Dissecting the non-specific and specific components of the initial folding reaction of barstar by multi-site FRET measurements

Initial polypeptide chain collapse plays a major role in the development of subsequent structure during protein folding, but it has been difficult to elucidate the coupling between its cooperativity and specificity. To better understand this important aspect of protein folding, nine different intramolecular distances in the protein have been measured by fluorescence resonance energy transfer (FRET) in the product(s) of the initial, sub-millisecond collapse reaction during the folding of barstar, under different folding conditions. All nine distances contract in these initial folding products, when the denaturant concentration is reduced. Two of these distances were also measured in peptides corresponding to sequence segments 38-55 and 51-69 of the protein. Surprisingly, both distances do not contract in the peptides which remain fully unfolded when the denaturant concentration is reduced. This suggests that the contraction of at least some segments of the polypeptide chain may be facilitated only by contraction of other segments. In the case of the initial product of folding of the protein, the dependence on denaturant concentration of the relative change in each distance suggests that there are two components to the initial folding reaction. One is a nonspecific component, which appears to be driven by the change in denaturant concentration that is used to initiate refolding. This component corresponds to the collapse of completely unfolded protein (U) to unfolded protein in refolding conditions (U(C)). The extent of nonspecific collapse can be predicted by the response of completely unfolded protein to a change in denaturant concentration. All distances undergo such solvent-induced contraction, but each distance contracts to a different extent. There is also a specific component to initial sub-millisecond folding, in which some distances (but not all) contract more than that predicted by solvent-induced contraction. The observation that only some of the distances undergo contraction over and above solvent-induced contraction, suggest that this specific component is associated with the formation of a specific intermediate (I(E)). FRET efficiency and distance change differently for the different donor-acceptor pairs, with a change in denaturant concentration, indicating that the formation or dissolution of structure in U(C) and I(E) does not happen in a synchronized manner across different regions of the protein molecule. Also, all nine FRET efficiencies and intramolecular distances in the product(s) of sub-ms folding, change continuously with a change in denaturant concentration. Hence, it appears that the transitions from U to U(C) and to I(E) are gradual transformations, and not all-or-none structural transitions. Nevertheless, the product of these gradual transitions, I(E), possesses specific structure.     

Kinetic studies of the folding of heterodimeric monellin: evidence for switching between alternative parallel pathways

Determining whether or not a protein uses multiple pathways to fold is an important goal in protein folding studies. When multiple pathways are present, defined by transition states that differ in their compactness and structure but not significantly in energy, they may manifest themselves by causing the dependence on denaturant concentration of the logarithm of the observed rate constant of folding to have an upward curvature. In this study, the folding mechanism of heterodimeric monellin [double-chain monellin (dcMN)] has been studied over a range of protein and guanidine hydrochloride (GdnHCl) concentrations, using the intrinsic tryptophan fluorescence of the protein as the probe for the folding reaction. Refolding is shown to occur in multiple kinetic phases. In the first stage of refolding, which is silent to any change in intrinsic fluorescence, the two chains of monellin bind to one another to form an encounter complex. Interrupted folding experiments show that the initial encounter complex folds to native dcMN via two folding routes. A productive folding intermediate population is identified on one route but not on both of these routes. Two intermediate subpopulations appear to form in a fast kinetic phase, and native dcMN forms in a slow kinetic phase. The chevron arms for both the fast and slow phases of refolding are shown to have upward curvatures, suggesting that at least two pathways each defined by a different intermediate are operational during these kinetic phases of structure formation. Refolding switches from one pathway to the other as the GdnHCl concentration is increased.     

Estimating the fraction of invariable codons with a capture-recapture method

Summary A codon-based approach to estimating the number of variable sites in a protein is presented. When first and second positions of codons are assumed to be replacement positions, a capture-recapture model can be used to estimate the number of variable codons from every pair of homologous and aligned sequences. The capture-recapture estimate is compared to a maximum likelihood estimate of the number of variable codons and to previous approaches that estimate the number of variable sites (not codons) in a sequence. Computer simulations are presented that show under which circumstances the capture-recapture estimate can be used to correct biases in distance matrices. Analysis of published sequences of two genes, calmodulin and serum albumin, shows that distance corrections that employ a capture-recapture estimate of the number of variable sites may be considerably different from corrections that assume that the number of variable sites is equal to the total number of positions in the sequence. Offprint requests to: A. Sidow     

Mapping the molecular structure of the voltage-dependent sodium channel. Distances between the tetrodotoxin and Leiurus quinquestriatus quinquestriatus scorpion toxin receptors

The Leiurus quinquestriatus quinquestriatus receptor site of the voltage-dependent sodium channel has been characterized using several fluorescent scorpion toxins. The derivatives show fluorescence enhancements upon binding to the receptor site on the channel together with blue shifts. The fluorescence properties of the bound probes indicate a conformationally flexible, hydrophobic site. Binding of tetrodotoxin has no effect on the fluorescence spectra of the bound derivatives, whereas binding of the allosteric activator batrachotoxin enhances the fluorescence about 2-fold and causes a red shift in the emission spectra, suggesting a batrachotoxin-induced conformational change in the scorpion toxin receptor. The distance between the tetrodotoxin receptor and the Leiurus scorpion toxin receptor on the channel was measured by fluorescence resonance energy transfer. Five different chromophoric scorpion toxin derivatives were used as energy transfer acceptors or donors with anthraniloyltetrodotoxin or N-methylanthraniloylglycine-tetrodotoxin as the energy donor or acceptor. Because of the presence of three tetrodotoxin receptors for each Leiurus receptor, the positions of the donors and acceptors were exchanged. Efficiencies of transfer were measured by both donor quenching and sensitized emission. The average distance of separation between these sites is 35 A. Upon batrachotoxin addition, this distance changes to 42 A indicating a conformational change in one subunit of the channel or a change in the interaction between two subunits coupled to the batrachotoxin-binding site. On the basis of these studies, we present a model suggesting that tetrodotoxin binds to a subunit/site which is extracellularly placed and is 35 A from the Leiurus subunit/site which is located in a protein cleft of the channel which extends partly into the membrane, and undergoes a neurotoxin and voltage-dependent conformational change.     

Deciphering the language of cells

Long distance cell-to-cell or organism-to-organism communications may be accomplished by transmission and reception of electromagnetic signals through membrane receptors or enzymes. Consistent with this idea is the observation that membrane ATPases are capable of absorbing energy from oscillating electric fields of defined frequency and amplitude and using it to perform chemical work. The concept of the 'electroconformational coupling' is used to explain how an electric signal can modulate the activity of a membrane protein, and conversely, how an energy-dissipating reaction can produce an electric signal.     

Exploiting symmetry properties of the discretizable molecular distance geometry problem

The Discretizable Molecular Distance Geometry Problem (DMDGP) involves a subset of instances of the distance geometry problem for which some assumptions allowing for discretization are satisfied. The search domain for the DMDGP is a binary tree that can be efficiently explored by employing a Branch & Prune (BP) algorithm. We showed in recent works that this binary tree may contain several symmetries, which are directly related to the total number of solutions of DMDGP instances. In this paper, we study the possibility of exploiting these symmetries for speeding up the solution of DMDGPs, and propose an extension of the BP algorithm that we named symmetry-driven BP (symBP). Computational experiments on artificial and protein instances are presented.     

Time-resolved fluorescence spectroscopy of lumazine protein from Photobacterium phosphoreum using synchrotron radiation

Time-resolved fluorescence on lumazine protein from Photobacterium phosphoreum was performed with synchrotron radiation as a source of continuously tunable excitation. The experiments yielded structural and dynamic details from which two aspects became apparent. From fluorescence anisotropy decay monitoring of lumazine fluorescence with different excitation wavelengths, the average correlation times were shown to change, which must indicate the presence of anisotropic motion of the protein. A similar study with 7-oxolumazine as the fluorescent ligand led to comparable results. The other remarkable observation dealt with the buildup of acceptor fluorescence, also observed with 7-oxolumazine although much less pronounced, which is caused by the finite energy transfer process between the single donor tryptophan and the energy accepting lumazine derivatives. Global analytical approaches in data analysis were used to yield realistic correlation times and reciprocal transfer rate constants. It was found that the tryptophan residue has a large motional freedom as also reported previously for this protein and for the related protein from P. leiognathi (Lee et al. 1985; Kulinski et al. 1987). The average distance between the tryptophan residue and the ligand donor-acceptor couple has been determined to be 2.7 nm for the same donor and two different acceptors.     

Resolvability of free energy changes for oxygen binding and subunit association by human hemoglobin.

Probability distributions of the free energy changes for oxygen binding, subunit association, and quaternary enhancement by human hemoglobin were obtained from Monte Carlo simulations performed on two independent sets of variable protein concentration equilibrium oxygen-binding data. Uncertainties in unliganded and fully liganded dimer to tetramer association free energy changes (0 delta G'2 and 4 delta G'2) were accounted for in the simulations. Distributions of the dimer to tetramer association free energy changes for forming singly and triply liganded tetramers (1 delta G'2 and 3 delta G'2) are well defined and quite symmetric, whereas that for forming doubly liganded tetramers (2 delta G'2) is poorly defined and highly asymmetric. The distribution of the dimer stepwise oxygen-binding free-energy change (delta g'2i) is well defined and quite symmetric as are those of the tetramer stepwise oxygen-binding free-energy changes for binding the first and last oxygens to tetramers (delta g'41 and delta g'44). Distributions of the intermediate tetramer stepwise oxygen-binding free-energy changes (delta g'42 and delta g'43) are poorly defined and highly asymmetric, but are compensatory in that their sum (delta g'4[2 + 3]) is again well defined and nearly symmetric. Distributions of the free energy changes corresponding to the tetramer product Adair oxygen binding constants (delta G'4i) are well defined and quite symmetric for i = 1, 3, 4 but not for i = 2. The distribution of delta g'44 - delta g'2i (the quaternary enhancement free energy change) is relatively narrow, nearly symmetric, and confined to the negative free-energy domain. This suggests that the quaternary enhancement free energy change (a) may be resolved with good confidence from this data and (b) is finite and negative under the conditions of these experiments. Our results also suggest two different four-state combinatorial switch models that provide accurate characterization of hemoglobin's functional behavior.     

Dependence of the size of the initially collapsed form during the refolding of barstar on denaturant concentration: evidence for a continuous transition

Two-site fluorescence resonance energy transfer (FRET) measurements have been made to determine how two intra-molecular distances contract in the sub-millisecond collapse reaction that occurs initially during the refolding of the small protein barstar. FRET measurements were made on two, single-Cys and single-Trp-containing mutant forms of barstar, Cys25 and Cys62, in each of which a thionitrobenzoate (TNB) adduct was attached to the cysteine thiol. In each protein, the core tryptophan, Trp53, acted as the FRET donor, and the TNB adduct, located either at C25 or at C62, acted as the FRET acceptor. The stabilities as well as observable folding kinetics of the Cys25 and Cys62 mutant proteins were found to be identical. The presence of the TNB adduct on the cysteine did not alter the stability or folding kinetics of either protein. Thus, the FRET-monitored changes in the two labeled mutant proteins, Cys25-TNB and Cys62-TNB, could be compared directly. Refolding was commenced from unfolded protein in 8M urea, and both the Trp53 to C25-TNB distance and the Trp53 to C62-TNB distance were found to contract upon dilution of urea. The extent of contraction of each distance, which was measured at a few milliseconds of refolding, was dependent continuously on the concentration of urea present during refolding, and was different for the two distances. For either FRET pair, the gradual contraction of distance with a decrease in the concentration of urea in which refolding occurs, was continuous with the contraction of the polypeptide chain that is seen with a decrease in the concentration of urea in the range in which the protein remains completely unfolded. It therefore appears that the products of the initial sub-millisecond refolding reaction of barstar are collapsed forms, whose dimensions do not change cooperatively in an all-or-none manner, but instead, change gradually with a change in concentration of urea. Thus, the sub-millisecond polypeptide chain collapse reaction of barstar upon denaturant dilution, appears to be a continuous structural transition.     

Design of a thermocontrollable protein complex

It is widely recognized that stimuli-responsive nanostructures play a promising role in nanodevices for medical treatments and experimental tools. We have designed and constructed a basic structure which controls the distance between two termini domains through temperature reversibility. Our structure, shaped like a bouquet, is composed of two proteins, alpha-helix and elastin-like protein (ELP). Alpha-helices align and bundle the ELP while ELP twists and forms a fiber-like structure at warm temperatures. This ELP conformational change alters the distance between the structure termini at the site opposite the alpha-helix. We connected enhanced yellow fluorescent protein (EYFP) and enhanced cyan fluorescent protein (ECFP) at the structure's two termini to evaluate the distance using fluorescence resonance energy transfer (FRET) efficiency. These proteins spontaneously formed a complex which decreased the distance between the two fluorescent proteins located at its termini, at physiologically relevant temperatures. This change was repeated with complete reversibility (n = 5).     

Fluorescence energy transfer detects changes in fibronectin structure upon surface binding

We report here the changes in intramolecular distances in human plasma fibronectin (Fn) detected, upon adsorption of the protein to the surface of the Cytodex dextran microcarrier, using a fluorescence energy transfer technique. The glutamine-3 residue, near the amino terminus of each chain, was labeled enzymatically with either monodansylcadaverine (dansyl) or monofluoresceinyl-cadaverine (fluorescein) by use of coagulation factor XIIIa. Using this donor (dansyl)-acceptor (fluorescein) pair, and steady-state measurements, we demonstrated previously that the two amino termini of plasma fibronectin in solution were juxtaposed and separated by 23 A (C. Wolff and C.-S. Lai (1988) Biochemistry 27, 3483-3487). Upon adsorption to the microcarrier, the energy transfer was found to be completely abolished, suggesting that the surface binding induces a conformational change by which the distance between the two amino termini is increased to more than 70 A. Moreover, we have labeled the amino terminus of each chain with fluorescein and the two free sulfhydryl groups of each chain with coumarinyl-phenylmaleimide which serves as an energy donor. The emission spectra of the double-labeled protein in solution showed the occurrence of energy transfer, indicating that the relative distances between the amino termini and the free sulfhydryl group(s) are within 70 A. Upon surface binding, a decrease in the energy transfer between this donor-acceptor pair was also noted. The results presented here are consistent with the notion that plasma Fn undergoes a drastic conformational change upon surface binding, perhaps changing from a compact form to an extended form. This process may be important for the surface activation of the fibronectin molecule.     

A likelihood approach to character weighting and what it tells us about parsimony and compatibility

The statistical framework of maximum likelihood estimation is used to examine character weighting in inferring phylogenies. A simple probabilistic model of evolution is used, in which each character evolves independently among two states, and different lineages evolve independently. When different characters have different known probabilities of change, all sufficiently small, the proper maximum likelihood method of estimating phylogenies is a weighted parsimony method in which the weights are logarithmically related to the rates of change. When rates of change are taken extremely small, the weights become more equal and unweighted parsimony methods are obtained. When it is known that a few characters have very high rates of change and the rest very low rates, but it is not known which characters are the ones having the high rates, the maximum likelihood criterion supports use of compatibility methods. By varying the fraction of characters believed to have high rates of change one obtains a ‘threshold method’ whose behavior depends on the value of a parameter. By altering this parameter the method changes smoothly from being a parsimony method to being a compatibility method. This provides us with a spectrum of intermediates between these methods. These intermediate methods may be of use in analysing real data.     

Conformational dynamics of two histidine-binding proteins of Salmonella typhimurium.

The Salmonella typhimurium periplasmic histidine-binding J-protein is one of four proteins encoded by the histidine transport operon. Mutant J-protein hisJ5625 binds L-histidine, but does not transport it. The tertiary structure and conformational dynamics of native and mutant J-protein have been compared using steady state fluorescence, fluorescence polarization, and fluorescence energy transfer measurements. The two proteins have different three-dimensional structures and exhibit different responses to histidine binding. Ligand-induced conformational changes were demonstrated in both J-proteins using fluorescence energy transfer (distant reporter method) between the single tryptophan residue per mole of protein and a fluorescein-labeled methionine residue. However, the conformational change of the mutant protein is qualitatively and quantitatively different from that of the wild-type protein. Moreover, the microenvironment of the tryptophan and its distance from the labeled methionine (44A for the wild type, 60A for the mutant J-protein) are different in the two proteins. In conclusion, these results indicate that the specific conformational change induced in the wild type J-protein is a necessary requirement for the transport of L-histidine.     

Pulse fluorimetry study of energy transfers between tryptophan residues and NADPH in beef liver glutamate dehydrogenase complexes.

A method is proposed to determine the rates of singlet energy transfers in an array of chromophores containing a finite number of donors and fluorescent acceptors. This method is based on measurements of transfer efficiency coupled with pulse fluorimetry. Three classes of donors can be distinguished which differ in their energy transfer rate. The rates of the first, the second and the third class are respectively greater than, of the order of, and smaller than the emission rate. The method is applied to the study of the energy transfers from tryptophan residues to NADPH, in ternary and quaternary glutamate dehydrogenase complexes. Practically, all these tryptophan residues belong to the first class. They can be divided into two subclasses having different transfer rate values. The distance between these residues and the NADPH site are of the order of 2.5 nm. In addition, the ligand binding induces a protein conformation change, leading to a fluorescence quenching of the tryptophanyl emission.     

Protein stability curves

The stability curve of a protein is defined as the plot of the free energy of unfolding as a function of temperature. For most proteins the change in heat capacity on denaturation, or unfolding, is large but approximately constant. When unfolding is s two-state process, most of the salient features of the stability curves of proteins can be derived from this fact. A number of relations are obtained, including the special features of low-temperature denaturation, the properties of the maximum in stability, and the interrelationships of the characteristic temperatures of the protein. The paper closes with a formula that permits one to calculate small changes in stabilization free energy from changes in the melting temperature of the protein.     

Protein-leverage in mice: the geometry of macronutrient balancing and consequences for fat deposition

Objective: The Protein‐Leverage Hypothesis proposes that humans regulate their intake of macronutrients and that protein intake is prioritized over fat and carbohydrate intake, causing excess energy ingestion when diets contain low %protein. Here we test in a model animal, the mouse: (i) the extent to which intakes of protein and carbohydrate are regulated; (ii) if protein intake has priority over carbohydrates so that unbalanced foods low in %protein leads to increased energy intake; and (iii) how such variations in energy intake are converted into growth and storage. Methods and Procedures: We fed mice one of five isocaloric foods having different protein to carbohydrate composition, or a combination of two of these foods (N = 15). Nutrient intake and corresponding growth in lean body mass and lipid mass were measured. Data were analyzed using a geometric approach for analyzing intake of multiple nutrients. Results: (i) Mice fed different combinations of complementary foods regulated their intake of protein and carbohydrate toward a relatively well‐defined intake target. (ii) When mice were offered diets with fixed protein to carbohydrate ratio, they regulated the intake of protein more strongly than carbohydrate. This protein‐leverage resulted in higher energy consumption when diets had lower %protein and led to increased lipid storage in mice fed the diet containing the lowest %protein. Discussion: Although the protein‐leverage in mice was less than what has been proposed for humans, energy intakes were clearly higher on diets containing low %protein. This result indicates that tight protein regulation can be responsible for excess energy ingestion and higher fat deposition when the diet contains low %protein.     

The retrieval of visuo-spatial memories by honeybees

Summary In order to explore how honeybees manage to retrieve the right landmark-memory in the right place, we trained bees along a short foraging route which consisted of two identical huts 33 m apart. Bees entered each hut to collect a drop of sucrose on the floor. The location of the drop was defined by the same arrangement of four blue and yellow cylindrical landmarks. However, in one hut the drop was between two yellow cylinders and in two other it was to the east of the blue cylinders. On tests with the sucrose missing, bees tended to search in the appropriate area in each hut (Fig. 1), thus showing that they used cues other than the sight of the local landmarks to select the appropriate memory.In a second experiment, the position of the sucrose was specified by yellow cylinders in one hut and by blue triangles in the other. When the arrays were swapped between huts, bees searched in the position specified by the array they encountered (Fig. 2). Thus, memories can be triggered by visual features of local landmarks.Bees were also trained outside to collect food from two platforms 40 m apart. The location of sucrose on one platform was defined by yellow cylinders, and on the other it was defined by blue triangles. When these arrays were exchanged between platforms, bees searched on each platform as though the landmarks had not been swapped. It seems that the more distant surroundings, which fill most of the visual field, may be more potent than the local landmarks in deciding which memory should be retrieved.It is argued that one role of distant landmarks and other contextual cues is to ensure that bees retrieve the correct memory of a constellation of local landmarks while the bees are still some distance away from their goal. Even at a short distance, a bee's current image of local landmarks may differ considerably from its stored representation of those landmarks as seen from the goal. Accurate recall of the appropriate memory will be more certain if it is primed by relatively distant landmarks which present a more constant image as a bee moves in the vicinity of its goal.     

Distance dependent centroid to centroid force fields using high resolution decoys

Simplified force fields play an important role in protein structure prediction and de novo protein design by requiring less computational effort than detailed atomistic potentials. A side chain centroid based, distance dependent pairwise interaction potential has been developed. A linear programming based formulation was used in which non-native "decoy" conformers are forced to take a higher energy compared with the corresponding native structure. This model was trained on an enhanced and diverse protein set. High quality decoy structures were generated for approximately 1400 nonhomologous proteins using torsion angle dynamics along with restricted variations of the hydrophobic cores of the native structure. The resulting decoy set was used to train the model yielding two different side chain centroid based force fields that differ in the way distance dependence has been used to calculate energy parameters. These force fields were tested on an independent set of 148 test proteins with 500 decoy structures for each protein. The side chain centroid force fields were successful in correctly identifying approximately 86% native structures. The Z-scores produced by the proposed centroid-centroid distance dependent force fields improved compared with other distance dependent C(alpha)-C(alpha) or side chain based force fields.