Global Fold Research Articles

Mapping the Conformational Stability of Maltose Binding Protein at the Residue Scale Using Nuclear Magnetic Resonance Hydrogen Exchange Experiments

Being able to differentiate local fluctuations from global folding-unfolding dynamics of a protein is of major interest for improving our understanding of structure-function determinants. The maltose binding protein (MBP), a protein that belongs to the maltose transport system, has a structure composed of two globular domains separated by a rigid-body "hinge bending". Here we determined, by using hydrogen exchange (HX) nuclear magnetic resonance experiments, the apparent stabilization free energies of 101 residues of MBP bound to β-cyclodextrin (MBP-βCD) under native conditions. We observed that the last helix of MBP (helix α14) has a lower protection factor than the rest of the protein. Further, HX experiments were performed using guanidine hydrochloride under subdenaturing conditions to discriminate between local fluctuations and global unfolding events and to determine the MBP-βCD energy landscape. The results show that helix α4 and a part of helices α5 and α6 are clearly grouped into a subdenaturing folding unit and represent a partially folded intermediate under native conditions. In addition, we observed that amide protons located in the hinge between the two globular domains share similar ΔG(gu)(app) and m values and should unfold simultaneously. These observations provide new points of view for improving our understanding of the thermodynamic stability and the mechanisms that drive folding-unfolding dynamics of proteins.

Rapid protein global fold determination using ultrasparse sampling, high-dynamic range artifact suppression, and time-shared NOESY.

In structural studies of large proteins by NMR, global fold determination plays an increasingly important role in providing a first look at a target's topology and reducing assignment ambiguity in NOESY spectra of fully protonated samples. In this work, we demonstrate the use of ultrasparse sampling, a new data processing algorithm, and a 4-D time-shared NOESY experiment (1) to collect all NOEs in (2)H/(13)C/(15)N-labeled protein samples with selectively protonated amide and ILV methyl groups at high resolution in only four days, and (2) to calculate global folds from this data using fully automated resonance assignment. The new algorithm, SCRUB, incorporates the CLEAN method for iterative artifact removal but applies an additional level of iteration, permitting real signals to be distinguished from noise and allowing nearly all artifacts generated by real signals to be eliminated. In simulations with 1.2% of the data required by Nyquist sampling, SCRUB achieves a dynamic range over 10000:1 (250× better artifact suppression than CLEAN) and completely quantitative reproduction of signal intensities, volumes, and line shapes. Applied to 4-D time-shared NOESY data, SCRUB processing dramatically reduces aliasing noise from strong diagonal signals, enabling the identification of weak NOE crosspeaks with intensities 100× less than those of diagonal signals. Nearly all of the expected peaks for interproton distances under 5 Å were observed. The practical benefit of this method is demonstrated with structure calculations for 23 kDa and 29 kDa test proteins using the automated assignment protocol of CYANA, in which unassigned 4-D time-shared NOESY peak lists produce accurate and well-converged global fold ensembles, whereas 3-D peak lists either fail to converge or produce significantly less accurate folds. The approach presented here succeeds with an order of magnitude less sampling than required by alternative methods for processing sparse 4-D data.

F09 The study on molecular changes related to energy metabolism in Huntington's disease subjects: looking for biomarkers

Huntington's disease (HD) is a neurodegenerative disorder characterised by a progressive motor and cognitive decline and psychiatric symptoms. The origin of molecular and biochemical disturbances in HD is the genetic...

Analyte-Driven Switching of DNA Charge Transport: De Novo Creation of Electronic Sensors for an Early Lung Cancer Biomarker

A general approach is described for the de novo design and construction of aptamer-based electrochemical biosensors, for potentially any analyte of interest (ranging from small ligands to biological macromolecules). As a demonstration of the approach, we report the rapid development of a made-to-order electronic sensor for a newly reported early biomarker for lung cancer (CTAP III/NAP2). The steps include the in vitro selection and characterization of DNA aptamer sequences, design and biochemical testing of wholly DNA sensor constructs, and translation to a functional electrode-bound sensor format. The working principle of this distinct class of electronic biosensors is the enhancement of DNA-mediated charge transport in response to analyte binding. We first verify such analyte-responsive charge transport switching in solution, using biochemical methods; successful sensor variants were then immobilized on gold electrodes. We show that using these sensor-modified electrodes, CTAP III/NAP2 can be detected with both high specificity and sensitivity (K(d) ~1 nM) through a direct electrochemical reading. To investigate the underlying basis of analyte binding-induced conductivity switching, we carried out Förster Resonance Energy Transfer (FRET) experiments. The FRET data establish that analyte binding-induced conductivity switching in these sensors results from very subtle structural/conformational changes, rather than large scale, global folding events. The implications of this finding are discussed with respect to possible charge transport switching mechanisms in electrode-bound sensors. Overall, the approach we describe here represents a unique design principle for aptamer-based electrochemical sensors; its application should enable rapid, on-demand access to a class of portable biosensors that offer robust, inexpensive, and operationally simplified alternatives to conventional antibody-based immunoassays.

Characterization and optimization of ArtinM lectin expression in Escherichia coli

BackgroundArtinM is a d-mannose-specific lectin from Artocarpus integrifolia seeds that induces neutrophil migration and activation, degranulation of mast cells, acceleration of wound healing, induction of interleukin-12 production by macrophages and dendritic cells, and protective T helper 1 immune response against Leishmania major, Leishmania amazonensis and Paracoccidioides brasiliensis infections. Considering the important biological properties of ArtinM and its therapeutic applicability, this study was designed to produce high-level expression of active recombinant ArtinM (rArtinM) in Escherichia coli system.ResultsThe ArtinM coding region was inserted in pET29a(+) vector and expressed in E. coli BL21(DE3)-Codon Plus-RP. The conditions for overexpression of soluble ArtinM were optimized testing different parameters: temperatures (20, 25, 30 or 37°C) and shaking speeds (130, 200 or 220 rpm) during induction, concentrations of the induction agent IPTG (0.01-4 mM) and periods of induction (1-19 h). BL21-CodonPlus(DE3)-RP cells induced under the optimized conditions (incubation at 20°C, at a shaking speed of 130 rpm, induction with 0.4 mM IPTG for 19 h) resulted in the accumulation of large amounts of soluble rArtinM. The culture provided 22.4 mg/L of rArtinM, which activity was determined by its one-step purification through affinity chromatography on immobilized d-mannose and glycoarray analysis. Gel filtration showed that rArtinM is monomeric, contrasting with the tetrameric form of the plant native protein (jArtinM). The analysis of intact rArtinM by mass spectrometry revealed a 16,099.5 Da molecular mass, and the peptide mass fingerprint and esi-cid-ms/ms of amino acid sequences of peptides from a tryptic digest covered 41% of the total ArtinM amino acid sequence. In addition, circular dichroism and fluorescence spectroscopy of rArtinM indicated that its global fold comprises β-sheet structure.ConclusionsOverall, the optimized process to express rArtinM in E. coli provided high amounts of soluble, correctly folded and active recombinant protein, compatible with large scale production of the lectin.

Monitoring cotranslational protein folding in mammalian cells at codon resolution

How the ribosome-bound nascent chain folds to assume its functional tertiary structure remains a central puzzle in biology. In contrast to refolding of a denatured protein, cotranslational folding is complicated by the vectorial nature of nascent chains, the frequent ribosome pausing, and the cellular crowdedness. Here, we present a strategy called folding-associated cotranslational sequencing that enables monitoring of the folding competency of nascent chains during elongation at codon resolution. By using an engineered multidomain fusion protein, we demonstrate an efficient cotranslational folding immediately after the emergence of the full domain sequence. We also apply folding-associated cotranslational sequencing to track cotranslational folding of hemagglutinin in influenza A virus-infected cells. In contrast to sequential formation of distinct epitopes, the receptor binding domain of hemagglutinin follows a global folding route by displaying two epitopes simultaneously when the full sequence is available. Our results provide direct evidence of domain-wise global folding that occurs cotranslationally in mammalian cells.

Fibrillation precursor of superoxide dismutase 1 revealed by gradual tuning of the protein-folding equilibrium

Although superoxide dismutase 1 (SOD1) stands out as a relatively soluble protein in vitro, it can be made to fibrillate by mechanical agitation. The mechanism of this fibrillation process is yet poorly understood, but attains considerable interest due to SOD1's involvement in the neurodegenerative disease amyotrophic lateral sclerosis (ALS). In this study, we map out the apoSOD1 fibrillation process from how it competes with the global folding events at increasing concentrations of urea: We determine how the fibrillation lag time (τ(lag)) and maximum growth rate (ν(max)) depend on gradual titration of the folding equilibrium, from the native to the unfolded state. The results show that the agitation-induced fibrillation of apoSOD1 uses globally unfolded precursors and relies on fragmentation-assisted growth. Mutational screening and fibrillation m-values (∂logτ(lag)/∂[urea] and ∂logν(max)/∂[urea]) indicate moreover that the fibrillation pathway proceeds via a diffusely bound transient complex that responds to the global physiochemical properties of the SOD1 sequence. Fibrillation of apoSOD1, as it bifurcates from the denatured ensemble, seems thus mechanistically analogous to that of disordered peptides, save the competing folding transition to the native state. Finally, we examine by comparison with in vivo data to what extent this mode of fibrillation, originating from selective amplification of mechanically brittle aggregates by sample agitation, captures the mechanism of pathological SOD1 aggregation in ALS.

Protein conformational switches: from nature to design.

Protein conformational switches alter their shape upon receiving an input signal, such as ligand binding, chemical modification, or change in environment. The apparent simplicity of this transformation--which can be carried out by a molecule as small as a thousand atoms or so--belies its critical importance to the life of the cell as well as its capacity for engineering by humans. In the realm of molecular switches, proteins are unique because they are capable of performing a variety of biological functions. Switchable proteins are therefore of high interest to the fields of biology, biotechnology, and medicine. These molecules are beginning to be exploited as the core machinery behind a new generation of biosensors, functionally regulated enzymes, and "smart" biomaterials that react to their surroundings. As inspirations for these designs, researchers continue to analyze existing examples of allosteric proteins. Recent years have also witnessed the development of new methodologies for introducing conformational change into proteins that previously had none. Herein we review examples of both natural and engineered protein switches in the context of four basic modes of conformational change: rigid-body domain movement, limited structural rearrangement, global fold switching, and folding-unfolding. Our purpose is to highlight examples that can potentially serve as platforms for the design of custom switches. Accordingly, we focus on inducible conformational changes that are substantial enough to produce a functional response (e.g., in a second protein to which it is fused), yet are relatively simple, structurally well-characterized, and amenable to protein engineering efforts.

Theory of the Molecular Transfer Model for Proteins with Applications to the Folding of the src-SH3 Domain

A theoretical basis for the molecular transfer model (MTM), which takes into account the effects of denaturants by combining experimental data and molecular models for proteins, is provided. We show that the MTM is a mean field-like model that implicitly takes into account denaturant-induced many body interactions. The MTM in conjunction with the coarse-grained self organized polymer model with side chains (SOP-SC) for polypeptide chains is used to simulate the folding of the src-SH3 domain as a function of temperature (T) and guanidine hydrochloride (GdmCl) concentration [C]. Besides reproducing the thermodynamic aspects of SH3 folding, the SOP-SC also captures the cooperativity of the folding transitions. A number of experimentally testable predictions are also made. First, we predict that the melting temperature T(m)([C]) decreases linearly as [C] increases. Second, we show that the midpoints C(m,i) and melting temperatures T(m,i) at which individual residues acquire 50% of their native contacts differ from the global midpoint (C(m) ≈ 2.5 M) and melting temperature (T(m) = 355 K) at which the folded and unfolded states coexist. Dispersion in C(m,i) is greater than that found for T(m,i). Third, folding kinetics at [C] = 0 M shows that the acquisition of contacts between all the secondary structural elements and global folding occur nearly simultaneously. Finally, from the free energy profiles as a function of the structural overlap function and the radius of gyration of the protein, we find that at a fixed T the transition state moves toward the folded state as [C] increases in accord with the Hammond postulate. In contrast, we predict that along the locus of points T(m)([C]) the location of the transition state does not change. The theory and the models used here are sufficiently general for studying the folding of other single domain proteins.

Mapping of unfolding states of integral helical membrane proteins by GPS-NMR and scattering techniques: TFE-induced unfolding of KcsA in DDM surfactant

Membrane proteins are vital for biological function, and their action is governed by structural properties critically depending on their interactions with the membranes. This has motivated considerable interest in studies of membrane protein folding and unfolding. Here the structural changes induced by unfolding of an integral membrane protein, namely TFE-induced unfolding of KcsA solubilized by the n-dodecyl β-d-maltoside (DDM) surfactant is investigated by the recently introduced GPS-NMR (Global Protein folding State mapping by multivariate NMR) (Malmendal et al., PlosONE 5, e10262 (2010)) along with dynamic light scattering (DLS) and small-angle X-ray scattering (SAXS). GPS-NMR is used as a tool for fast analysis of the protein unfolding processes upon external perturbation, and DLS and SAXS are used for further structural characterization of the unfolding states. The combination allows addressing detergent properties and protein conformations at the same time. The mapping of the states reveals that KcsA undergoes a series of rearrangements which include expansion of the tetramer in several steps followed by dissociation into monomers at 29% TFE. Supplementary studies of DDM and TFE in the absence of KcsA suggest that the disintegration of the tetramer at 29% TFE is caused by TFE dissolving the surrounding DDM rim. Above 34% TFE, KcsA collapses to a new structure that is fully formed at 44% TFE.

Protein fold determined by paramagnetic magic-angle spinning solid-state NMR spectroscopy

Biomacromolecules that are challenging for the usual structural techniques can be studied with atomic resolution by solid-state nuclear magnetic resonance. However, the paucity of >5 Å distance restraints, traditionally derived from measurements of magnetic dipole-dipole couplings between protein nuclei, is a major bottleneck that hampers such structure elucidation efforts. Here we describe a general approach that enables the rapid determination of global protein fold in the solid phase via measurements of nuclear paramagnetic relaxation enhancements (PREs) in several analogs of the protein of interest containing covalently-attached paramagnetic tags, without the use of conventional internuclear distance restraints. The method is demonstrated using six cysteine-EDTA-Cu2+ mutants of the 56-residue B1 immunoglobulin-binding domain of protein G, for which ~230 longitudinal backbone 15N PREs corresponding to ~10-20 Å distances were obtained. The mean protein fold determined in this manner agrees with the X-ray structure with a backbone atom root-mean-square deviation of 1.8 Å.

Efficient acquisition of high-resolution 4-D diagonal-suppressed methyl–methyl NOESY for large proteins

The methyl–methyl NOESY experiment plays an important role in determining the global folds of large proteins. Despite the high sensitivity of this experiment, the analysis of methyl–methyl NOEs is frequently hindered by the limited chemical shift dispersion of methyl groups, particularly methyl protons. This makes it difficult to unambiguously assign all of the methyl–methyl NOE crosspeaks using 3-D spectroscopy. The recent development of sparse sampling methods enables highly efficient acquisition of high-resolution 4-D spectra, which provides an excellent solution to resolving the degeneracy of methyl signals. However, many reconstruction algorithms for processing sparsely-sampled NMR data do not provide adequate suppression of aliasing artifacts in the presence of strong NOE diagonal signals. In order to overcome this limitation, we present a 4-D diagonal-suppressed methyl–methyl NOESY experiment specifically optimized for ultrasparse sampling and evaluate it using a deuterated, ILV methyl-protonated sample of the 42kDa Escherichia coli maltose binding protein (MBP). Suppression of diagonal signals removes the dynamic range barrier of the methyl–methyl NOESY experiment such that residual aliasing artifacts in the CLEAN-reconstructed high-resolution 4-D spectrum can be further reduced. At an ultrasparse sampling rate of less than 1%, we were able to identify and unambiguously assign the vast majority of expected NOE crosspeaks between methyl groups separated by less than 5Å and to detect very weak NOE crosspeaks from methyl groups that are over 7Å apart.

Functional Prediction of Binding Pockets

Putative function for targets with no known ligands has typically been determined from liganded homologous proteins using sequence and structure comparisons. However, it is debatable what percentage of sequence identity implies similar function, whereas structural similarity is focused on global folds and could miss divergent structures and novel global folds. The present study describes an approach to classify a diverse set of proteins and predict their function. Descriptors corresponding to structural, physicochemical, and geometric properties of the ligand-binding cavities of a collection of 434 complexes (17 protein families) were calculated and analyzed by statistical methods. The best model using discriminant function analysis (DFA) consisted of 371 proteins (15 families) and had correct classification rates of 90% and cross-validation 86%. DFA with one protein and a random sample of the remaining proteins led to 100% correct prediction of putative protein function for 10 of the 15 protein families.

Simulations of Tryptophan Fluorescence Dynamics during Folding of the Villin Headpiece

Protein folding kinetics is commonly monitored by changes in tryptophan (Trp) fluorescence intensity. Considerable recent discussion has centered on whether the fluorescence of the single Trp in the much-studied, fast-folding villin headpiece C-terminal domain (HP35) accurately reflects folding kinetics, given the general view that quenching is by a histidine cation (His(+)) one turn away in an α-helix (helix III) that forms early in the folding process, according to published MD simulations. To help answer this question, we ran 1.0 μs MD simulations on HP35 (N27H) and a faster-folding variant in its folded form at 300 K and used the coordinates and force field charges with quantum calculations to simulate fluorescence quenching caused by electron transfer to the local amide and to the His(+). The simulations demonstrate that quenching by His(+) in the fully formed helix III is possible only during certain Trp and His(+) rotamer and solvent conformations, the propensity of which is a variable that can allow Trp fluorescence to report the global folding rate, as recent experiments imply.

Fast and accurate modeling of protein-protein interactions by combining template-interface-based docking with flexible refinement.

The similarity between folding and binding led us to posit the concept that the number of protein-protein interface motifs in nature is limited, and interacting protein pairs can use similar interface architectures repeatedly, even if their global folds completely vary. Thus, known protein-protein interface architectures can be used to model the complexes between two target proteins on the proteome scale, even if their global structures differ. This powerful concept is combined with a flexible refinement and global energy assessment tool. The accuracy of the method is highly dependent on the structural diversity of the interface architectures in the template dataset. Here, we validate this knowledge-based combinatorial method on the Docking Benchmark and show that it efficiently finds high-quality models for benchmark complexes and their binding regions even in the absence of template interfaces having sequence similarity to the targets. Compared to "classical" docking, it is computationally faster; as the number of target proteins increases, the difference becomes more dramatic. Further, it is able to distinguish binders from nonbinders. These features allow performing large-scale network modeling. The results on an independent target set (proteins in the p53 molecular interaction map) show that current method can be used to predict whether a given protein pair interacts. Overall, while constrained by the diversity of the template set, this approach efficiently produces high-quality models of protein-protein complexes. We expect that with the growing number of known interface architectures, this type of knowledge-based methods will be increasingly used by the broad proteomics community.

An energetically beneficial leader–linker interaction abolishes ligand-binding cooperativity in glycine riboswitches

Comprised of two aptamers connected by a short nucleotide linker, the glycine riboswitch was the first example of naturally occurring RNA elements reported to bind small organic molecules cooperatively. Earlier works have shown binding of glycine to the second aptamer allows tertiary interactions to be made between the two aptamers, which facilitates binding of a separate glycine molecule to the first aptamer, leading to glycine-binding cooperativity. Prompted by a distinctive protection pattern in the linker region of a minimal glycine riboswitch construct, we have identified a highly conserved (>90%) leader-linker duplex involving leader nucleotides upstream of the previously reported consensus glycine riboswitch sequences. In >50% of the glycine riboswitches, the leader-linker interaction forms a kink-turn motif. Characterization of three glycine ribsowitches showed that the leader-linker interaction improved the glycine-binding affinities by 4.5- to 86-fold. In-line probing and native gel assays with two aptamers in trans suggested synergistic action between glycine-binding and interaptamer interaction during global folding of the glycine riboswitch. Mutational analysis showed that there appeared to be no ligand-binding cooperativity in the glycine riboswitch when the leader-linker interaction is present, and the previously measured cooperativity is simply an artifact of a truncated construct missing the leader sequence.

Folding of the hammerhead ribozyme: Pyrrolo-cytosine fluorescence separates core folding from global folding and reveals a pH-dependent conformational change

The catalytic activity of the hammerhead ribozyme is limited by its ability to fold into the native tertiary structure. Analysis of folding has been hampered by a lack of assays that can independently monitor the environment of nucleobases throughout the ribozyme-substrate complex in real time. Here, we report the development and application of a new folding assay in which we use pyrrolo-cytosine (pyC) fluorescence to (1) probe active-site formation, (2) examine the ability of peripheral ribozyme domains to support native folding, (3) identify a pH-dependent conformational change within the ribozyme, and (4) explore its influence on the equilibrium between the folded and unfolded core of the hammerhead ribozyme. We conclude that the natural ribozyme folds in two distinct noncooperative steps and the pH-dependent correlation between core folding and activity is linked to formation of the G8-C3 base pair.

Strategies for the Solution NMR Structure Determination of Beta-Barrel Membrane Proteins

To date, a handful of β-barrel membrane proteins have been determined with solution nuclear magnetic spectroscopy (NMR) with the first reported in 2001. The bottlenecks seem to be protein sample preparation and obtaining near-complete assignment. In the process of determining the structures of two β-barrel membrane proteins, Opa50 and Opa60 from Neisseria gonorrhoeae, strategies for obtaining quality NMR spectra and backbone assignments were established. The effects of detergents on the global fold and NMR spectral quality were determined. Although all detergents stabilized the overall fold (after refolding in dodecyphosphocholine), there were variable effects on the NMR spectra. In particular, the resonances corresponding to the β-barrel were less affected than the loop regions indicating an interaction between some detergent micelles and the Opa extracellular loops. Once quality NMR spectra are obtained, the next task for NMR structure determination is the backbone assignment. For the Opa proteins, the amino acid content and the crowding in the random coil region of the spectrum (>50% of the protein is comprised by the extracellular loops) has created difficulties in obtaining a comprehensive assignment for structure determination. Several strategies that circumvent these difficulties will be presented.

Computational Predictions of Exponential and Non-Exponential Tryptophan Fluorescence Decay in NATA, the Villin Headpiece Subdomain, and other Proteins

Decay of fluorescence from a single Tryptophan (Trp) in a peptide or protein is sometimes exponential and sometimes non-exponential. Understanding the details of this behavior has proven elusive. We have had qualitative success predicting average fluorescence quantum yields for a variety of proteins based on the stabilization of the charge transfer (CT) state arising from electron transfer from the aromatic ring to a backbone amide by the protein electrostatic environment. Here we report 100ns - 1 microsecond MD simulations, augmented by quantum mechanical (ZINDO) computations of the fluorescing and CT states, on N-acetyl-tryptophan-amide (NATA) and 20 single Trp proteins, including the highly studied fast folding villin headpiece. Although the environment for NATA in water is very similar to that of solvent exposed Trps on the surface of a protein, which almost always exhibit non-exponential decay, NATA shows a surprisingly pure single-exponential decay. In our simulations, all possible rotamer states are well represented, and transitions between rotamers happen at a rate of about 1 per 5 ns. Preliminary results indicate that rare conformations in which an amide carbonyl is H-bonded by 2-3 waters produce spikes of high quenching, more or less independent of rotamer state. Survival curve averaging of the 600 ns trajectory yields a single exponential decay of near 3 ns. For folded villin at 300 K, we find that rotamer transitions on Trp occur only every ∼100 ns and that quenching by the nearby His+ happens only during these transient events, although the helix is always intact. Villin is therefore predicted to show extreme heterogeneity in lifetimes. This provides a mechanism for Trp fluorescence to report the global folding rate. Heterogeneity for the other proteins will also be discussed.

PoSSuM: a database of similar protein-ligand binding and putative pockets

Numerous potential ligand-binding sites are available today, along with hundreds of thousands of known binding sites observed in the PDB. Exhaustive similarity search for such vastly numerous binding site pairs is useful to predict protein functions and to enable rapid screening of target proteins for drug design. Existing databases of ligand-binding sites offer databases of limited scale. For example, SitesBase covers only ∼33 000 known binding sites. Inferring protein function and drug discovery purposes, however, demands a much more comprehensive database including known and putative-binding sites. Using a novel algorithm, we conducted a large-scale all-pairs similarity search for 1.8 million known and potential binding sites in the PDB, and discovered over 14 million similar pairs of binding sites. Here, we present the results as a relational database Pocket Similarity Search using Multiple-sketches (PoSSuM) including all the discovered pairs with annotations of various types. PoSSuM enables rapid exploration of similar binding sites among structures with different global folds as well as similar ones. Moreover, PoSSuM is useful for predicting the binding ligand for unbound structures, which provides important clues for characterizing protein structures with unclear functions. The PoSSuM database is freely available at http://possum.cbrc.jp/PoSSuM/.