier Inc. All rights reserved.)

The prevalence of double-stranded RNA (dsRNA) in eukaryotic cells has only recently been appreciated. Of interest here, RNA silencing begins with dsRNA substrates that are bound by the dsRNA-binding domains (dsRBDs) of their processing proteins. Specifically, processing of microRNA (miRNA) in the nucleus minimally requires the enzyme Drosha and its dsRBD-containing cofactor protein, DGCR8. The smallest recombinant construct of DGCR8 that is sufficient for in vitro dsRNA binding, referred to as DGCR8-Core, consists of its two dsRBDs and a C-terminal tail. As dsRBDs rarely recognize the nucleotide sequence of dsRNA, it is reasonable to hypothesize that DGCR8 function is dependent on the recognition of specific structural features in the miRNA precursor. Previously, we demonstrated that noncanonical structural elements that promote RNA flexibility within the stem of miRNA precursors are necessary for efficient in vitro cleavage by reconstituted Microprocessor complexes. Here, we combine gel shift assays with in vitro processing assays to demonstrate that neither the N-terminal dsRBD of DGCR8 in isolation nor the DGCR8-Core construct is sensitive to the presence of noncanonical structural elements within the stem of miRNA precursors, or to single-stranded segments flanking the stem. Extending DGCR8-Core to include an N-terminal heme-binding region does not change our conclusions. Thus, our data suggest that although the DGCR8-Core region is necessary for dsRNA binding and recruitment to the Microprocessor, it is not sufficient to establish the previously observed connection between RNA flexibility and processing efficiency., (© 2015 Wiley Periodicals, Inc.)

Intrinsically disordered proteins (IDPs) are broadly defined as protein regions that do not cooperatively fold into a spatially or temporally stable structure. Recent research strongly supports the hypothesis that a conserved functional role for structural disorder renders IDPs uniquely capable of functioning in biological processes such as cellular signaling and transcription. Recently, the frequency of application of rigorous mechanistic biochemistry and quantitative biophysics to disordered systems has increased dramatically. For example, the launch of the Protein Ensemble Database (pE-DB) demonstrates that the potential now exists to refine models for the native state structure of IDPs using experimental data. However, rigorous assessment of which observables place the strongest and least biased constraints on those ensembles is now needed. Most importantly, the past few years have seen strong growth in the number of biochemical and biophysical studies attempting to connect structural disorder with function. From the perspective of equilibrium thermodynamics, there is a clear need to assess the relative significance of hydrophobic versus electrostatic forces in IDP interactions, if it is possible to generalize at all. Finally, kinetic mechanisms that invoke conformational selection and/or induced fit are often used to characterize coupled IDP folding and binding, although application of these models is typically built upon thermodynamic observations. Recently, the reaction rates and kinetic mechanisms of more intrinsically disordered systems have been tested through rigorous kinetic experiments. Motivated by these exciting advances, here we provide a review and prospectus for the quantitative study of IDP structure, thermodynamics, and kinetics.

Background: MicroRNAs (miRNAs) are critical post-transcriptional regulators of gene expression. Their precursors have a globally A-form helical geometry, which prevents most proteins from identifying their nucleotide sequence. This suggests the hypothesis that local structural features (e.g., bulges, internal loops) play a central role in specific double-stranded RNA (dsRNA) selection from cellular RNA pools by dsRNA binding domain (dsRBD) containing proteins. Furthermore, the processing enzymes in the miRNA maturation pathway require tandem-dsRBD cofactor proteins for optimal function, suggesting that dsRBDs play a key role in the molecular mechanism for precise positioning of the RNA within these multi-protein complexes. Here, we focus on the tandem-dsRBDs of TRBP, which have been shown to bind dsRNA tightly., Methodology/principal Findings: We present a combination of dsRNA binding assays demonstrating that TRBP binds dsRNA in an RNA-length dependent manner. Moreover, circular dichroism data shows that the number of dsRBD moieties bound to RNA at saturation is different for a tandem-dsRBD construct than for constructs with only one dsRBD per polypeptide, revealing another reason for the selective pressure to maintain multiple domains within a polypeptide chain. Finally, we show that helical defects in precursor miRNA alter the apparent dsRNA size, demonstrating that imperfections in RNA structure influence the strength of TRBP binding., Conclusion/significance: We conclude that TRBP is responsible for recognizing structural imperfections in miRNA precursors, in the sense that TRBP is unable to bind imperfections efficiently and thus is positioned around them. We propose that once positioned around structural defects, TRBP assists Dicer and the rest of the RNA-induced silencing complex (RISC) in providing efficient and homogenous conversion of substrate precursor miRNA into mature miRNA downstream.

The human proteome contains myriad intrinsically disordered proteins. Within intrinsically disordered proteins, polyproline-II motifs are often located near sites of phosphorylation. We have used an unconventional experimental paradigm to discover that phosphorylation by protein kinase A (PKA) occurs in the intrinsically disordered domain of hepatitis C virus non-structural protein 5A (NS5A) on Thr-2332 near one of its polyproline-II motifs. Phosphorylation shifts the conformational ensemble of the NS5A intrinsically disordered domain to a state that permits detection of the polyproline motif by using (15)N-, (13)C-based multidimensional NMR spectroscopy. PKA-dependent proline resonances were lost in the presence of the Src homology 3 domain of c-Src, consistent with formation of a complex. Changing Thr-2332 to alanine in hepatitis C virus genotype 1b reduced the steady-state level of RNA by 10-fold; this change was lethal for genotype 2a. The lethal phenotype could be rescued by changing Thr-2332 to glutamic acid, a phosphomimetic substitution. Immunofluorescence and transmission electron microscopy showed that the inability to produce Thr(P)-2332-NS5A caused loss of integrity of the virus-induced membranous web/replication organelle. An even more extreme phenotype was observed in the presence of small molecule inhibitors of PKA. We conclude that the PKA-phosphorylated form of NS5A exhibits unique structure and function relative to the unphosphorylated protein. We suggest that post-translational modification of viral proteins containing intrinsic disorder may be a general mechanism to expand the viral proteome without a corresponding expansion of the genome., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

There is an extraordinary need to describe the structures of intrinsically disordered proteins (IDPs) due to their role in various biological processes involved in signaling and transcription. However, general study of IDPs by NMR spectroscopy is limited by the poor (1)H amide chemical shift dispersion typically observed in their spectra. Recently, (13)C direct-detected NMR spectroscopy has been recognized as enabling broad structural study of IDPs. Most notably, multidimensional experiments based on the (15)N,(13)C CON spectrum make complete chemical shift assignment feasible. Here we document a collection of NMR-based tools that efficiently lead to chemical shift assignment of IDPs, motivated by a case study of the C-terminal disordered region from the human pancreatic transcription factor Pdx1. Our strategy builds on the combination of two three-dimensional (3D) experiments, (HN-flip)N(CA)CON and 3D (HN-flip)N(CA)NCO, that enable daisy chain connections to be built along the IDP backbone, facilitated by acquisition of amino acid-specific (15)N,(13)C CON-detected experiments. Assignments are completed through carbon-detected, total correlation spectroscopy (TOCSY)-based side chain chemical shift measurement. Conducting our study required producing valuable modifications to many previously published pulse sequences, motivating us to announce the creation of a database of our pulse programs, which we make freely available through our website., (Copyright © 2013 Elsevier Inc. All rights reserved.)

In this work, we quantitatively investigate the thermodynamic analogy between the folding of monomeric proteins and the interactions of intrinsically disordered proteins (IDPs). Motivated by the hypothesis that similar hydrophobic forces guide both globular protein folding and also IDP interactions, we present a unified experimental and computational investigation of the coupling between the folding and binding of the intrinsically disordered tail of FCP1 when interacting with the cooperatively folding winged-helix domain of Rap74. Our calorimetric measurements quantitatively demonstrate the significance of hydrophobic interactions for this binding event. Our computational studies indicate that IDPs relieve frustration at the surface of ordered proteins to generate a minimally frustrated complex that is strikingly similar to a globular monomeric protein. In summary, these results not only quantify the thermodynamic forces driving disordered protein interactions but also highlight the role of ordered proteins for IDP function.

In adult mammals, the production of insulin and other peptide hormones, such as the islet amyloid polypeptide (IAPP), is limited to β-cells due to tissue-specific expression of a set of transcription factors, the best known of which is pancreatic duodenal homeobox protein 1 (Pdx1). Like many homeodomain transcription factors, Pdx1 binds to a core DNA recognition sequence containing the tetranucleotide 5'-TAAT-3'; its consensus recognition element is 5'-CTCTAAT(T/G)AG-3'. Currently, a complete thermodynamic profile of Pdx1 binding to near-consensus and native promoter sequences has not been established, obscuring the mechanism of target site selection by this critical transcription factor. Strikingly, while Pdx1 responsive elements in the human insulin promoter conform to the pentanucleotide 5'-CTAAT-3' sequence, the Pdx1 responsive elements in the human iapp promoter all contain a substitution to 5'-TTAAT-3'. The crystal structure of Pdx1 bound to the consensus nucleotide sequence does not explain how Pdx1 identifies this natural variation, if it does at all. Here we report a combination of isothermal calorimetric titrations, NMR spectroscopy, and extensive multi-microsecond molecular dynamics calculations of Pdx1 that define its interactions with a panel of natural promoter elements and consensus-derived sequences. Our results show a small preference of Pdx1 for a C base 5' relative to the core TAAT promoter element. Molecular mechanics calculations, corroborated by experimental NMR data, lead to a rational explanation for sequence discrimination at this position. Taken together, our results suggest a molecular mechanism for differential Pdx1 affinity to elements from the insulin and iapp promoter sequences., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

By dephosphorylating the C-terminal domain (CTD) of RNA polymerase II (Pol II), the Transcription Factor IIF (TFIIF)-associating CTD phosphatase (FCP1) performs an essential function in recycling Pol II for subsequent rounds of transcription. The interaction between FCP1 and TFIIF is mediated by the disordered C-terminal tail of FCP1, which folds to form an α-helix upon binding the RAP74 subunit of TFIIF. The present work reports a structure-based simulation study of this interaction between the folded winged-helix domain of RAP74 and the disordered C-terminal tail of FCP1. The comparison of measured and simulated chemical shifts suggests that the FCP1 peptide samples 40-60% of its native helical structure in the unbound disordered ensemble. Free energy calculations suggest that productive binding begins when RAP74 makes hydrophobic contacts with the C-terminal region of the FCP1 peptide. The FCP1 peptide then folds into an amphipathic helix by zipping up the binding interface. The relative plasticity of FCP1 results in a more cooperative binding mechanism, allows for a greater diversity of pathways leading to the bound complex, and may also eliminate the need for "backtracking" from contacts that form out of sequence.

Most noncoding RNAs function properly only when folded into complex three-dimensional (3D) structures, but the experimental determination of these structures remains challenging. Understanding of primary microRNA (miRNA) maturation is currently limited by a lack of determined structures for nonprocessed forms of the RNA. SHAPE chemistry efficiently determines RNA secondary structural information with single-nucleotide resolution, providing constraints suitable for input into MC-Pipeline for refinement of 3D structure models. Here we combine these approaches to analyze three structurally diverse primary microRNAs, revealing deviations from canonical double-stranded RNA structure in the stem adjacent to the Drosha cut site for all three. The necessity of these deformable sites for efficient processing is demonstrated through Drosha processing assays. The structure models generated herein support the hypothesis that deformable sequences spaced roughly once per turn of A-form helix, created by noncanonical structure elements, combine with the necessary single-stranded RNA-double-stranded RNA junction to define the correct Drosha cleavage site.

Intrinsically disordered proteins (IDPs) lack unique 3D structures under native conditions and as such exist as highly dynamic ensembles in solution. We present two (13)C-direct detection experiments for the measurement of (15)N NMR spin relaxation called the CON(T1)-IPAP and CON(T2)-IPAP that quantify backbone dynamics on a per-residue basis for IDPs in solution. These experiments have been applied to the intrinsically disordered C-terminal of FCP1, both free in solution and while bound to the RAP74 winged-helix domain. The results provide evidence that most of FCP1 remains highly dynamic in both states, while the 20 residues forming direct contact with RAP74 become more ordered in the complex. Parallel analysis of RAP74 backbone (15)N NMR spin relaxation reveals only very limited ordering of RAP74 upon FCP1 binding. Taken together, these data show that folding-upon-binding is highly local in this system, with disorder prevailing even in the complex.

One of the most exciting recent developments in RNA biology has been the discovery of small non-coding RNAs that affect gene expression through the RNA interference (RNAi) mechanism. Two major classes of RNAs involved in RNAi are small interfering RNA (siRNA) and microRNA (miRNA). Dicer, an RNase III enzyme, plays a central role in the RNAi pathway by cleaving precursors of both of these classes of RNAs to form mature siRNAs and miRNAs, which are then loaded into the RNA-induced silencing complex (RISC). miRNA and siRNA precursors are quite structurally distinct; miRNA precursors are short, imperfect hairpins while siRNA precursors are long, perfect duplexes. Nonetheless, Dicer is able to process both. Dicer, like the majority of RNase III enzymes, contains a dsRNA binding domain (dsRBD), but the data are sparse on the exact role this domain plays in the mechanism of Dicer binding and cleavage. To further explore the role of human Dicer-dsRBD in the RNAi pathway, we determined its binding affinity to various RNAs modeling both miRNA and siRNA precursors. Our study shows that Dicer-dsRBD is an avid binder of dsRNA, but its binding is only minimally influenced by a single-stranded - double-stranded junction caused by large terminal loops observed in miRNA precursors. Thus, the Dicer-dsRBD contributes directly to substrate binding but not to the mechanism of differentiating between pre-miRNA and pre-siRNA. In addition, NMR spin relaxation and MD simulations provide an overview of the role that dynamics contribute to the binding mechanism. We compare this current study with our previous studies of the dsRBDs from Drosha and DGCR8 to give a dynamic profile of dsRBDs in their apo-state and a mechanistic view of dsRNA binding by dsRBDs in general.

We report atomically detailed molecular dynamics simulations characterizing the interaction of the RAP74 winged helix domain with the intrinsically disordered C-terminal of FCP1. The RAP74-FCP1 complex promotes the essential dephosphorylation of RNA polymerase II prior to initiation of transcription. Although disordered in solution, the C-terminal of FCP1 forms an amphipathic helix when bound to RAP74. Our simulations demonstrate that this interaction also reorganizes and stabilizes RAP74. These simulations illuminate the significance of hydrophobic contacts for stabilizing disordered protein complexes, provide new insight into the mechanism of protein binding by winged helix domains, and also reveal "dynamic fuzziness" in the complex as FCP1 retains significant flexibility after binding. In conjunction with our recent NMR experiments identifying residual structure in unbound FCP1, these simulations suggest that FCP1 loses relatively little conformational entropy upon binding and that the associated coupled folding-binding transition may be less sharp than expected., (© 2011 American Chemical Society)

Intrinsically disordered proteins (IDPs) lack unique 3D structures under native conditions and yet retain critical functions. Recycling of RNA Polymerase II after transcription is promoted by an interaction between the winged helix domain of RAP74, a component of the general transcription factor IIF (TFIIF), and the C-terminus of the TFIIF-associating CTD phosphatase (FCP1). Sixteen residues from the C-terminus of FCP1 form an α-helix in the complex, but the protein is otherwise agreed in the literature to be intrinsically disordered. Here we show through CD and recently developed carbon-detected NMR that, although FCP1 is intrinsically disordered, the above 16 residues composing the RAP74 binding surface form nascent α-helical structure in the unbound state. We further show retention of general FCP1 disorder and the nascent helical content in HeLa extract, establishing cellular relevance. The conformational bias observed leads to a mechanistic proposal for FCP1's transition from a disordered ensemble to an ordered conformation upon binding., (Copyright © 2011 Elsevier Inc. All rights reserved.)

MicroRNAs (miRNAs) affect gene regulation by base pairing with mRNA and contribute to the control of cellular homeostasis. The first step in miRNA maturation is conducted in the nucleus by the "microprocessor" complex made up of an RNase III enzyme, Drosha, that contains one dsRNA binding domain (dsRBD), and DGCR8, that contains two dsRBDs in tandem. The crystal structure of DGCR8-Core (493-720), containing both dsRBDs, and the NMR solution structure of Drosha-dsRBD (1259-1337) have been reported, but the solution dynamics have not been explored for any of these dsRBDs. To better define the mechanism of dsRNA binding and thus the nuclear maturation step of miRNA processing, we report NMR spin relaxation and MD simulations of Drosha-dsRBD (1259-1337) and DGCR8-dsRBD1 (505-583). The study was motivated by electrophoretic mobility shift assays (EMSAs) of the two dsRBDs, which showed that Drosha-dsRBD does not bind a representative miRNA but isolated DGCR8-dsRBD1 does (K(d) = 9.4 ± 0.4 μM). Our results show that loop 2 in both dsRBDs is highly dynamic but the pattern of the correlations observed in MD is different for the two proteins. Additionally, the extended loop 1 of Drosha-dsRBD is more flexible than the corresponding loop in DGCR8-dsRBD1 but shows no correlation with loop 2, which potentially explains the lack of dsRNA binding by Drosha-dsRBD in the absence of the RNase III domains. The results presented in this study provide key structural and dynamic features of dsRBDs that contribute to the binding mechanism of these domains to dsRNA.

The configurational entropy of a protein is under physiological conditions a major contributor to the free energy. Its quantitative characterization is therefore an important step toward the understanding of protein function. The configurational entropy of the oncoprotein MDM2, whose determination is a challenge by experiment alone, is studied here by means of 0.4 μs molecular dynamics computer simulations in both the presence and absence of the p53-peptide ligand. By characterizing protein motions in dihedral angle space, it is found that the motional amplitudes change considerably upon ligand binding while correlations between dihedral angle motions are remarkably well conserved. This applies for backbone and side-chain dihedral angle pairs at both short- and long-range distance to the binding site. As a direct consequence, the change of the configurational entropy can be decomposed into a sum of local contributions. This significantly facilitates the understanding of the relationship between protein dynamics and thermodynamics, which is important, for example, in the context of protein-ligand and protein-protein interactions. The findings also have implications for the direct derivation of entropy changes from site-specific dynamics measurements as afforded by NMR spectroscopy.

Over the past decade, microRNAs (miRNAs) have been shown to affect gene regulation by basepairing with messenger RNA, and their misregulation has been directly linked with cancer. DGCR8, a protein that contains two dsRNA-binding domains (dsRBDs) in tandem, is vital for nuclear maturation of primary miRNAs (pri-miRNAs) in connection with the RNase III enzyme Drosha. The crystal structure of the DGCR8 Core (493-720) shows a unique, well-ordered structure of the linker region between the two dsRBDs that differs from the flexible linker connecting the two dsRBDs in the antiviral response protein, PKR. To better understand the interfacial interactions between the two dsRBDs, we ran extensive MD simulations of isolated dsRBDs (505-583 and 614-691) and the Core. The simulations reveal correlated reorientations of the two domains relative to one another, with the well-ordered linker and C-terminus serving as a pivot. The results demonstrate that motions at the domain interface dynamically impact the conformation of the RNA-binding surface and may provide an adaptive separation distance that is necessary to allow interactions with a variety of different pri-miRNAs with heterogeneous structures. These results thus provide an entry point for further in vitro studies of the potentially unique RNA-binding mode of DGCR8., (Copyright 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The phosphorylation state of the RNA polymerase II C-terminal repeat domain (CTD) regulates progression through the mRNA biogenesis cycle. Termination of transcription and recycling of RNA polymerase II is promoted by an interaction between the general transcription factor IIF (TFIIF) and the TFIIF-associating CTD phosphatase (FCP1). The acidic C-terminal region of FCP1 is disordered in the free state, but adopts an alpha-helical conformation upon binding to the heavy chain of TFIIF. Here we report (1)H, (13)C, and (15)N resonance assignments for the intrinsically disordered unbound form of human C-terminal FCP1 (residues 879-961). The use of recently developed (13)C direct detected "protonless" NMR experiments allowed the nearly complete assignment of FCP1 reported here and is likely to be a generally effective strategy for the chemical shift assignment of disordered proteins.

Exclusively heteronuclear (13)C-detected NMR spectroscopy of proteins in solution has seen resurgence in the past several years. For disordered or unfolded proteins, which tend to have poor (1)H-amide chemical shift dispersion, these experiments offer enhanced resolution and the possibility of complete heteronuclear resonance assignment at the cost of leaving the (1)H resonances unassigned. Here we report two novel (13)C-detected NMR experiments which incorporate a (1)H chemical shift evolution period followed by (13)C-TOCSY mixing for aliphatic (1)H resonance assignment without reliance on (1)H detection.

(3)J scalar couplings report on the conformational averaging of backbone phi angles in peptides and proteins, and therefore represent a potentially powerful tool for studying the details of both structure and dynamics in solution. We have compared an extensive experimental dataset with J-couplings predicted from unrestrained molecular dynamics simulation using enhanced sampling available from accelerated molecular dynamics or using long timescale trajectories (200 ns). The dynamic fluctuations predicted to be present along the backbone, in agreement with residual dipolar coupling analysis, are compatible with the experimental (3)J scalar couplings providing a slightly better reproduction of these experimental parameters than a high-resolution static structure.

The oncoprotein MDM2 regulates the activity and stability of the tumor suppressor p53 through protein-protein interaction involving their N-terminal domains. The N-terminal lid of MDM2 has been implicated in p53 regulation; however, due to its flexible nature, limited data are available concerning its role in ligand binding. The quantitative dynamics study using NMR reported here shows, for the first time, that the lid in apo-MDM2 slowly interconverts between a "closed" state that is associated with the p53-binding cleft and an "open" state that is highly flexible. Our results reveal that apo-MDM2 predominantly populates the closed state, whereas the p53-bound MDM2 exclusively populates the open state. Unlike p53 binding, the small molecule MDM2 antagonist nutlin-3 binds to the cleft essentially without perturbing the closed lid state. The lid dynamics thereby represents a signature for the experimental and virtual screening of therapeutic antagonists that target the p53-MDM2 interaction.

The model-free approach (Lipari, G.; Szabo, A. J. Am. Chem. Soc. 1982, 104, 4546-4559; 4559-4570) is the standard method for the analysis of NMR relaxation data from proteins. The method assigns to each interaction vector a generalized order parameter S(2), which is a measure of the spatial restriction that the vector experiences in a molecular reference frame. A variety of techniques for the interpretation of S(2) values and their underlying assumptions are tested here for a dynamic alpha-helix represented by an ensemble of peptide structures that satisfies a set of predefined constraints. A self-consistent picture is developed that characterizes the influence of major determinants, including backbone dihedral angle fluctuations and their correlations, separability of internal and overall motion, the preservation of hydrogen bonds, restricted end-to-end distance fluctuations, locally anisotropic dynamics, and local contacts between interaction vector atoms and their environment. Many of the features parallel experimental NMR and computational observations of helices in proteins. The understanding gained from this model system is expected to contribute toward the ever more detailed interpretation of experimental order parameter profiles.