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PubMed2002

Polyproline II helical structure in protein unfolded states: lysine peptides revisited.

The left-handed polyproline II (PPII) helix gives rise to a circular dichroism spectrum that is remarkably similar to that of unfolded proteins. This similarity has been used as the basis for the hypothesis that unfolded proteins possess considerable PPII helical content. It has long been known that homopolymers of lysine adopt the PPII helical conformation at neutral pH, presumably a result of electrostatic repulsion between side chains. It is shown here that a seven-residue lysine peptide also adopts the PPII conformation. In contrast with homopolymers of lysine, this short peptide is shown to retain PPII helical character under conditions in which side-chain charges are heavily screened or even neutralized. The most plausible explanation for these observations is that the peptide backbone favors the PPII conformation to maximize favorable interactions with solvent. These data are evidence that unfolded proteins do indeed possess PPII content, indicating that the ensemble of unfolded states is significantly smaller than is commonly assumed.

Circular Dichroism↗

PubMed2001

The PD...(D/E)XK motif in restriction enzymes: a link between function and conformation.

The active sites of Mg(II)-dependent nucleases feature a cluster of conserved charged residues which includes both acidic (Asp and Glu) and basic (Lys) side chains. In restriction enzymes, these side chains are part of the conserved PD...(D/E)XK functional sequence motif which has been implicated as being important in metal ion binding and catalytic steps. Recent work revealing the unusual behavior of the active site variant D58A of the representative PvuII endonuclease prompted speculation that the array of charged groups in the nuclease active site may also be linked to conformational behavior [Dupureur, C. M., and Conlan, L. H. (2000) Biochemistry 39, 10921-10927]. To address this issue, we analyzed the conformational behavior of active site variants of PvuII endonuclease using both NMR spectroscopic and thermodynamic methods. NMR spectroscopic analysis via (19)F and (1)H-(15)N HSQC experiments indicates that a number of side chain and backbone amide groups are perturbed upon Ala substitution at conserved active site residues Asp58, Glu68, and Lys70. Spectral changes are particularly pronounced for the lowest-activity mutants (D58A and K70A). These changes are accompanied by perturbations in conformational stability. Ala substitution at each of these positions results in 2-5 kcal/mol of stabilization over the wild-type enzyme at pH 7.7, changes which constitute increases in DeltaG(d)(H2O) of 20-50%. The pH dependencies of mutant enzyme stabilities are distinct from those of the wild type, results which confirm that these ionizable groups strongly influence stability. Wild-type enzyme stability is correlated with the ionization of groups shown to be important to metal ion binding and orientation. Correlations between spectral changes and conformational stability indicate that the latter measurements may prove useful in the evaluation of site-directed mutant restriction enzymes. More importantly, these results indicate that structure-function relationships in restriction enzyme active sites can be complex, and that the ensemble of conserved charged residues which mediate DNA hydrolysis in Mg(II)-dependent nucleases constitutes a critical link between function and conformation.

Alanine↗

PubMed1998

Dynamics of fluorescence fluctuations in green fluorescent protein observed by fluorescence correlation spectroscopy.

We have investigated the pH dependence of the dynamics of conformational fluctuations of green fluorescent protein mutants EGFP (F64L/S65T) and GFP-S65T in small ensembles of molecules in solution by using fluorescence correlation spectroscopy (FCS). FCS utilizes time-resolved measurements of fluctuations in the molecular fluorescence emission for determination of the intrinsic dynamics and thermodynamics of all processes that affect the fluorescence. Fluorescence excitation of a bulk solution of EGFP decreases to zero at low pH (pKa = 5.8) paralleled by a decrease of the absorption at 488 nm and an increase at 400 nm. Protonation of the hydroxyl group of Tyr-66, which is part of the chromophore, induces these changes. When FCS is used the fluctuations in the protonation state of the chromophore are time resolved. The autocorrelation function of fluorescence emission shows contributions from two chemical relaxation processes as well as diffusional concentration fluctuations. The time constant of the fast, pH-dependent chemical process decreases with pH from 300 microseconds at pH 7 to 45 microseconds at pH 5, while the time-average fraction of molecules in a nonfluorescent state increases to 80% in the same range. A second, pH-independent, process with a time constant of 340 microseconds and an associated fraction of 13% nonfluorescent molecules is observed between pH 8 and 11, possibly representing an internal proton transfer process and associated conformational rearrangements. The FCS data provide direct measures of the dynamics and the equilibrium properties of the protonation processes. Thus FCS is a convenient, intrinsically calibrated method for pH measurements in subfemtoliter volumes with nanomolar concentrations of EGFP.

Fluorescence↗

PubMed1997

The nucleation-collapse mechanism in protein folding: evidence for the non-uniqueness of the folding nucleus.

BACKGROUND: Recent experimental and theoretical studies have shown that several small proteins reach the native state by a nucleation-collapse mechanism. Studies based on lattice models have been used to suggest that the critical nucleus is specific, leading to the notion that the transition state may be unique. On the other hand, results of studies using off-lattice models show that the critical nuclei should be viewed as fluctuating mobile structures, thus implying non-unique transition states. RESULTS: The microscopic underpinnings of the nucleation-collapse mechanism in protein folding are probed using minimal off-lattice models and Langevin dynamics. We consider a 46-mer continuum model which has a native beta-barrel-like structure. The fast-folding trajectories reach the native state by a nucleation-collapse process. An algorithm based on the self-organized neural nets is used to identify the critical nuclei for a large number of rapidly folding trajectories. This method, which reduces the determination of the critical nucleus to one of 'pattern recognition', unambiguously shows that the folding nucleus is not unique. The only common characteristics of the mobile critical nuclei are that they are small (containing on average 15-22 residues) and are largely composed of residues near the loop regions of the molecule. The structures of the transition states, corresponding to the critical nuclei, show the existence of spatially localized ordered regions that are largely made up of residues that are close to each other. These structures are stabilized by a few long-range contacts. The structures in the ensemble of transition states exhibit a rather diverse degree of similarity to the native conformation. CONCLUSIONS: The multiplicity of delocalized nucleation regions can explain the two-state folding by a nucleation-collapse mechanism for small single-domain proteins (such as chymotrypsin inhibitor 2) and their mutants. Because there are many distinct critical nuclei, we predict that the folding kinetics of fast-folding proteins will not be drastically changed even if some of the residues in a 'typical' nucleus are altered.

Algorithms↗

PubMed2000

Structure-based optimization of peptide inhibitors of mammalian ribonucleotide reductase.

Mammalian ribonucleotide reductase (mRR), a potential target for cancer intervention, is composed of two subunits, mR1 and mR2, whose association is critical for enzyme activity. In this article we describe the structural features of the mRR-inhibitor Ac-F-c[ELAK]-DF (Peptide 3) while bound to the mR1 subunit as determined by transferred NOEs. Peptide 3 is a cyclic analogue of the N-acetylated form of the heptapeptide C-terminus of the mR2 subunit (Ac-FTLDADF), which is the link between the two subunits and previously shown to be the minimal sequence inhibitor mRR by competing with mR2 for binding to mR1. Structural refinement employing an ensemble-based, full-relaxation matrix approach resulted in two structures varying in the conformations of F(1) and the cyclic lactam side chains of E(2) and K(5). The remainder of the molecule, both backbone and side chains, is extremely well-defined, with an RMSD of 0.54 A. The structural features of this conformationally constrained analogue provide unique insight into the requirements for binding to mR1, critical for further inhibitor development.

Animals↗

PubMed2005

NMR dynamics-derived insights into the binding properties of a peptide interacting with an SH2 domain.

The signal transduction protein phospholipase C-gamma1 (PLC-gamma1) is activated when its C-terminal SH2 domain (PLCC) binds the phosphorylated Tyr-1021 site (pTyr-1021) in the beta-platelet-derived growth factor receptor (PDGFR). To better understand the contributions that dynamics make to binding, we have used NMR relaxation experiments to investigate the motional properties of backbone amide and side chain methyl groups in a peptide derived from the pTyr-1021 site of PDGFR, both free and in complex with the PLCC SH2 domain. The free peptide has relaxation properties that are typical for a small, unstructured polymer, while the backbone of the bound peptide is least flexible for residues in the central portion of the binding site with the amplitude of pico- to nanosecond time scale motions increasing toward the C-terminus of the peptide. The increase in large amplitude motion toward the end of the pY1021 peptide is consistent with the bound peptide existing as an ensemble of states with C-terminal residues having the broadest distribution of backbone conformations, while residues in the central binding site are the most restricted. Deuterium spin relaxation experiments establish that the protein-peptide interface is highly dynamic, and this mobility may play an important role in modulating the affinity of the interaction.

Amides↗

PubMed2005

Dopamine receptor oligomerization visualized in living cells.

G protein-coupled receptors occur as dimers within arrays of oligomers. We visualized ensembles of dopamine receptor oligomers in living cells and evaluated the contributions of receptor conformation to the dynamics of oligomer association and dissociation, using a strategy of trafficking a receptor to another cellular compartment. We incorporated a nuclear localization sequence into the D1 dopamine receptor, which translocated from the cell surface to the nucleus. Receptor inverse agonists blocked this translocation, retaining the modified receptor, D1-nuclear localization signal (NLS), at the cell surface. D1 co-translocated with D1-NLS to the nucleus, indicating formation of homooligomers. (+)-Butaclamol retained both receptors at the cell surface, and removal of the drug allowed translocation of both receptors to the nucleus. Agonist-nonbinding D1(S198A/S199A)-NLS, containing two substituted serine residues in transmembrane 5 also oligomerized with D1, and both were retained on the cell surface by (+)-butaclamol. Drug removal disrupted these oligomerized receptors so that D1 remained at the cell surface while D1(S198A/S199A)-NLS trafficked to the nucleus. Thus, receptor conformational differences permitted oligomer disruption and showed that ligand-binding pocket occupancy by the inverse agonist induced a conformational change. We demonstrated robust heterooligomerization between the D2 dopamine receptor and the D1 receptor. The heterooligomers could not be disrupted by inverse agonists targeting either one of the receptor constituents. However, D2 did not heterooligomerize with the structurally modified D1(S198A/S199A), indicating an impaired interface for their interaction. Thus, we describe a novel method showing that a homogeneous receptor conformation maintains the structural integrity of oligomers, whereas conformational heterogeneity disrupts it.

Butaclamol↗

PubMed1996

Secondary structural elements as a basis for antibody recognition in the immunodominant region of human immunodeficiency viruses 1 and 2.

Synthetic peptide antigens corresponding to the entire third variable region V3, the principal neutralizing determinant of the human immunodeficiency virus (HIV) envelope glycoprotein of HIV-1 subtype B (1), HIV-2 subtype A (5), and HIV-2 subtype B (7) were synthesized by solid-phase peptide synthesis (Table 1). 1 and 5 were also prepared as their GlcNAc-glycosylated forms at the natural N-glycosylation site NXT (positions 6-8; peptides 4 and 6). Additionally, the proposed beta-turn region of 1 (GPGR; positions 15-18) was altered by introducing D-Ala17 (2) and D-Pro16 (3). All compounds have been studied by two-dimensional NMR techniques. Interproton distances and 3JNH/H alpha coupling constants derived from NMR data are used as restraints in distance geometry and ENSEMBLE-Distance and angle-bound driven dynamics calculations. The stimulations led to disordered conformations except for a high propensity of a beta II-turn in the region GPXR (positions 15-18) in 1, 2, and 4. In 3 (G-D-ProGR, positions 15-18), a type beta I'-turn was mainly found instead. For peptide 7, the consensus sequence of HIV-2 subtype B, a type beta II-turn was also found although the primary structure (VSGL; positions 15-18) differs grossly from the HIV-1 peptide 1. With the exception of 2, all beta II-turns were able to form a canonically opened beta-turn by a 180 degree rotation of phi(G17). Surprisingly, compounds 5 and 6 that are highly similar to 7 showed no beta II-type turn within MSGL (positions 15-18). They form a type beta VIII-turn across the tetrapeptide SGLV (positions 16-19) together with a non-canonical turn conformation across LMSG (positions 14-17) leading to an S-conformation. The reaction of the peptides with HIV-positive sera from patients infected with different subtypes of HIV-1 and HIV-2 was tested in enzyme-linked immunosorbent assays (ELISA reactions). No HIV-2 sera reacted peptide 1 and no HIV-1 sera showed reactivity to peptide 5. We propose that certain amino acid exchanges within the V3 domain lead to altered conformations of the V3 loop resulting in antibodies that show altered binding properties to the peptide antigens used in the ELISA reactions.

Amino Acid Sequence↗

PubMed1998

Essential spaces defined by NMR structure ensembles and molecular dynamics simulation show significant overlap.

Large concerted motions of proteins which span its "essential space," are an important component of protein dynamics. We investigate to what extent structure ensembles generated with standard structure calculation techniques such as simulated annealing can capture these motions by comparing them to long-time molecular dynamics (MD) trajectories. The motions are analyzed by principal component analysis and compared using inner products of eigenvectors of the respective covariance matrices. Two very different systems are studied, the beta-spectrin PH domain and the single-stranded DNA binding protein (ssDBP) from the filamentous phage Pf3. A comparison of the ensembles from NMR and MD shows significant overlap of the essential spaces, which in the case of ssDBP is extraordinarily high. The influence of variations in the specifications of distance restraints is investigated. We also study the influence of the selection criterion for the final structure ensemble on the definition of mobility. The results suggest a modified criterion that improves conformational sampling in terms of amplitudes of correlated motion.

Computer Simulation↗

PubMed2000

Towards a complete description of the structural and dynamic properties of the denatured state of barnase and the role of residual structure in folding.

The detailed characterization of denatured proteins remains elusive due to their mobility and conformational heterogeneity. NMR studies are beginning to provide clues regarding residual structure in the denatured state but the resulting data are too sparse to be transformed into molecular models using conventional techniques. Molecular dynamics simulations can complement NMR by providing detailed structural information for components of the denatured ensemble. Here, we describe three independent 4 ns high-temperature molecular dynamics simulations of barnase in water. The simulated denatured state was conformationally heterogeneous with respect to the conformations populated both within a single simulation and between simulations. Nonetheless, there were some persistent interactions that occurred to varying degrees in all simulations and primarily involved the formation of fluid hydrophobic clusters with participating residues changing over time. The region of the beta(3-4) hairpin contained a particularly high degree of such side-chain interactions but it lacked beta-structure in two of the three denatured ensembles: beta(3-4) was the only portion of the beta-structure to contain significant residual structure in the denatured state. The two principal alpha-helices (alpha1 and alpha2) adopted dynamic helical structure. In addition, there were persistent contacts that pinched off core 2 from the body of the protein. The rest of the protein was unstructured, aside from transient and mostly local side-chain interactions. Overall, the simulated denatured state contains residual structure in the form of dynamic, fluctuating secondary structure in alpha1 and alpha2, as well as fluctuating tertiary contacts in the beta(3-4) region, and between alpha1 and beta(3-4), in agreement with previous NMR studies. Here, we also show that these regions containing residual structure display impaired mobility by both molecular dynamics and NMR relaxation experiments. The residual structure was important in decreasing the conformational states available to the chain and in repairing disrupted regions. For example, tertiary contacts between beta(3-4) and alpha1 assisted in the refolding of alpha1. This contact-assisted helix formation was confirmed in fragment simulations of beta(3-4) and alpha1 alone and complexed, and, as such, alpha1 and beta(3-4) appear to be folding initiation sites. The role of these sites in folding was investigated by working backwards and considering the simulation in reverse, noting that earlier time-points from the simulations provide models of the major intermediate and transition states in quantitative agreement with data from both unfolding and refolding experiments. Both beta(3-4) and alpha1 are dynamic in the denatured state but when they collide and make enough contacts, they provide a loose structural scaffold onto which further beta-strands pack. The beta-structure condenses about beta(3-4), while alpha1 aids in stabilizing beta(3-4) and maintaining its orientation. The resulting beta-structure is relatively planar and loose in the major intermediate. Further packing ensues, and as a result the beta-sheet twists, leading to the major transition state. The structure is still expanded and loops are not well formed at this point. Fine-tuning of the packing interactions and the final condensation of the structure then occurs to yield the native state.

Amino Acid Sequence↗

PubMed2004

High-resolution molecular structure of a peptide in an amyloid fibril determined by magic angle spinning NMR spectroscopy.

Amyloid fibrils are self-assembled filamentous structures associated with protein deposition conditions including Alzheimer's disease and the transmissible spongiform encephalopathies. Despite the immense medical importance of amyloid fibrils, no atomic-resolution structures are available for these materials, because the intact fibrils are insoluble and do not form diffraction-quality 3D crystals. Here we report the high-resolution structure of a peptide fragment of the amyloidogenic protein transthyretin, TTR(105-115), in its fibrillar form, determined by magic angle spinning NMR spectroscopy. The structure resolves not only the backbone fold but also the precise conformation of the side chains. Nearly complete (13)C and (15)N resonance assignments for TTR(105-115) formed the basis for the extraction of a set of distance and dihedral angle restraints. A total of 76 self-consistent experimental measurements, including 41 restraints on 19 backbone dihedral angles and 35 (13)C-(15)N distances between 3 and 6 A were obtained from 2D and 3D NMR spectra recorded on three fibril samples uniformly (13)C, (15)N-labeled in consecutive stretches of four amino acids and used to calculate an ensemble of peptide structures. Our results indicate that TTR(105-115) adopts an extended beta-strand conformation in the amyloid fibrils such that both the main- and side-chain torsion angles are close to their optimal values. Moreover, the structure of this peptide in the fibrillar form has a degree of long-range order that is generally associated only with crystalline materials. These findings provide an explanation of the unusual stability and characteristic properties of this form of polypeptide assembly.

Amino Acid Sequence↗

PubMed1995

Structure and dynamics of ferrocytochrome c553 from Desulfovibrio vulgaris studied by NMR spectroscopy and restrained molecular dynamics.

The solution structure of Desulfovibrio vulgaris Hildenborough (DvH) ferrocytochrome c553 has been determined by nuclear magnetic resonance spectroscopy and combined simulated annealing/high temperature restrained molecular dynamics calculations. This three-stage protocol consists of an initial determination of overall fold from randomised co-ordinates, followed by a 20 picosecond exploratory stage, during which the non-bonded terms are simplified to facilitate as broad a sampling of conformational space as possible, and a 26 picosecond refinement stage, using the full AMBER force field. This latter stage systematically improved the energetic and convergence characteristics of the ensemble, while still satisfying the experimental restraints. Forty structures have been obtained from a total of 875 distance constraints for this protein of 79 amino acid residues. The root-mean-square deviation over all residues with respect to the mean is 0.70(+/- 0.12)A for the backbone (N, C alpha and C') atoms. Two conformations of the turn motif at the solvent/heme cleft interface have been identified, both fulfilling the experimental data and having equally viable energetic characteristics. The stability of the ensemble and the dynamic characteristics have been further investigated by subjecting ten of the structures to constraint-free molecular dynamics calculations (130 picoseconds) in vacuo. The structures were found to be stable to within 1.5 A of the initial backbone conformation. Comparison with the dynamic behaviour of the restrained molecular dynamics calculations has been used to identify regions of inherent flexibility in the molecule.

Cytochrome c Group↗

PubMed2004

Role of solvent in determining conformational preferences of alanine dipeptide in water.

Evidence from a variety of spectroscopic probes indicates that (phi, psi) values corresponding to the left-handed polyproline II helix (P(II)) are preferred for short alanine-based peptides in water. On the basis of results from theoretical studies, it is believed that the observed preference is dictated by favorable peptide-solvent interactions, which are realized through formation of optimal hydrogen-bonding water bridges between peptide donor and acceptor groups. In the present study, we address this issue explicitly by analyzing the hydration structure and thermodynamics of 16 low-energy conformers of the alanine dipeptide (N-acetylalanine-N'-methylamide) in liquid water. Monte Carlo simulations in the canonical ensemble were performed under ambient conditions with all-atom OPLS parameters for the alanine dipeptide and the TIP5P model for water. We find that the number of hydrogen-bonded water molecules connecting the peptide group donor and acceptor atoms has no effect on the solvation thermodynamics. Instead, the latter are determined by the work done to fully hydrate the peptide. This work is minimal for conformations that are characterized by a minimal overlap of the primary hydration shells around the peptide donor and acceptor atoms. As a result, peptide-solvent interactions favor "compact" conformations that do not include P(II)-like geometries. Our main conclusion is that the experimentally observed preference for P(II) does not arise due to favorable direct interactions between the peptide and water molecules. Instead, the latter act to unmask underlying conformational preferences that are a consequence of minimizing intrapeptide steric conflicts.

Dipeptides↗

PubMed2002

Engineering of metallothionein-3 neuroinhibitory activity into the inactive isoform metallothionein-1.

The third isoform of mammalian metallothioneins (MT-3), mainly expressed in brain and down-regulated in Alzheimer's disease, exhibits neuroinhibitory activity in vitro and a highly flexible structure that distinguishes it from the widely expressed MT-1/-2 isoforms. Previously, we showed that two conserved prolyl residues of MT-3 are crucial for both the bioactivity and cluster dynamics of this isoform. We have now used genetic engineering to introduce these residues into mouse MT-1. The S6P,S8P MT-1 mutant is inactive in neuronal survival assays. However, the additional introduction of the unique Thr5 insert of MT-3 resulted in a bioactive MT-1 form. Temperature-dependent and saturation transfer (113)Cd NMR experiments performed on the (113)Cd-reconstituted wild-type and mutant Cd(7)-MT-1 forms revealed that the gain of MT-3-like neuronal inhibitory activity is paralleled by an increase in conformational flexibility and intersite metal exchange in the N-terminal Cd(3)-thiolate cluster. The observed correlation suggests that structure/cluster dynamics are critical for the biological activity of MT-3. We propose that the interplay between the specific Pro-induced conformational requirements and those of the metal-thiolate bonds gives rise to an alternate and highly fluctuating cluster ensemble kinetically trapped by the presence of the (5)TCPCP(9) motif. The functional significance of such heterogeneous cluster ensemble is discussed.

Amino Acid Sequence↗

PubMed2006

NMR analysis of the conformational properties of the trapped on-pathway folding intermediate of the bacterial immunity protein Im7.

Previous work shows that the transiently populated, on-pathway intermediate in Im7 folding contains three of the four native alpha-helices docked around a core stabilised by native and non-native interactions. To determine the structure and dynamic properties of this species in more detail, we have used protein engineering to trap the intermediate at equilibrium and analysed the resulting proteins using NMR spectroscopy and small angle X-ray scattering. Four variants were created. In L53AI54A, two hydrophobic residues within helix III are truncated, preventing helix III from docking stably onto the developing hydrophobic core. In two other variants, the six residues encompassing the native helix III were replaced with three (H3G3) or six (H3G6) glycine residues. In the fourth variant, YY, two native tyrosine residues (Tyr55 and Tyr56) were re-introduced into H3G6 to examine their role in determining the properties of the intermediate ensemble. All four variants show variable peak intensities and broad peak widths, consistent with these proteins being conformationally dynamic. Chemical shift analyses demonstrated that L53AI54A and YY contain native-like secondary structure in helices I and IV, while helix II is partly formed and helix III is absent. Lack of NOEs and rapid NH exchange for L53AI54A, combined with detailed analysis of the backbone dynamics, indicated that the hydrophobic core of this variant is not uniquely structured, but fluctuates on the NMR timescale. The results demonstrate that though much of the native-like secondary structure of Im7 is present in the variants, their hydrophobic cores remain relatively fluid. The comparison of H3G3/H3G6 and L53AI54A/YY suggests that Tyr55 and/or Tyr56 interact with the three-helix core, leading other residues in this region of the protein to dock with the core as folding progresses. In this respect, the three-helix bundle acts as a template for formation of helix III and the creation of the native fold.

Amino Acid Sequence↗

PubMed2004

Single-molecule spectroscopy studies of conformational change dynamics in enzymatic reactions.

Protein conformational dynamics, often associated with static and dynamic inhomogeneities, plays a crucial role in biomolecular functions. It is extremely difficult to characterize such inhomogeneous dynamics in an ensemble-averaged measurement, especially when the protein involves in a multiple-step complex chemical reaction, such as an enzymatic reaction. Single-molecule spectroscopy is a powerful approach to analyze protein conformational dynamics in real time under physiological conditions, providing dynamic perspectives on a molecular-level understanding of protein structure-function mechanisms. In this minireview, we discuss our recent studies on single-molecule enzymatic reaction dynamics and protein conformational dynamics of the T4 lysozyme hydrolyzation reaction of a polysaccharide by a combined approach of single-molecule spectroscopy, molecular dynamics simulation, and theoretical modeling. Detailed characterization of the complex enzymatic reaction dynamics identified the nature of the inhomogeneity and revealed multiple intermediate conformational states associated with the enzyme-substrate complex formation in the multiple-step enzymatic reaction.

Animals↗

PubMed2005

DNA molecular motor driven micromechanical cantilever arrays.

The unique ability of living systems to translate biochemical reactions into mechanical work has inspired the design of synthetic DNA motors which generate nanoscale motion via controlled conformational change. However, while Nature has evolved intricate mechanisms to convert molecular shape change into specific micrometer-scale mechanical cellular responses, the integration of artificial DNA motors with mechanical devices presents a major challenge. Here we report the direct integration between an ensemble of DNA motors and an array of microfabricated silicon cantilevers. The forces exerted by the precise duplex to nonclassical i-motif conformational change were probed via differential measurements using an in-situ reference cantilever coated with a nonspecific sequence of DNA. Fueled by the addition of protons, the open to close stroke of the motor induced 32 +/- 3 mN/m compressive surface stress, which corresponds to a single motor force of approximately 11 pN/m, an order of magnitude larger than previous classical hybridization studies. Furthermore, the surface-tethered conformational change was found to be highly reversible, in contrast to classical DNA motors which typically suffer rapid system poisoning. The direction and amplitude of motor-induced cantilever motion was tuneable via control of buffer pH and ionic strength, indicating that electrostatic forces play an important role in stress generation. Hybrid devices which directly harness the multiple accessible conformational states of dynamic oligonucleotides and aptamers, translating biochemical energy into micromechanical work, present a radical new approach to the construction of "smart" nanoscale machinery and mechano-biosensors.

Biomechanical Phenomena↗

PubMed2006

Ensemble and single-molecule fluorescence spectroscopy of a calcium-ion indicator dye.

The spectroscopic properties of Calcium Green 2 (CG-2), a dual-fluorophore Ca(2+) indicator dye, were characterized by a combination of steady state and time-resolved ensemble spectroscopic measurements, molecular mechanics calculations and single-molecule fluorescence spectroscopy. It was found that in Ca(2+) free solutions, CG-2 exists primarily as a highly quenched intramolecular dimer, but when bound to Ca(2+), the molecule adopts an extended, fluorescent conformation. The difference in emission properties of these two CG-2 conformations is explained in terms of simple exciton theory. Through single-molecule fluorescence measurements, we have shown that the bulk increase in ensemble fluorescence intensity correlates with a simple statistical increase in the number of fluorescent molecules in solution. In addition, we have also observed that the majority of CG-2 molecules photobleach in a single step, despite the molecule possessing two distinct fluorophores. A small fraction of molecules photobleach in multiple steps or show a series of transitions between emissive and nonemissive fluorescent states ("blinking"). We rationalize these photophysical phenomena using a simple model based on dipole-dipole Förster coupling between fluorophores in conjunction with irreversible photodamage to one of the constituent chromophores.

Algorithms↗