Tests of Kramers' Theory at the Single-Molecule Level: Evidence for Folding of an Isolated RNA Tertiary Interaction at the Viscous Speed Limit.

Dissipation and friction influence the conformational dynamics of biological polymers as they traverse barriers on rugged free energy surfaces. It is well established that the "speed limit" for macromolecular folding is dictated by a combination of (i) solvent friction, which depends on solvent viscosity, η, and (ii) internal friction, which is independent of solvent and depends solely on the molecular folding pathway. In this work, single-molecule Förster resonance energy transfer (FRET) confocal spectroscopy is used to study viscosity-dependent folding kinetics of an isolated RNA tertiary motif, that of the GAAA tetraloop receptor, allowing both solvent and internal frictional contributions to be investigated and extracted independently for both flexible PEG- and RNA-based (rU7, rA7) linkers in the unimolecular construct. Specifically, our single-molecule data reveal that (i) folding rate constants scale linearly with the inverse solvent viscosity (η), which supports Kramers'/Grote-Hynes' rate theory for η-dependent RNA folding and that (ii) they provide quantitative upper limits for the intrinsic viscosity, [ηint ≈ 0.1(2) cP], arising from internal friction associated with folding/unfolding of an isolated RNA tertiary interaction. Furthermore, in contrast to strong viscosity-induced shifts in the folding/unfolding rate constants, temperature-dependent studies demonstrate that the enthalpic, entropic, and free energy contributions to the transition state barrier are largely insensitive to the solvent viscosity. This supports a very simple picture for the conformational kinetics of isolated RNA tertiary interactions wherein rate constants for folding/unfolding are both inversely dependent on viscosity and limited by diffusional access to the transition state region on a multidimensional free energy surface. Particularly under cellular conditions, where ηsolv > 1 cp, this suggests that RNAs fold/unfold at a "speed limit" dictated by solvent viscosity and transition-state barrier thermodynamics rather than internal molecular friction.

Evaluation of the VeriStrat® serum protein test in patients with advanced squamous cell carcinoma of the lung treated with second-line afatinib or erlotinib in the phase III LUX-Lung 8 study.

OBJECTIVES: Identification of biomarkers associated with clinical benefit may be crucial in establishing optimal treatment choice for patients with squamous cell carcinoma (SCC) of the lung after first-line chemotherapy. In this study, the ability of the VeriStrat serum protein test to predict differential clinical benefit with afatinib versus erlotinib, and the association of VeriStrat status with clinical outcomes irrespective of EGFR-TKI used, was assessed in a retrospective analysis of the phase III LUX-Lung 8 trial. MATERIALS AND METHODS: Pretreatment plasma samples were analyzed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Spectra were evaluated to assign a VeriStrat 'Good' (VS-G) or VeriStrat 'Poor' (VS-P) classification. Overall survival (OS), progression-free survival, and other endpoints were assessed with respect to pretreatment VeriStrat status; OS was the primary efficacy variable. Outcomes with other efficacy endpoints were similar. RESULTS: Of 795 patients randomized in LUX-Lung 8, 675 were classified (VS-G: 412; VS-P: 263). In the VS-G group, OS was significantly longer with afatinib versus erlotinib (HR 0.79 [95% CI: 0.63-0.98]). In the VS-P group, there was no significant difference in OS between afatinib and erlotinib (HR 0.90 [0.70-1.16]). However, there was no interaction between VeriStrat classification and treatment group for OS (pinteraction=0.5303). OS was significantly longer in VS-G versus VS-P patients, both in the overall VeriStrat-classified population (HR 0.41 [0.35-0.49]) and afatinib-treated patients (HR 0.40 [0.31-0.51]). Multivariate analysis showed that VeriStrat was an independent predictor of OS in afatinib-treated patients, regardless of ECOG PS or best response to first-line chemotherapy. CONCLUSION: VS-G classification is strongly associated with favorable survival outcomes with either afatinib or erlotinib compared with VS-P classification. In VS-G patients, survival outcomes with afatinib are superior to those with erlotinib. VeriStrat classification may guide treatment decisions in patients with SCC of the lung. ClinicalTrials.gov registration number: NCT01523587.

Development and Clinical Utility of a Blood-Based Test Service for the Rapid Identification of Actionable Mutations in Non-Small Cell Lung Carcinoma.

Nearly 80% of cancer patients do not have genetic mutation results available at initial oncology consultation; up to 25% of patients begin treatment before receiving their results. These factors hinder the ability to pursue optimal treatment strategies. This study validates a blood-based genome-testing service that provides accurate results within 72 hours. We focused on targetable variants in advanced non-small cell lung carcinoma-epidermal growth factor receptor gene (EGFR) variant L858R, exon 19 deletion (ΔE746-A750), and T790M; GTPase Kirsten ras gene (KRAS) variants G12C/D/V; and echinoderm microtubule associated protein like and 4 anaplastic lymphoma receptor tyrosine kinase fusion (EML4-ALK) transcripts 1/2/3. Test development included method and clinical validation using samples from donors with (n = 219) or without (n = 30) cancer. Clinical sensitivity and specificity for each variant ranged from 78.6% to 100% and 94.2% to 100%, respectively. We also report on 1643 non-small cell lung carcinoma samples processed in our CLIA-certified laboratory. Mutation results were available within 72 hours for 94% of the tests evaluated. We detected 10.5% mutations for EGFR sensitizing (n = 2801 samples tested), 13.8% mutations for EGFR resistance (n = 1055), 13.2% mutations in KRAS (n = 3477), and 2% mutations for EML4-ALK fusion (n = 304). This rapid, highly sensitive, and actionable blood-based assay service expands testing options and supports faster treatment decisions.

Kinetic and thermodynamic origins of osmolyte-influenced nucleic acid folding.

The influential role of monovalent and divalent metal cations in facilitating conformational transitions in both RNA and DNA has been a target of intense biophysical research efforts. However, organic neutrally charged cosolutes can also significantly alter nucleic acid conformational transitions. For example, highly soluble small molecules such as trimethylamine N-oxide (TMAO) and urea are occasionally utilized by organisms to regulate cellular osmotic pressure. Ensemble studies have revealed that these so-called osmolytes can substantially influence the thermodynamics of nucleic acid conformational transitions. In the present work, we exploit single-molecule FRET (smFRET) techniques to measure, for first time, the kinetic origins of these osmolyte-induced changes to the folding free energy. In particular, we focus on smFRET RNA and DNA constructs designed as model systems for secondary and tertiary structure formation. These findings reveal that TMAO preferentially stabilizes both secondary and tertiary interactions by increasing kfold and decreasing kunfold, whereas urea destabilizes both conformational transitions, resulting in the exact opposite shift in kinetic rate constants (i.e., decreasing kfold and increasing kunfold). Complementary temperature-dependent smFRET experiments highlight a thermodynamic distinction between the two different mechanisms responsible for TMAO-facilitated conformational transitions, while only a single mechanism is seen for the destabilizing osmolyte urea. Finally, these results are interpreted in the context of preferential interactions between osmolytes, and the solvent accessible surface area (SASA) associated with the (i) nucleobase, (ii) sugar, and (iii) phosphate groups of nucleic acids in order to map out structural changes that occur during the conformational transitions.

Defining the molecular basis of amyloid inhibitors: human islet amyloid polypeptide-insulin interactions.

Human islet amyloid polypeptide (hIAPP or Amylin) is a 37 residue hormone that is cosecreted with insulin from the pancreatic islets. The aggregation of hIAPP plays a role in the progression of type 2 diabetes and contributes to the failure of islet cell grafts. Despite considerable effort, little is known about the mode of action of IAPP amyloid inhibitors, and this has limited rational drug design. Insulin is one of the most potent inhibitors of hIAPP fibril formation, but its inhibition mechanism is not understood. In this study, the aggregation of mixtures of hIAPP with insulin, as well as with the separate A and B chains of insulin, were characterized using ion mobility spectrometry-based mass spectrometry and atomic force microscopy. Insulin and the insulin B chain target the hIAPP monomer in its compact isoform and shift the equilibrium away from its extended isoform, an aggregation-prone conformation, and thus inhibit hIAPP from forming ß-sheets and subsequently amyloid fibrils. All-atom molecular modeling supports these conclusions.

NaV1.5 sodium channel window currents contribute to spontaneous firing in olfactory sensory neurons.

Olfactory sensory neurons (OSNs) fire spontaneously as well as in response to odor; both forms of firing are physiologically important. We studied voltage-gated Na(+) channels in OSNs to assess their role in spontaneous activity. Whole cell patch-clamp recordings from OSNs demonstrated both tetrodotoxin-sensitive and tetrodotoxin-resistant components of Na(+) current. RT-PCR showed mRNAs for five of the nine different Na(+) channel α-subunits in olfactory tissue; only one was tetrodotoxin resistant, the so-called cardiac subtype NaV1.5. Immunohistochemical analysis indicated that NaV1.5 is present in the apical knob of OSN dendrites but not in the axon. The NaV1.5 channels in OSNs exhibited two important features: 1) a half-inactivation potential near -100 mV, well below the resting potential, and 2) a window current centered near the resting potential. The negative half-inactivation potential renders most NaV1.5 channels in OSNs inactivated at the resting potential, while the window current indicates that the minor fraction of noninactivated NaV1.5 channels have a small probability of opening spontaneously at the resting potential. When the tetrodotoxin-sensitive Na(+) channels were blocked by nanomolar tetrodotoxin at the resting potential, spontaneous firing was suppressed as expected. Furthermore, selectively blocking NaV1.5 channels with Zn(2+) in the absence of tetrodotoxin also suppressed spontaneous firing, indicating that NaV1.5 channels are required for spontaneous activity despite resting inactivation. We propose that window currents produced by noninactivated NaV1.5 channels are one source of the generator potentials that trigger spontaneous firing, while the upstroke and propagation of action potentials in OSNs are borne by the tetrodotoxin-sensitive Na(+) channel subtypes.

Molecular-crowding effects on single-molecule RNA folding/unfolding thermodynamics and kinetics.

The effects of "molecular crowding" on elementary biochemical processes due to high solute concentrations are poorly understood and yet clearly essential to the folding of nucleic acids and proteins into correct, native structures. The present work presents, to our knowledge, first results on the single-molecule kinetics of solute molecular crowding, specifically focusing on GAAA tetraloop-receptor folding to isolate a single RNA tertiary interaction using time-correlated single-photon counting and confocal single-molecule FRET microscopy. The impact of crowding by high-molecular-weight polyethylene glycol on the RNA folding thermodynamics is dramatic, with up to ΔΔG° ∼ -2.5 kcal/mol changes in free energy and thus >60-fold increase in the folding equilibrium constant (Keq) for excluded volume fractions of 15%. Most importantly, time-correlated single-molecule methods permit crowding effects on the kinetics of RNA folding/unfolding to be explored for the first time (to our knowledge), which reveal that this large jump in Keq is dominated by a 35-fold increase in tetraloop-receptor folding rate, with only a modest decrease in the corresponding unfolding rate. This is further explored with temperature-dependent single-molecule RNA folding measurements, which identify that crowding effects are dominated by entropic rather than enthalpic contributions to the overall free energy change. Finally, a simple "hard-sphere" treatment of the solute excluded volume is invoked to model the observed kinetic trends, and which predict ΔΔG° ∼ -5 kcal/mol free-energy stabilization at excluded volume fractions of 30%.

Pulsed IR heating studies of single-molecule DNA duplex dissociation kinetics and thermodynamics.

Single-molecule fluorescence spectroscopy is a powerful technique that makes it possible to observe the conformational dynamics associated with biomolecular processes. The addition of precise temperature control to these experiments can yield valuable thermodynamic information about equilibrium and kinetic rate constants. To accomplish this, we have developed a microscopy technique based on infrared laser overtone/combination band absorption to heat small (≈10(-11) liter) volumes of water. Detailed experimental characterization of this technique reveals three major advantages over conventional stage heating methods: 1), a larger range of steady-state temperatures (20-100°C); 2), substantially superior spatial (≤20 µm) control; and 3), substantially superior temporal (≈1 ms) control. The flexibility and breadth of this spatial and temporally resolved laser-heating approach is demonstrated in single-molecule fluorescence assays designed to probe the dissociation of a 21 bp DNA duplex. These studies are used to support a kinetic model based on nucleic acid end fraying that describes dissociation for both short (<10 bp) and long (>10 bp) DNA duplexes. These measurements have been extended to explore temperature-dependent kinetics for the 21 bp construct, which permit determination of single-molecule activation enthalpies and entropies for DNA duplex dissociation.

Single-molecule kinetics reveal cation-promoted DNA duplex formation through ordering of single-stranded helices.

In this work, the kinetics of short, fully complementary oligonucleotides are investigated at the single-molecule level. Constructs 6-9 bp in length exhibit single exponential kinetics over 2 orders of magnitude time for both forward (kon, association) and reverse (koff, dissociation) processes. Bimolecular rate constants for association are weakly sensitive to the number of basepairs in the duplex, with a 2.5-fold increase between 9 bp (k'on = 2.1(1) × 10(6) M(-1) s(-1)) and 6 bp (k'on = 5.0(1) × 10(6) M(-1) s(-1)) sequences. In sharp contrast, however, dissociation rate constants prove to be exponentially sensitive to sequence length, varying by nearly 600-fold over the same 9 bp (koff = 0.024 s(-1)) to 6 bp (koff = 14 s(-1)) range. The 8 bp sequence is explored in more detail, and the NaCl dependence of kon and koff is measured. Interestingly, kon increases by >40-fold (kon = 0.10(1) s(-1) to 4.0(4) s(-1) between [NaCl] = 25 mM and 1 M), whereas in contrast, koff decreases by fourfold (0.72(3) s(-1) to 0.17(7) s(-1)) over the same range of conditions. Thus, the equilibrium constant (Keq) increases by ≈160, largely due to changes in the association rate, kon. Finally, temperature-dependent measurements reveal that increased [NaCl] reduces the overall exothermicity (ΔΔH° > 0) of duplex formation, albeit by an amount smaller than the reduction in entropic penalty (-TΔΔS° < 0). This reduced entropic cost is attributed to a cation-facilitated preordering of the two single-stranded species, which lowers the association free-energy barrier and in turn accelerates the rate of duplex formation.

Dimerization of chirally mutated Enkephalin neurotransmitters: implications for peptide and protein aggregation mechanisms.

We have probed the structures and aggregation propensities of chirally substituted [Ala(2)]-Leu-Enkephalin peptides (i.e., Leu-Enkephalin G2A) with a combination of ion-mobility spectrometry/mass spectrometry and techniques of computational chemistry. Our IMS/MS data reveal a strong correlation between the propensity to form peptide dimers and the subsequent aggregation propensity. This correlation indicates that the dimerization process is fundamental to the overall self-assembly process. Our computational data correlate a conformational conversion during the peptide association process with a reduced experimental dimer formation and subsequent aggregation propensity. Furthermore, our analysis indicates that monomer activation does not precede peptide association and thus suggests that the entire-refolding or gain-in-interaction models are more realistic accounts of the peptide self-assembly process than the monomer-conversion model. In sum, our results suggest that conformational transitions of early peptide oligomers represent bottlenecks of the peptide self-assembly process and thus highlight the importance of structurally characterizing this reaction during amyloid formation.

The amyloid formation mechanism in human IAPP: dimers have ß-strand monomer-monomer interfaces.

Early oligomerization of human IAPP (hIAPP) is responsible for ß-cell death in the pancreas and is increasingly considered a primary pathological process linked to Type II Diabetes (T2D). Yet, the assembly mechanism remains poorly understood, largely due to the inability of conventional techniques to probe distributions or detailed structures of early oligomeric species. Here, we describe the first experimental data on the isolated and unmodified dimers of human (hIAPP) and nonamyloidogenic rat IAPP (rIAPP). The experiments reveal that the human IAPP dimers are more extended than those formed by rat IAPP and likely descend from extended monomers. Independent all-atom molecular dynamics simulations show that rIAPP forms compact helix and coil rich dimers, whereas hIAPP forms ß-strand rich dimers that are generally more extended. Also, the simulations reveal that the monomer-monomer interfaces of the hIAPP dimers are dominated by ß-strands and that ß-strands can recruit coil or helix structured regions during the dimerization process. Our ß-rich interface contrasts with an N-terminal helix-to-helix interface proposed in the literature but is consistent with existing experimental data on the self-interaction pattern of hIAPP, mutation effects, and inhibition effects of the N-methylation in the mutation region.

Ion mobility-mass spectrometry reveals a conformational conversion from random assembly to ß-sheet in amyloid fibril formation.

Amyloid cascades that lead to peptide ß-sheet fibrils and plaques are central to many important diseases. Recently, intermediate assemblies of these cascades were identified as the toxic agents that interact with cellular machinery. The location and cause of the transformation from a natively unstructured assembly to the ß-sheet oligomers found in all fibrils is important in understanding disease onset and the development of therapeutic agents. Largely, research on this early oligomeric region was unsuccessful because all the traditional techniques measure only the average oligomer properties of the ensemble. We utilized ion-mobility methods to deduce the peptide self-assembly mechanism and examined a series of amyloid-forming peptides clipped from larger peptides or proteins associated with disease. We provide unambiguous evidence for structural transitions in each of these fibril-forming peptide systems and establish the potential of this method for the development of therapeutic agents and drug evaluation.

Human islet amyloid polypeptide monomers form ordered beta-hairpins: a possible direct amyloidogenic precursor.

Oligomerization of human islet amyloid polypeptide (IAPP) has been increasingly considered a pathogenic process in type II diabetes. Here structural features of the IAPP monomer have been probed using a combination of ion mobility mass spectrometry (IMS-MS) and all-atom replica exchange molecular dynamics (REMD) simulations. Three distinct conformational families of human IAPP monomer are observed in IMS experiments, and two of them are identified as dehydrated solution structures on the basis of our simulation results: one is an extended beta-hairpin structural family, and the second is a compact helix-coil structural family. The extended beta-hairpin family is topologically similar to the peptide conformation in the solid-state NMR fibril structure published by Tycko and co-workers. It is absent in both experiments and simulations performed on the non-amyloidogenic rat IAPP, suggesting it may play an important role in the fibrillation pathway of human IAPP. In addition, pH dependence studies show that the relative abundance of the beta-hairpin structural family is significantly enhanced at pH 8.0. This observation is consistent with the increased rate of fibrillation at high pH in vitro and offers a possible explanation of the pH dependent fibrillation in vivo. This paper, to the best of our knowledge, presents the first experimental evidence of a significant population of beta-hairpin conformers for the IAPP peptide. It is consistent with a previous suggestion in the literature that beta-sheet-rich oligomers are assembled from ordered beta-hairpins rather than from coiled structures.

DNA hairpin, pseudoknot, and cruciform stability in a solvent-free environment.

The secondary structures of DNA hairpins, pseudoknots and cruciforms are of great interest because of their possible role in materials applications and biological functions such as regulating transcription. To determine the stability of these structures, DNA sequences capable of forming each were analyzed with mass spectrometry, ion mobility, and molecular dynamics calculations. Nano-ESI mass spectra indicated that stoichiometries compatible with hairpin, pseudoknot, and cruciform structures were present. Ion mobility spectrometry (IMS) was utilized to obtain experimental collision cross sections for all complexes. These cross sections were compared with structures from molecular dynamics, and in all cases, the lowest-charge states could be matched with a structure for an intact hairpin, pseudoknot, or cruciform. However, as the charge states of the single-stranded hairpins and pseudoknots increased, their structures elongated, and all Watson-Crick pairs were broken.

Amyloid-ß protein oligomerization and the importance of tetramers and dodecamers in the aetiology of Alzheimer's disease.

In recent years, small protein oligomers have been implicated in the aetiology of a number of important amyloid diseases, such as type 2 diabetes, Parkinson's disease and Alzheimer's disease. As a consequence, research efforts are being directed away from traditional targets, such as amyloid plaques, and towards characterization of early oligomer states. Here we present a new analysis method, ion mobility coupled with mass spectrometry, for this challenging problem, which allows determination of in vitro oligomer distributions and the qualitative structure of each of the aggregates. We applied these methods to a number of the amyloid-ß protein isoforms of Aß40 and Aß42 and showed that their oligomer-size distributions are very different. Our results are consistent with previous observations that Aß40 and Aß42 self-assemble via different pathways and provide a candidate in the Aß42 dodecamer for the primary toxic species in Alzheimer's disease.

Spermine binding to Parkinson's protein alpha-synuclein and its disease-related A30P and A53T mutants.

Aggregation of alpha-synuclein (alpha-syn), a protein implicated in Parkinson's disease (PD), is believed to progress through formation of a partially folded intermediate. Using nanoelectrospray ionization (nano-ESI) mass spectrometry combined with ion mobility measurements we found evidence for a highly compact partially folded family of structures for alpha-syn and its disease-related A53T mutant with net charges of -6, -7, and -8. For the other early onset PD mutant, A30P, this highly compact population was only evident when the protein had a net charge of -6. When bound to spermine near physiologic pH, all three proteins underwent a charge reduction from the favored solution charge state of -10 to a net charge of -6. This charge reduction is accompanied by a dramatic size reduction of about a factor of 2 (cross section of 2600 A2 (-10 charge state) down to 1430 A2 (-6 charge state)). We conclude that spermine increases the aggregation rate of alpha-syn by inducing a collapsed conformation, which then proceeds to form aggregates.

Probing shapes of bichromophoric metal-organic complexes using ion mobility mass spectrometry.

Ion mobility mass spectrometry (IM-MS) was used to probe the structures of several metal complexes carrying pendant chromophores. The three complexes investigated were the copper(II) complex Cu(DAC)2+ (DAC = 1,8-bis(9-methylanthracyl)cyclam, cyclam = 1,4,8,11-tetraazacyclotetradecane), the N-nitrosylated ligand DAC-NO, and the Roussin's red salt ester (mu-S,mu-S')-protoporphyrin-IX-bis(2-thioethyl ester)tetranitrosyldiiron (PPIX-RSE). From the IM-MS data coupled with theoretical calculations, it was found that [Cu(II)(DAC - H)]+ exists as a single conformer, with one anthracenyl group above the cyclam and the other below, similar to the crystal structure of Cu(II)(DAC)2+. The metal-free N-nitrosylated ligand (DAC-NO + H)+ has two conformations: one family of structures has one anthracenyl group above the cyclam and one below, while the other has both anthracenyl groups on the same side of the cyclam. These observations are consistent with 1H NMR data for the neutral DAC-NO complex that indicate the presence of two geometric isomers in solution. The third species, PPIX-RSE, has a porphyrin chromophore covalently linked to an Fe2S2(NO)4 cluster for use as a precursor for the photochemical delivery of nitric oxide in single- and two-photon excitation processes. Ion mobility indicates the presence of two (PPIX-RSE + H)+ conformations, consistent with the previous interpretation of the bimodal fluorescence lifetime decay seen for PPIX-RSE. DFT structures, in good agreement with the IM-MS cross sections, indicate two "bent" conformations with the planes of the porphyrin and Fe2S2 rings at different angles with respect to each other.

Laser control of product electronic state: desorption from alkali halides.

We demonstrate laser control of the electronic product state distribution of photodesorbed halogen atoms from alkali halide crystals. Our general model of surface exciton desorption dynamics is developed into a simple method for laser control of the relative halogen atom spin-orbit laser desorption yield. By tuning the excitation laser photon energy in a narrow region of the absorption threshold, the yield of excited state chorine atoms, Cl(2P(1/2)), can be made to vary from near 0 to 80% for KCl and from near 0 to 50% for NaCl relative to the total yield of Cl atoms. We describe the physical properties necessary to obtain a high degree of product state control and the limitation induced when these requirements are not met. These results demonstrate that laser control can be applied to solid state surface reactions and provide strong support for surface exciton-based desorption models.