Controlled unfolding and refolding of a single sodium-proton antiporter using atomic force microscopy

Single-molecule force-spectroscopy was employed to unfold and refold single sodium-proton antiporters (NhaA) of Escherichia coli from membrane patches. Although transmembrane alpha-helices and extracellular polypeptide loops exhibited sufficient stability to individually establish potential barriers against unfolding, two helices predominantly unfolded pairwise, thereby acting as one structural unit. Many of the potential barriers were detected unfolding NhaA either from the C-terminal or the N-terminal end. It was found that some molecular interactions stabilizing secondary structural elements were directional, while others were not. Additionally, some interactions appeared to occur between the secondary structural elements. After unfolding ten of the 12 helices, the extracted polypeptide was allowed to refold back into the membrane. After five seconds, the refolded polypeptide established all secondary structure elements of the native protein. One helical pair showed a characteristic spring like "snap in" into its folded conformation, while the refolding process of other helices was not detected in particular. Additionally, individual helices required characteristic periods of time to fold. Correlating these results with the primary structure of NhaA allowed us to obtain the first insights into how potential barriers establish and determine the folding kinetics of the secondary structure elements.     

Differentiating ligand and inhibitor interactions of a single antiporter

Regulatory mechanisms of ion and solute transporters are in focus of biomedical and biochemical studies and build a key for disease therapies. Inhibition of sodium/proton exchangers efficiently prevents ischemic heart disease and reperfusion development in humans, but molecular mechanisms behind are not clear. Using single-molecule force spectroscopy we observe the binding of the inhibitor 2-aminoperimidine (AP) to sodium/proton antiporters NhaA from Escherichia coli. Deactivating interactions were significantly suppressed at enhanced sodium concentrations of 200 mM as well as in the pH-locked inactive conformation of NhaA. New molecular interactions were quantified and localized within the protein occurring upon a competitive inhibitor binding. The inhibitor, which was targeted and bound to the ligand-binding pocket, altered interactions established at alpha-helix IX. These molecular mechanisms deactivating the antiporter were different to those established upon ligand binding and activation of NhaA.     

Detecting molecular interactions that stabilize native bovine rhodopsin

Using single-molecule force spectroscopy we probed molecular interactions within native bovine rhodopsin and discovered structural segments of well-defined mechanical stability. Highly conserved residues among G protein-coupled receptors were located at the interior of individual structural segments, suggesting a dual role for these segments in rhodopsin. Firstly, structural segments stabilize secondary structure elements of the native protein, and secondly, they position and hold the highly conserved residues at functionally important environments. Two main classes of force curves were observed. One class corresponded to the unfolding of rhodopsin with the highly conserved Cys110-Cys187 disulfide bond remaining intact and the other class corresponded to the unfolding of the entire rhodopsin polypeptide chain. In the absence of the Cys110-Cys187 bond, the nature of certain molecular interactions within folded rhodopsin was altered. These changes highlight the structural importance of this disulfide bond and may form the basis of dysfunctions associated with its absence.     

Role of a solvent-exposed aromatic cluster in the folding of Escherichia coli CspA

Escherichia coli CspA is a member of the cold shock protein family. All cold shock proteins studied to date fold rapidly by an apparent two-state mechanism. CspA contains an unusual cluster of aromatic amino acids on its surface that is necessary for nucleic acid binding and also provides stability to CspA (Hillier et al., 1998). To elucidate the role this aromatic cluster plays in the determining the folding rate and pathway of CspA, we have studied the folding kinetics of mutants containing either leucine or serine substituted for Phe 18, Phe20, and/or Phe31. The leucine substitutions are found to accelerate folding and the serine substitutions to decelerate folding. Because these residues exert effects on the free energy of the folding transition state, they may be necessary for nucleating folding. They are not responsible, however, for the very compact, native-like transition state ensemble seen in the cold shock proteins, as the refolding rates of the mutants all show a similar, weak dependence of unfolding rate on denaturant concentration. Using mutant cycle analysis, we show that there is energetic coupling among the three residues between the unfolded and transition states, suggesting that the cooperative nature of these interactions helps to determine the unfolding rate. Overall, our results suggest that separate evolutionary pressures can act simultaneously on the same group of residues to maintain function, stability, and folding rate.     

Peptide fragment studies on the folding elements of dihydrofolate reductase from Escherichia coli

One of the necessary conditions for a protein to be foldable is the presence of a complete set of “folding elements” (FEs) that are short, contiguous peptide segments distributed over an amino acid sequence. The FE‐assembly model of protein folding has been proposed, in which the FEs play a role in guiding structure formation through FE–FE interactions early in folding. However, two major issues remain to be clarified regarding the roles of the FEs in determining protein foldability. Are the FEs AFUs that can form nativelike structures in isolation? Is the presence of only the FEs without mutual connections a sufficient condition for a protein to be foldable? Here, we address these questions using peptide fragments corresponding to the FEs of DHFR from Escherichia coli. We show by CD measurement that the FE peptides are unfolded under the native conditions, and some of them have the propensities toward non‐native helices. MD simulations also show the non‐native helical propensities of the peptides, and the helix contents estimated from the simulations are well correlated with those estimated from the CD in TFE. Thus, the FEs of DHFR are not AFUs, suggesting the importance of the FEs in nonlocal interactions. We also show that equimolar mixtures of the FE peptides do not induce any structural formation. Therefore, mutual connections between the FEs, which should strengthen the nonlocal FE–FE interactions, are also one of the necessary conditions for a protein to be foldable. Proteins 2006. © 2005 Wiley‐Liss, Inc.     

Single-molecule mechanical unfolding and folding of a pseudoknot in human telomerase RNA

RNA unfolding and folding reactions in physiological conditions can be facilitated by mechanical force one molecule at a time. By using force-measuring optical tweezers, we studied the mechanical unfolding and folding of a hairpin-type pseudoknot in human telomerase RNA in a near-physiological solution, and at room temperature. Discrete two-state folding transitions of the pseudoknot are seen at approximately 10 and approximately 5 piconewtons (pN), with ensemble rate constants of approximately 0.1 sec(-1), by stepwise force-drop experiments. Folding studies of the isolated 5'-hairpin construct suggested that the 5'-hairpin within the pseudoknot forms first, followed by formation of the 3'-stem. Stepwise formation of the pseudoknot structure at low forces are in contrast with the one-step unfolding at high forces of approximately 46 pN, at an average rate of approximately 0.05 sec(-1). In the constant-force folding trajectories at approximately 10 pN and approximately 5 pN, transient formation of nonnative structures were observed, which is direct experimental evidence that folding of both the hairpin and pseudoknot takes complex pathways. Possible nonnative structures and folding pathways are discussed.     

Thermal folding and mechanical unfolding pathways of protein secondary structures

Mechanical stretching of secondary structures is studied through molecular dynamics simulations of a Go-like model. Force versus displacement curves are studied as a function of the stiffness and velocity of the pulling device. The succession of stretching events, as measured by the order in which contacts are ruptured, is compared to the sequencing of events during thermal folding and unfolding. Opposite cross-correlations are found for an alpha-helix and a beta-hairpin structure. In a tandem of two alpha-helices, the two constituent helices unravel nearly simultaneously. A simple condition for simultaneous versus sequential unraveling of repeat units is presented.     

Early intermediates in the folding of dihydrofolate reductase from Escherichia coli detected by hydrogen exchange and NMR.

The kinetic folding mechanism for Escherichia coli dihydrofolate reductase postulates two distinct types of transient intermediates. The first forms within 5 ms and has substantial secondary structure but little stability. The second is a set of four species that appear over the course of several hundred milliseconds and have secondary structure, specific tertiary structure, and significant stability (Jennings PA, Finn BE, Jones BE, Matthews CR, 1993, Biochemistry 32:3783-3789). Pulse labeling hydrogen exchange experiments were performed to determine the specific amide hydrogens in alpha-helices and beta-strands that become protected from exchange through the formation of stable hydrogen bonds during this time period. A significant degree of protection was observed for two subsets of the amide hydrogens within the dead time of this experiment (6 ms). The side chains of one subset form a continuous nonpolar strip linking six of the eight strands in the beta-sheet. The other subset corresponds to a nonpolar cluster on the opposite face of the sheet and links three of the strands and two alpha-helices. Taken together, these data demonstrate that the complex strand topology of this eight-stranded sheet can be formed correctly within 6 ms. Measurement of the protection factors at three different folding times (13 ms, 141 ms, and 500 ms) indicates that, of the 13 amide hydrogens displaying significant protection within 6 ms, 8 exhibit an increase in their protection factors from approximately 5 to approximately 50 over this time range; the remaining five exhibit protection factors > 100 at 13 ms. Only approximately half of the population of molecules form this set of stable hydrogen bonds. Thirteen additional hydrogens in the beta-sheet become protected from exchange as the set of native conformers appear, suggesting that the stabilization of this network reflects the global cooperativity of the folding reaction.     

Introduction of a Na+/H+ antiporter gene from Atriplex gmelini confers salt tolerance to rice

We engineered a salt-sensitive rice cultivar (Oryza sativa cv. Kinuhikari) to express a vacuolar-type Na+/H+ antiporter gene from a halophytic plant, Atriplex gmelini (AgNHX1). The activity of the vacuolar-type Na+/H+ antiporter in the transgenic rice plants was eight-fold higher than that in wild-type rice plants. Salt tolerance assays followed by non-stress treatments showed that the transgenic plants overexpressing AgNHX1 could survive under conditions of 300 mM NaCl for 3 days while the wild-type rice plants could not. These results indicate that overexpression of the Na+/H+ antiporter gene in rice plants significantly improves their salt tolerance.     

The folding mechanism of a two-domain protein: folding kinetics and domain docking of the gene-3 protein of phage fd

The gene-3 protein (G3P) of filamentous phages is essential for the infection of Escherichia coli. The carboxy-terminal domain anchors this protein in the phage coat, whereas the two amino-terminal domains N1 and N2 protrude from the phage surface. We analyzed the folding mechanism of the two-domain fragment N1-N2 of G3P (G3P(*)) and the interplay between folding and domain assembly. For this analysis, a variant of G3P(*) was used that contained four stabilizing mutations (IIHY-G3P(*)). The observed refolding kinetics extend from 10 ms to several hours. Domain N1 refolds very rapidly (with a time constant of 9.4 ms at 0.5 M guanidinium chloride, 25 degrees C) both as a part of IIHY-G3P(*) and as an isolated protein fragment. The refolding of domain N2 is slower and involves two reactions with time constants of seven seconds and 42 seconds. These folding reactions of the individual domains are followed by a very slow, spectroscopically silent docking process, which shows a time constant of 6200 seconds. This reaction was detected by a kinetic unfolding assay for native molecules. Before docking, N1 and N2 unfold fast and independently, after docking they unfold slowly in a correlated fashion. A high energy barrier is thus created by domain docking, which protects G3P kinetically against unfolding. The slow domain docking is possibly important for the infection of E.coli by the phage. Upon binding to the F pilus, the N2 domain separates from N1 and the binding site for TolA on domain N1 is exposed. Since domain reassembly is so slow, this binding site remains accessible until pilus retraction has brought N1 close to TolA on the bacterial surface.     

Kinetic studies of folding of the B-domain of staphylococcal protein A with molecular dynamics and a united-residue (UNRES) model of polypeptide chains

Langevin dynamics is used with our physics-based united-residue (UNRES) force field to study the folding pathways of the B-domain of staphylococcal protein A (1BDD (alpha; 46 residues)). With 400 trajectories of protein A started from the extended state (to gather meaningful statistics), and simulated for more than 35 ns each, 380 of them folded to the native structure. The simulations were carried out at the optimal folding temperature of protein A with this force field. To the best of our knowledge, this is the first simulation study of protein-folding kinetics with a physics-based force field in which reliable statistics can be gathered. In all the simulations, the C-terminal alpha-helix forms first. The ensemble of the native basin has an average RMSD value of 4 A from the native structure. There is a stable intermediate along the folding pathway, in which the N-terminal alpha-helix is unfolded; this intermediate appears on the way to the native structure in less than one-fourth of the folding pathways, while the remaining ones proceed directly to the native state. Non-native structures persist until the end of the simulations, but the native-like structures dominate. To express the kinetics of protein A folding quantitatively, two observables were used: (i) the average alpha-helix content (averaged over all trajectories within a given time window); and (ii) the fraction of conformations (averaged over all trajectories within a given time window) with Calpha RMSD values from the native structure less than 5 A (fraction of completely folded structures). The alpha-helix content grows quickly with time, and its variation fits well to a single-exponential term, suggesting fast two-state kinetics. On the other hand, the fraction of folded structures changes more slowly with time and fits to a sum of two exponentials, in agreement with the appearance of the intermediate, found when analyzing the folding pathways. This observation demonstrates that different qualitative and quantitative conclusions about folding kinetics can be drawn depending on which observable is monitored.     

Stability and interactions of the amino-terminal domain of ClpB from Escherichia coli

ClpB is a member of a multichaperone system in Escherichia coli (with DnaK, DnaJ, and GrpE) that reactivates aggregated proteins. The sequence of ClpB contains two ATP-binding regions that are enclosed between the N- and C-terminal extensions. Whereas it has been found that the N-terminal region of ClpB is essential for the chaperone activity, the structure of this region is not known, and its biochemical properties have not been studied. We expressed and purified the N-terminal fragment of ClpB (residues 1-147). Circular dichroism of the isolated N-terminal region showed a high content of alpha-helical structure. Differential scanning calorimetry showed that the N-terminal region of ClpB is thermodynamically stable and contains a single folding domain. The N-terminal domain is monomeric, as determined by gel-filtration chromatography, and the elution profile of the N-terminal domain does not change in the presence of the N-terminally truncated ClpB (ClpBDeltaN). This indicates that the N-terminal domain does not form strong contacts with ClpBDeltaN. Consistently, addition of the separated N-terminal domain does not reverse an inhibition of ATPase activity of ClpBDeltaN in the presence of casein. As shown by ELISA measurements, full-length ClpB and ClpBDeltaN bind protein substrates (casein, inactivated luciferase) with similar affinity. We also found that the isolated N-terminal domain of ClpB interacts with heat-inactivated luciferase. Taken together, our results indicate that the N-terminal fragment of ClpB forms a distinct domain that is not strongly associated with the ClpB core and is not required for ClpB interactions with other proteins, but may be involved in recognition of protein substrates.     

Testing the relationship between foldability and the early folding events of dihydrofolate reductase from Escherichia coli

A "folding element" is a contiguous peptide segment crucial for a protein to be foldable and is a new concept that could assist in our understanding of the protein-folding problem. It is known that the presence of the complete set of folding elements of dihydrofolate reductase (DHFR) from Escherichia coli is essential for the protein to be foldable. Since almost all of the amino acid residues known to be involved in the early folding events of DHFR are located within the folding elements, a close relationship between the folding elements and early folding events is hypothesized. In order to test this hypothesis, we have investigated whether or not the early folding events are preserved in circular permutants and topological mutants of DHFR, in which the order of the folding elements is changed but the complete set of folding elements is present. The stopped-flow circular dichroism (CD) measurements show that the CD spectra at the early stages of folding are similar among the mutants and the wild-type DHFR, indicating that the presence of the complete set of folding elements is sufficient to preserve the early folding events. We have further examined whether or not sequence perturbation on the folding elements by a single amino acid substitution affects the early folding events of DHFR. The results show that the amino acid substitutions inside of the folding elements can affect the burst-phase CD spectra, whereas the substitutions outside do not. Taken together, these results indicate that the above hypothesis is true, suggesting a close relationship between the foldability of a protein and the early folding events. We propose that the folding elements interact with each other and coalesce to form a productive intermediate(s) early in the folding, and these early folding events are important for a protein to be foldable.     

Thermodynamics and kinetics of non-native interactions in protein folding: a single point mutant significantly stabilizes the N-terminal domain of L9 by modulating non-native interactions in the denatured state

Comparatively little is known about the role of non-native interactions in protein folding and their role in both folding and stability is controversial. We demonstrate that non-native electrostatic interactions involving specific residues in the denatured state can have a significant effect upon protein stability and can persist in the transition state for folding. Mutation of a single surface exposed residue, Lys12 to Met, in the N-terminal domain of the ribosomal protein L9 (NTL9), significantly increased the stability of the protein and led to faster folding. Structural and energetic studies of the wild-type and K12M mutant show that the 1.9 kcal mol(-1) increase in stability is not due to native state effects, but rather is caused by modulation of specific non-native electrostatic interactions in the denatured state. pH dependent stability measurements confirm that the increased stability of the K12M is due to the elimination of favorable non-native interactions in the denatured state. Kinetic studies show that the non-native electrostatic interactions involving K12 persist in the transition state. The analysis demonstrates that canonical Phi-values can arise from the disruption of non-native interactions as well as from the development of native interactions.     

Role of electrostatic interactions for the stability and folding behavior of cold shock protein

The impacts of three charged‐residue‐involved mutations, E46A, R3E, and R3E/L66E, on the thermostability and folding behavior of the cold shock protein from the themophile Bacillus caldolyticus (Bc‐Csp) were investigated by using a modified Gō‐like model, in which the nonspecific electrostatic interactions of charged residues were taken into account. Our simulation results show that the wild‐type Bc‐Csp and its three mutants are all two‐sate folders, which is consistent with the experimental observations. It is found that these three mutations all lead to a decrease of protein thermodynamical stability, and the effect of R3E mutation is the strongest. The lower stability of these three mutants is due to the increase of the enthalpy of the folded state and the entropy of the unfolded state. Using this model, we also studied the folding kinetics and the folding/unfolding pathway of the wild‐type Bc‐Csp as well as its three mutants and then discussed the effects of electrostatic interactions on the folding kinetics. The results indicate that the substitutions at positions 3 and 46 largely decrease the folding kinetics, whereas the mutation of residue 66 only slightly decreases the folding rate. This result agrees well with the experimental observations. It is also found that these mutations have little effects on the folding transition state and the folding pathway, in which the N‐terminal β sheet folds earlier than the C‐terminal region. We also investigated the detailed unfolding pathway and found that it is really the reverse of the folding pathway, providing the validity of our simulation results. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Role of cranberry juice on molecular-scale surface characteristics and adhesion behavior of Escherichia coli

Cranberry juice has long been believed to benefit the prevention and treatment of urinary tract infections (UTIs). As the first step in the development of infection, bacterial adhesion is of great research interest, yet few studies have addressed molecular level adhesion in this context. P-fimbriated Escherichia coli play a major role in the development of a serious type of UTI, acute pyelonephritis. Experiments were conducted to investigate the molecular-scale effects of cranberry juice on two E. coli strains: HB101, which has no fimbriae, and the mutant HB101pDC1 which expresses P-fimbriae. Atomic force microscopy (AFM) was used to investigate both bacterial surface characteristics and adhesion forces between a probe surface (silicon nitride) and the bacteria, providing a direct evaluation of bacterial adhesion and interaction forces. Cranberry juice affected bacterial surface polymer and adhesion behavior after a short exposure period (<3 h). Cranberry juice affected the P-fimbriated bacteria by decreasing the adhesion forces between the bacterium and tip and by altering the conformation of the surface macromolecules on E. coli HB101pDC1. The equilibrium length of polymer (P-fimbriae) on this bacterium decreased from approximately 148 to approximately 48 nm upon being exposed to cranberry juice. Highly acidic conditions were not necessary for the prevention of bacterial adhesion, since neutralization of cranberry juice solutions to pH = 7.0 allowed us to observe differences in adhesion between the E. coli strains. Our results demonstrate molecular-level changes in the surfaces of P-fimbriated E. coli upon exposure to neutralized cranberry juice.     

Fluorescence and folding properties of Tyr mutant tryptophan synthase alpha-subunits from Escherichia coli

The fluorescence of tyrosine has been used to monitor a folding process of tryptophan synthase alpha-subunit from Escherichia coli, because this protein has 7 tyrosines, but not tryptophan. Here to assess the contribution of each Tyr to fluorescence properties of this protein during folding, mutant proteins in which Tyr was replaced with Phe were analyzed. The result shows that a change of Tyr fluorescence occurring during folding of this protein is contributed to approximately 40% each by Tyr(4) and Tyr(115), and to the remaining approximately 20% by Tyr(173) and Tyr(175). Y173F and Y175F mutant proteins showed an increase in their fluorescence intensity by approximately 40% and approximately 10%, respectively. These increases appear to be due to multiple effects of increased hydrophobicity, quenching effect of nearby residue Glu(49), and/or energy transfer between Tyrs. Two data for Y173F alpha-subunit of urea-induced unfolding equilibrium monitored by UV and fluorescence were different. This result, together with ANS binding and far UV CD, shows that folding intermediate(s) of Y173F alpha-subunit, contrary to that of wild-type, may contain self-inconsistent properties such as more buried hydrophobicity, highly quenched fluorescence, and different dependencies on urea of UV absorbance, suggesting an ensemble of heterogeneous structures.     

Folding and stretching in a Go-like model of titin

Mechanical stretching of the I27 domain of titin and of its double and triple repeats are studied through molecular dynamics simulations of a Go-like model with Lennard-Jones contact interactions. We provide a thorough characterization of the system and correlate the sequencing of the folding and unraveling events with each other and with the contact order. The roles of cantilever stiffness and pulling rate are studied. Unraveling of tandem titin structures has a serial nature. The force-displacement curves in this coarse-grained model are similar to those obtained through all atom calculations.     

Two-dimensional kinetics of inter-connexin interactions from single-molecule force spectroscopy

Gap junction channels are intercellular channels that form by docking the extracellular loops of connexin protein subunits. While the structure and function of gap junctions as intercellular channels have been characterized using different techniques, the physics of the inter-connexin interaction remain unknown. Moreover, as far as we know, the capacity of gap junction channels to work as adhesion complexes supporting pulling forces has not yet been quantitatively addressed. We report the first quantitative characterization of the kinetics and binding strength of the interaction of a short peptide mimicking extracellular loop 2 of Cx26 with membrane-reconstituted Cx26, combining the imaging and force spectroscopy capabilities of atomic force microscopy. The fast dissociation rate inferred a dynamic bond, while the slow association rate reflected the reduced flexibility and small size of extracellular loops. Our results propose the gap junction channel as an adhesion complex that associates slowly and dissociates fast at low force but is able to support important pulling forces in its native, hexameric form.