Unfolded Domain Research Articles

The Unstructured N-terminal Tail of ParG Modulates Assembly of a Quaternary Nucleoprotein Complex in Transcription Repression

ParG is the prototype of a group of small (<10 kDa) proteins involved in accurate plasmid segregation. The protein is a dimeric DNA binding factor, which consists of symmetric paired C-terminal domains that interleave into a ribbon-helix-helix fold that is crucial for the interaction with DNA, and unstructured N-terminal domains of previously unknown function. Here the ParG protein is shown to be a transcriptional repressor of the parFG genes. The protein assembles on its operator site initially as a tetramer (dimer of dimers) and, at elevated protein concentrations, as a pair of tetramers. Progressive deletion of the mobile N-terminal tails concomitantly decreased transcriptional repression by ParG and perturbed the DNA binding kinetics of the protein. The flexible tails are not necessary for ParG dimerization but instead modulate the organization of a higher order nucleoprotein complex that is crucial for proper transcriptional repression. This is achieved by transient associations between the flexible and folded domains in complex with the target DNA. Numerous ParG homologs encoded by plasmids of Gram-negative bacteria similarly are predicted to possess N-terminal disordered tails, suggesting that this is a common feature of partition operon autoregulation. The results provide new insights into the role of natively unfolded domains in protein function, the molecular mechanisms of transcription regulation, and the control of plasmid segregation.

Bio-inspired Design of Modular Multi-domain Polymers for Advanced Biomaterials

AbstractNative load-bearing proteins, such as the muscle protein titin, exhibit a remarkable degree of combined toughness, strength, and elasticity which have yet to be matched by synthetic materials.Single molecule nanomechanical studies on titin and other modular proteins suggest that these exceptional properties arise from a modular elongation mechanism. The sequential unfolding allows modular biopolymers to sustain a large force over the whole extension of the chain, which makes the polymer strong, along with a large area under the force-extension curve, making it tough as well. In addition, when the external force is removed, the unfolded domains of modular proteins will refold automatically, making them elastic. Inspired by nature, one research effort in my group is aimed at designing synthetic macromolecules that form high order structures by programming non-covalent interactions into polymer chain. The goal is to achieve synthetic biomaterials with combined strength, toughness and elasticity. Three classes of well-defined modular polymers have been synthesized in our laboratory: (1) using quadruple hydrogen-bonding motif 2-ureidon-4-pyrimidone (Upy) to direct the formation loops along a polymer chain (J. Am. Chem. Soc.2004, 126, 2058); (2) using a peptidomimetic beta-sheet based double-closed loop (DCL) as module (J. Am. Chem. Soc.2004, 126, 14328); and (3) an engineered protein G domain III as module. Single molecule force-extension experiments revealed the sequential unfolding of the loops or domains as these modular polymers are stretched, resulting in sawtooth-patterned curves similar to those seen in titin and other biopolymers. In this paper, we will discuss our designs, syntheses and single-molecule studies of polymers having modular domain structures.

Natively unfolded domains in endocytosis: hooks, lines and linkers

It is commonly assumed that a protein must adopt a tertiary structure to achieve its active native state and that regions of a protein that are devoid of alpha-helix or beta-sheet structures are functionally inert. Although extended proline-rich regions are recognized as presenting binding motifs to, for example, Src homology 2 (SH2) and SH3 domains, the idea persists that natively unfolded regions in functional proteins are simply 'spacers' between the folded domains. Such a view has been challenged in recent years and the importance of natively unfolded proteins in biology is now being recognized. In this review, we highlight the role of natively unfolded domains in the field of endocytosis, and show that some important endocytic proteins lack a traditionally folded structure and harbour important binding motifs in their unstructured linker regions.

Improved stability of a protein vaccine through elimination of a partially unfolded state.

Ricin is a potent toxin presenting a threat as a biological weapon. The holotoxin consists of two disulfide-linked polypeptides: an enzymatically active A chain (RTA) and a galactose/N-acetylgalactosamine-binding B chain. Efforts to develop an inactivated version of the A chain as a vaccine have been hampered by limitations of stability and solubility. Previously, recombinant truncated versions of the 267-amino-acid A chain consisting of residues 1-33/44-198 or 1-198 were designed by protein engineering to overcome these limits and were shown to be effective and nontoxic as vaccines in mice. Herein we used CD, dynamic light scattering, fluorescence, and Fourier-transform infrared spectroscopy to examine the biophysical properties of these proteins. Although others have found that recombinant RTA (rRTA) adopts a partially unfolded, molten globule-like state at 45 degrees C, rRTA 1-33/44-198 and 1-198 are significantly more thermostable, remaining completely folded at temperatures up to 53 degrees C and 51 degrees C, respectively. Deleting both an exposed loop region (amino acids 34-43) and the C-terminal domain (199-267) contributed to increased thermostability. We found that chemically induced denaturation of rRTA, but not the truncated variants, proceeds through at least a three-state mechanism. The intermediate state in rRTA unfolding has a hydrophobic core accessible to ANS and an unfolded C-terminal domain. Removing the C-terminal domain changed the mechanism of rRTA unfolding, eliminating a tendency to adopt a partially unfolded state. Our results support the conclusion that these derivatives are superior candidates for development as vaccines against ricin and suggest an approach of reduction to minimum essential domains for design of more thermostable recombinant antigens.

Ultrafast hydration dynamics in protein unfolding: human serum albumin.

We report studies of unfolding and ultrafast hydration dynamics of the protein human serum albumin. Unique in this study is our ability to examine different domains of the same protein and the intermediate on the way to the unfolded state. With femtosecond resolution and site-selective labeling, we isolate the dynamics of domains I and II of the native protein, domain I of the intermediate at 2 M guanidine hydrochloride, and the unfolded state at 6 M of the denaturant. For studies of unfolding, we used the fluorophores, acrylodan (covalently bound to Cys-34 in domain I) and the intrinsic tryptophan (domain II), whereas for hydration dynamics, we probed acrylodan and prodan; the latter is bound to domain II. From the time-dependent spectra and the correlation functions, we obtained the time scale of dynamically ordered water: 57 ps for the more stable domain I and 32 ps for the less stable domain II, in contrast to approximately 0.8 ps for labile, bulk-type water. This trend suggests an increased hydrophilic residues-water interaction of domain I, contrary to some packing models. In the intermediate state, which is characterized by essentially intact domain I and unfolded domain II, the dynamics of ordered water around domain I is nearly the same (61 ps) as that of native state (57 ps), whereas that in the unfolded protein is much shorter (13 ps). We discuss the role of this fluidity in the correlation between stability and function of the protein.

Charge sequence coding in statistical modeling of unfolded proteins

Unfolded proteins recently attracted attention due to accumulation of experimental evidences for their significant role in different life processes. Modeling of electrostatic interactions (EI) in unfolded state of proteins is becoming increasingly important as well. In this paper, we stress on the importance of how the sequence of charged residues of a given protein is incorporated into the models for calculation of EI in the unfolded state. On the basis of the distributions of distances between titratable sites of charged residues calculated for polypeptide chains of various compositions, it was found that the distance distribution for a pair of residues, located close to each other along the sequence of a protein, depends on what residues constitute the pair in question. It was concluded that the consideration of these residue-specific distributions is essential for a statistical model to be accurate from the physical point of view. It was suggested that use of distance intervals in the spherical model of unfolded proteins accounts better for the charge sequence than the set of single distance values. This was illustrated by comparison of the p K values of the titratable groups of the unfolded N-terminal SH3 domain of the Drosophila protein drk to the available experimental data.

Interaction of SDS with Na+/K+-ATPase: SDS-SOLUBILIZED ENZYME RETAINS PARTIAL STRUCTURE AND FUNCTION

Because nearly all structure/function studies on Na(+)/K(+)-ATPase have been done on enzymes prepared in the presence of SDS, we have studied previously unrecognized consequences of SDS interaction with the enzyme. When the purified membrane-bound kidney enzyme was solubilized with SDS or TDS concentrations just sufficient to cause complete solubilization, but not at concentrations severalfold higher, the enzyme retained quaternary structure, exhibiting alpha,alpha-, alpha,beta-, beta,beta-, and alpha,gamma-associations as detected by chemical cross-linking. The presence of solubilized oligomers was confirmed by sucrose density gradient centrifugation. This solubilized enzyme had no ATPase activity and was not phosphorylated by ATP, but it retained the ability to occlude Rb(+) and Na(+). This, and comparison of cross-linking patterns obtained with different reagents, suggested that the transmembrane domains of the enzyme are more resistant to SDS-induced unfolding than its other domains. These findings (a). indicate that the partially unfolded oligomer(s) retaining partial function is the intermediate in the SDS-induced denaturation of the native membrane enzyme having the minimum oligomeric structure of (alpha,beta,gamma)(2) and (b). suggest potential functions for Na(+)/K(+)-ATPase with intrinsically unfolded domains. Mixtures of solubilized/partially unfolded enzyme and membrane-bound enzyme exhibited cross-linking patterns and Na(+) occlusion capacities different from those of either enzyme species, suggesting that the two interact. Formation of the partially unfolded enzyme during standard purification procedure for the preparation of the membrane-bound enzyme was shown, indicating that it is necessary to ensure the separation of the partially unfolded enzyme from the membrane-bound enzyme to avoid the distortion of the properties of the latter.

Coupling of functioning and folding: photoactive yellow protein as an example system

It has been recognized recently that a significant amount of proteins have partially or entirely disordered conformations in their functional state. Photoactive yellow protein (PYP), which is a model protein of photoreceptor and is a prototype of PAS domain superfamily, has the unfolded N-terminal domain in its signaling state. Using a simple coarse-grained model, we illustrate the free energy surface of the photocycle of PYP. The result implies the importance of cooperative effects between the local structural disturbance around the chromophore and the global loosening of the structure associated with unfolding of the N-terminal domain. This example offers a new scenario of photochemistry in which the photochemical kinetics is controlled by the designed energy landscape of protein folding.

Stabilization of antibody V H-domains by proteolytic selection

Single variable domains of antibodies represent the smallest antigen binding fragments but are less stable than when associated with their cognate variable domains. Here we have attempted to improve the thermodynamic stability of a model heavy chain variable domain (V H) by “proteolytic selection” a method whereby the protease-resistance of the displayed protein is coupled to the infectivity of a filamentous bacteriophage. The gene encoding the heavy chain variable domain was taken from the anti-lysozyme antibody HyHEL-10, mutated at random by error-prone PCR, and displayed on filamentous bacteriophage by fusion between the domains of the phage p3 protein. As the entire p3 protein is required for phage infectivity, treatment of the phage library with trypsin at an elevated temperature (which leads to cleavage of p3 fusions with unfolded variable domains) selects for infectious phages bearing the more stable variable domains. After several rounds of selection, a mutant (S65G/T70S/D99N) was obtained with improved stability ( T m=58.5 °C and ΔG 25 ° C =6.3 kcal/mol compared to 51.6 °C and 4.2 kcal/mol for the parent domain). These mutations are conservative and the mutant domain retains the ability to pair with its cognate light chain variable domain in an Fv fragment and to mediate binding to lysozyme. Our results show that the thermodynamic stability of antibody single domains can be improved by “proteolytic selection” and this may represent a step towards making useful antibody single domains for biotechnological application.

Checking folding inside and out

![][1] The location of the unfolded domain (star) is what matters at the ER. The ER is a site of both synthesis and quality control. Rejected, improperly folded proteins are destroyed by a well-characterized machinery (the ER-associated degradation [ERAD] pathway) in the cytosol and

Automated microscopy identifies estrogen receptor subdomains with large-scale chromatin structure unfolding activity.

Recently, several transcription factors were found to possess large-scale chromatin unfolding activity; these include the VP16 acidic activation domain, BRCA1, E2F1, p53, and the glucocorticoid and estrogen steroid receptors. In these studies, proteins were fluorescently labeled and targeted to a multimerized array of DNA sequences in mammalian cultured cells, and changes in the appearance and/or size of the array were observed. This type of experiment is impeded by the low throughput of traditional microscopy. We report the application of automated microscopy to provide unattended, rapid, quantitative measurements of fluorescently labeled chromosome regions. The automated image collection routine produced results comparable to results previously obtained by manual methods and was significantly faster. Using this approach, we identified two subdomains within the E domain of estrogen receptor alpha capable of inducing large-scale chromatin decondensation. This work confirms that, like BRCA1, the activation function 2 region of the estrogen receptor has more than one distinct chromatin unfolding domain. In addition, we demonstrate the feasibility of using automated microscopy as a high-throughput screen for identifying modulators of large-scale chromatin folding. The Supplementary Material referred to in this article can be found at the CYTO Part A website (http://www.interscience.wiley.com/jpages/0196-4763/suppmat/v58A.html)

Chaperone-mediated folding and assembly of myosin in striated muscle.

De novo folding and assembly of striated muscle myosin was analyzed by expressing a GFP-tagged embryonic myosin heavy chain (GFP-myosin) in post-mitotic C2C12 myocytes using replication defective adenoviruses. In the early stages of muscle differentiation, the GFP-myosin accumulates in bright globular foci and short filamentous structures that are later replaced by brightly fluorescent myofibrils. Time-lapse microscopy shows that the intermediates are dynamic and are present in elongating and fusing myocytes and in multinucleated myotubes. Immunostaining reveals the co-localization of the molecular chaperones Hsc70 and Hsp90 with the GFP-myosin in the intermediates, but not in the mature myofibrils. Uninfected cells have similar intermediates suggesting a common pathway for myosin maturation. Two conformation-sensitive antibodies that bind the unfolded motor domain and the coiled-coil conformation of the rod demonstrate that in the intermediates, the myosin rod is folded but the motor domain is not folded. Electron microscopy reveals that the intermediates contain loose filament bundles surrounded by a protein rich matrix. Geldanamycin, a specific inhibitor of Hsp90, reversibly blocks myofibril assembly and triggers accumulation of myosin folding intermediates. We conclude that multimeric complexes of nascent myosin filaments associated with Hsc70 and Hsp90 are intermediates in the folding and assembly pathway of muscle myosin.

Packing, specificity, and mutability at the binding interface between the p160 coactivator and CREB‐binding protein

Among the most common interaction motifs between nuclear proteins is the recognition of one or more amphipathic helices. In an effort to determine principles behind this recognition, we have investigated the interaction between the p160 coactivator protein ACTR and the ACTR-binding domain of the CREB-binding protein, CBP. The two proteins use relatively small portions of their primary sequences to form a single synergistically folded domain consisting of six intertwined alpha-helices, three from each protein. Neither of the component polypeptides forms a cooperatively folded domain in isolation. However, a considerable amount of residual secondary structure remains in the isolated CBP domain according to CD spectroscopy. Chemical denaturation, differential scanning calorimetry, and ANS binding experiments demonstrate that the isolated CBP domain is not entirely unfolded but forms a helical state with the characteristics of a molten globule. Mutations probing the functional and energetic significance of a buried intermolecular Arg-Asp salt bridge in the interface of the protein complex suggest that these residues are tuned for functional discrimination and not strictly for binding affinity or stability. These results suggest a mechanism for formation of the complex where the unfolded ACTR domain interacts with the partly folded CBP domain in a rapid and specific manner to form the final stable complex.

Structural Characterization by NMR of the Natively Unfolded Extracellular Domain of β-Dystroglycan: Toward the Identification of the Binding Epitope for α-Dystroglycan

Dystroglycan (DG) is an adhesion molecule playing a crucial role for tissue stability during both early embriogenesis and adulthood and is composed by two tightly interacting subunits: alpha-DG, membrane-associated and highly glycosylated, and the transmembrane beta-DG. Recently, by solid-phase binding assays and NMR experiments, we have shown that the C-terminal domain of alpha-DG interacts with a recombinant extracellular fragment of beta-DG (positions 654-750) independently from glycosylation and that the linear binding epitope is located between residues 550 and 565 of alpha-DG. In order to elucidate which moieties of beta-DG are specifically involved in the complex with alpha-DG, the ectodomain has been recombinantly expressed and purified in a labeled ((13)C,(15)N) form and studied by multidimensional NMR. Although it represents a natively unfolded protein domain, we obtained an almost complete backbone assignment. Chemical shift index, (1)H-(15)N heteronuclear single-quantum coherence and nuclear Overhauser effect (HSQC-NOESY) spectra and (3)J(HN,H)(alpha) coupling constant values confirm that this protein is highly disordered, but (1)H-(15)N steady-state NOE experiments indicate that the protein presents two regions of different mobility. The first one, between residues 659 and 722, is characterized by a limited degree of mobility, whereas the C-terminal portion, containing about 30 amino acids, is highly flexible. The binding of beta-DG(654-750) to the C-terminal region of the alpha subunit, alpha-DG(485-620), has been investigated, showing that the region of beta-DG(654-750) between residues 691 and 719 is involved in the interaction.

How Bacterial Ribosomal Protein L20 Assembles with 23 S Ribosomal RNA and Its Own Messenger RNA

In bacteria, the expression of ribosomal proteins is often feedback-regulated at the translational level by the binding of the protein to its own mRNA. This is the case for L20, which binds to two distinct sites of its mRNA that both resemble its binding site on 23 S rRNA. In the present work, we report an NMR analysis of the interaction between the C-terminal domain of L20 (L20C) and both its rRNA- and mRNA-binding sites. Changes in the NMR chemical shifts of the L20C backbone nuclei were used to show that the same set of residues are modified upon addition of either the rRNA or the mRNA fragments, suggesting a mimicry at the atomic level. In addition, small angle x-ray scattering experiments, performed with the rRNA fragment, demonstrated the formation of a complex made of two RNAs and two L20C molecules. A low resolution model of this complex was then calculated using (i) the rRNA/L20C structure in the 50 S context and (ii) NMR and small angle x-ray scattering results. The formation of this complex is interesting in the context of gene regulation because it suggests that translational repression could be performed by a complex of two proteins, each interacting with the two distinct L20-binding sites within the operator.

Modulation of the ATPase Cycle of BiP by Peptides and Proteins

BiP, the Hsp70 homologue of the endoplasmic reticulum, interacts with its non-native substrate proteins in an ATP-dependent manner. This interaction is coupled to the ATPase cycle of the chaperone. Binding of short, synthetic peptides stimulate the ATPase activity of BiP. In previous work, we showed that a stably unfolded antibody domain forms a binary complex with BiP. In this study we made use of this complex to analyse the effect of substrate proteins on the ATPase cycle of BiP. Kinetic constants of the partial reactions of the ATPase cycle were determined without substrate, in the presence of a short binding peptide and in the presence of the antibody domain. We show that, in contrast to smaller peptides, the non-native protein domain decelerates the rate limiting hydrolysis step of the ATPase cycle.

Concerted Folding and Binding of a Flexible Colicin Domain to Its Periplasmic Receptor TolA

Compared with folded structures, natively unfolded protein domains are over-represented in protein-protein and protein-DNA interactions. Such domains are common features of all colicins and are required for their translocation across the outer membrane of the target Escherichia coli cell. All of these domains bind to at least one periplasmic protein of the Tol or Ton family. Similar domains are found in Ton-dependent outer membrane transporters, indicating they may interact in a related manner. In this article we have studied binding of the colicin N translocation domain to its periplasmic receptor TolA, by fluorescence resonance energy transfer (FRET) using fluorescent probes attached to engineered cysteine residues and NMR techniques. The domain exhibits a random coil circular dichroism spectrum. However, FRET revealed that guanidinium hydrochloride denaturation caused increases in all measured intramolecular distances showing that, although natively unfolded, the domain is not extended. Furthermore NMR reported a compact hydrodynamic radius of 18 A. Nevertheless the FRET-derived distances changed upon binding to TolA indicating a significant structural rearrangement. Using 1H-15N NMR we show that, when bound, the peptide switches from a disordered state to an ordered state. The kinetics of binding and the associated structural change were measured by stopped-flow methods, and both events appear to occur simultaneously. The data therefore suggest that this molecular recognition involves the concerted binding and folding of a flexible but collapsed state.

Folding–unfolding of FN-III domains in tenascin: an elastically coupled two-state system

In a single-molecule atomic force microscopy (AFM) experiment, the tenascin molecule is stretched by an external force causing an elongation which is due to the unfolding of the FN-III modules. The features of the force-extension curves depend on the pulling speed and show a saw-tooth pattern (lower speeds) or a smooth pattern (higher speeds). In any case, the unfolded domains are elastically coupled to the unfolded modules, acting as transmitters of the external force. In this communication, the folding–unfolding process of the FN-III domains in tenascin is studied using reaction rate theory and a simple two-state model. The main hypothesis of the study is that, at microscopic level, the force needed to unfold a domain and the unfolding rate (unfolding velocity) can mimic the macroscopic process of measurement by AFM. As the external force is applied, the probability of unfolding increases as dictated by the reaction rate theory. Within this context, a relationship between the unfolding force and the unfolding velocity is obtained. The latter relation will describe microscopically the process in a phenomenological fashion. Moreover, while relating the results of this study with other experimental (AFM measurements) and theoretical (Monte Carlo simulations) data, we found that the graph of unfolding force-unfolding velocity is similar to that of external force-pulling velocity. The refolding process can also be studied within this model and the results show similar trends. The latter suggests a generic and universal behavior of such kind of molecular domains at least in the light of the proposed model.

Quaternary structure of aldolase leads to differences in its folding and unfolding intermediates.

Pulsed hydrogen exchange mass spectrometry has been used to investigate folding of rabbit muscle aldolase, an alpha/beta-barrel protein exhibiting the classic TIM structure. Aldolase unfolded in GdHCl refolded as the denaturant concentration was reduced by dialysis. Samples withdrawn during dialysis were pulse-labeled with deuterium to identify unfolded regions in structural forms highly populated during the folding process. Intact, labeled aldolase was digested into fragments, which were analyzed by HPLC electrospray ionization mass spectrometry to detect the H/D exchange along the aldolase backbone. For some concentrations of GdHCl, bimodal distributions of deuterium were found for most peptic fragments, indicating that regions represented by these fragments were either unfolded or folded in the intact polypeptide prior to labeling. The extent of folding was determined from these mass spectra, as well as by CD (220 nm) and enzymatic activity. These results show that folding to the active form involves three domains and two intermediates. Approximately 110 residues fold to highly compact forms in each step. These results also show that each folding domain includes widely separated regions of the backbone. When compared with the results of a previous study of aldolase unfolding, these results show that the folding and unfolding domains include most of the same residues. However, three short segments change domains depending on whether the process is folding or unfolding. These changes are attributed to the very stable quaternary structure of rabbit muscle aldolase.

Structural Analysis of Bacillus subtilis SPP1 Phage Helicase Loader Protein G39P

The Bacillus subtilis SPP1 phage-encoded protein G39P is a loader and inhibitor of the phage G40P replicative helicase involved in the initiation of DNA replication. We have carried out a full x-ray crystallographic and preliminary NMR analysis of G39P and functional studies of the protein, including assays for helicase binding by a number of truncated mutant forms, in an effort to improve our understanding of how it both interacts with the helicase and with the phage replisome organizer, G38P. Our structural analyses reveal that G39P has a completely unexpected bipartite structure comprising a folded N-terminal domain and an essentially unfolded C-terminal domain. Although G39P has been shown to bind its G40P target with a 6:6 stoichiometry, our crystal structure and other biophysical characterization data reveal that the protein probably exists predominantly as a monomer in solution. The G39P protein is proteolytically sensitive, and our binding assays show that the C-terminal domain is essential for helicase interaction and that removal of just the 14 C-terminal residues abolishes interaction with the helicase in vitro. We propose a number of possible scenarios in which the flexibility of the C-terminal domain of G39P and its proteolytic sensitivity may have important roles for the function of G39P in vivo that are consistent with other data on SPP1 phage DNA replication.