An in vivo platform for identifying inhibitors of protein aggregation.

Protein aggregation underlies an array of human diseases, yet only one small-molecule therapeutic targeting this process has been successfully developed to date. Here, we introduce an in vivo system, based on a ß-lactamase tripartite fusion construct, that is capable of identifying aggregation-prone sequences in the periplasm of Escherichia coli and inhibitors that prevent their aberrant self-assembly. We demonstrate the power of the system using a range of proteins, from small unstructured peptides (islet amyloid polypeptide and amyloid ß) to larger, folded immunoglobulin domains. Configured in a 48-well format, the split ß-lactamase sensor readily differentiates between aggregation-prone and soluble sequences. Performing the assay in the presence of 109 compounds enabled a rank ordering of inhibition and revealed a new inhibitor of islet amyloid polypeptide aggregation. This platform can be applied to both amyloidogenic and other aggregation-prone systems, independent of sequence or size, and can identify small molecules or other factors able to ameliorate or inhibit protein aggregation.

Thermal unfolding kinetics of ubiquitin in the microsecond-to-second time range probed by Tyr-59 fluorescence.

Thermal folding/unfolding kinetics of wild-type ubiquitin (wt-UBQ) was studied in a wide time range, from microseconds to seconds, by combining rapid-mixing T-jump and laser T-jump with fluorescence detection (MTJ-F and LTJ-F, respectively) to monitor the fluorescence changes of Tyr-59 located on the 310-helix. The kinetics occurs exclusively in the millisecond to second time range, and the decays are strictly single exponential. From global analysis of folding and unfolding decays, the kf and ku values were determined, without use of the equilibrium constant Ku. The activation enthalpy of folding is negative (DeltaHf(#)(Tm) = -10.8 kcal/mol), but the free energy of the transition state is substantially larger than that of the unfolded state (DeltaGf(#)(Tm) = 7.6 kcal/mol RTm). Thus, wt-UBQ behaves as a two-state folder, when folding is monitored by the fluorescence of Tyr-59. The observation of kinetics on the microsecond time scale, when folding is monitored by the disruption of hydrogen bonds between beta-strands, using nonlinear infrared spectroscopy of the amide I vibrations (LTJ-DVE) [Chung, H. S.; Tokmakoff, A. Proteins: Struct., Funct., Bioinf. 2008, 72, 474-487], seems to result from the fact that MTJ-F monitors the effective unfolding (backbone exposure to water) of the thermally excited protein alone, while LTJ-DVE also monitors preliminary events (hydrogen-bond breaking) and thermal re-equilibration of the thermally excited protein.

Single-molecule studies of the Im7 folding landscape.

Under appropriate conditions, the four-helical Im7 (immunity protein 7) folds from an ensemble of unfolded conformers to a highly compact native state via an on-pathway intermediate. Here, we investigate the unfolded, intermediate, and native states populated during folding using diffusion single-pair fluorescence resonance energy transfer by measuring the efficiency of energy transfer (or proximity or P ratio) between pairs of fluorophores introduced into the side chains of cysteine residues placed in the center of helices 1 and 4, 1 and 3, or 2 and 4. We show that while the native states of each variant give rise to a single narrow distribution with high P values, the distributions of the intermediates trapped at equilibrium (denoted I(eqm)) are fitted by two Gaussian distributions. Modulation of the folding conditions from those that stabilize the intermediate to those that destabilize the intermediate enabled the distribution of lower P value to be assigned to the population of the unfolded ensemble in equilibrium with the intermediate state. The reduced stability of the I(eqm) variants allowed analysis of the effect of denaturant concentration on the compaction and breadth of the unfolded state ensemble to be quantified from 0 to 6 M urea. Significant compaction is observed as the concentration of urea is decreased in both the presence and absence of sodium sulfate, as previously reported for a variety of proteins. In the presence of Na(2)SO(4) in 0 M urea, the P value of the unfolded state ensemble approaches that of the native state. Concurrent with compaction, the ensemble displays increased peak width of P values, possibly reflecting a reduction in the rate of conformational exchange among iso-energetic unfolded, but compact conformations. The results provide new insights into the initial stages of folding of Im7 and suggest that the unfolded state is highly conformationally constrained at the outset of folding.

Reduction of the damping on an AFM cantilever in fluid by the use of micropillars.

In single molecule force measurements with soft atomic force microscope (AFM) cantilevers, the force sensitivity is limited by the Brownian motion of the cantilever. When a cantilever is close to the surface, the hydrodynamic interaction between the cantilever beam and the surface, called the "squeezing effect", becomes significant, and the resonance peak of the thermal oscillation of the cantilever is heavily broadened and shifted to lower frequency which makes it difficult to eliminate the thermal noise by low-pass filtering. In this study, we propose an easy and low-cost method to improve the force sensitivity. We demonstrate that by bringing a tip of a cantilever onto the edge of a micropillar structure a significant reduction of the damping and an enhancement of force sensitivity are achieved.

Raman spectroscopy and advanced mathematical modelling in the discrimination of human thyroid cell lines.

Raman spectroscopy could offer non-invasive, rapid and an objective nature to cancer diagnostics. However, much work in this field has focused on resolving differences between cancerous and non-cancerous tissues, and lacks the reproducibility and interpretation to be put into clinical practice. Much work is needed on basic cellular differences between malignancy and normal. This would allow the establishment of a clinically relevant cellular based model to translate to tissue classification. Raman spectroscopy provides a very detailed biochemical analysis of the target material and to 'unlock' this potential requires sophisticated mathematical modelling such as neural networks as an adjunct to data interpretation. Commercially obtained cancerous and non-cancerous cells, cultured in the laboratory were used in Raman spectral measurements. Data trends were visualised through PCA and then subjected to neural network analysis based on self-organising maps; consisting of m maps, where m is the number of classes to be recognised. Each map approximates the statistical distribution of a given class. The neural network analysis provided a 95% accuracy for identification of the cancerous cell line and 92% accuracy for normal cell line. In this preliminary study we have demonstrated th ability to distinguish between "normal" and cancerous commercial cell lines. This encourages future work to establish the reasons underpinning these spectral differences and to move forward to more complex systems involving tissues. We have also shown that the use of sophisticated mathematical modelling allows a high degree of discrimination of 'raw' spectral data.

Potential for Raman spectroscopy to provide cancer screening using a peripheral blood sample.

Cancer poses a massive health burden with incidence rates expected to double globally over the next decade. In the United Kingdom screening programmes exists for cervical, breast, and colorectal cancer. The ability to screen individuals for solid malignant tumours using only a peripheral blood sample would revolutionise cancer services and permit early diagnosis and intervention. Raman spectroscopy interrogates native biochemistry through the interaction of light with matter, producing a high definition biochemical 'fingerprint' of the target material. This paper explores the possibility of using Raman spectroscopy to discriminate between cancer and non-cancer patients through a peripheral blood sample. Forty blood samples were obtained from patients with Head and Neck cancer and patients with respiratory illnesses to act as a positive control. Raman spectroscopy was carried out on all samples with the resulting spectra being used to build a classifier in order to distinguish between the cancer and respiratory patients' spectra; firstly using principal component analysis (PCA)/linear discriminant analysis (LDA), and secondly with a genetic evolutionary algorithm. The PCA/LDA classifier gave a 65% sensitivity and specificity for discrimination between the cancer and respiratory groups. A sensitivity score of 75% with a specificity of 75% was achieved with a 'trained' evolutionary algorithm. In conclusion this preliminary study has demonstrated the feasibility of using Raman spectroscopy in cancer screening and diagnostics of solid tumours through a peripheral blood sample. Further work needs to be carried out for this technique to be implemented in the clinical setting.

The mechanism of folding of Im7 reveals competition between functional and kinetic evolutionary constraints.

Many proteins reach their native state through pathways involving the presence of folding intermediates. It is not clear whether this type of folding landscape results from insufficient evolutionary pressure to optimize folding efficiency, or arises from a conflict between functional and folding constraints. Here, using protein-engineering, ultra-rapid mixing and stopped-flow experiments combined with restrained molecular dynamics simulations, we characterize the transition state for the formation of the intermediate populated during the folding of the bacterial immunity protein, Im7, and the subsequent molecular steps leading to the native state. The results provide a comprehensive view of the folding process of this small protein. An analysis of the contributions of native and non-native interactions at different stages of folding reveals how the complexity of the folding landscape arises from concomitant evolutionary pressures for function and folding efficiency.

Nanoscale probing reveals that reduced stiffness of clots from fibrinogen lacking 42 N-terminal Bbeta-chain residues is due to the formation of abnormal oligomers.

Removal of Bbetal-42 from fibrinogen by Crotalus atrox venom results in a molecule lacking fibrinopeptide B and part of a thrombin binding site. We investigated the mechanism of polymerization of desBbeta1-42 fibrin. Fibrinogen trinodular structure was clearly observed using high resolution noncontact atomic force microscopy. E-regions were smaller in desBbeta1-42 than normal fibrinogen (1.2 nm +/- 0.3 vs. 1.5 nm +/- 0.2), whereas there were no differences between the D-regions (1.7 nm +/- 0.4 vs. 1.7 nm +/- 0.3). Polymerization rate for desBbeta1-42 was slower than normal, resulting in clots with thinner fibers. Differences in oligomers were found, with predominantly lateral associations for desBbeta1-42 and longitudinal associations for normal fibrin. Clot elasticity as measured by magnetic tweezers showed a G' of approximately 1 Pa for desBbeta1-42 compared with approximately 8 Pa for normal fibrin. Spring constants of early stage desBbeta1-42 single fibers determined by atomic force microscopy were approximately 3 times less than normal fibers of comparable dimensions and development. We conclude that Bbeta1-42 plays an important role in fibrin oligomer formation. Absence of Bbeta1-42 influences oligomer structure, affects the structure and properties of the final clot, and markedly reduces stiffness of the whole clot as well as individual fibrin fibers.

Internal friction of single polypeptide chains at high stretch.

Experiments that measure the viscoelasticity of single molecules from the Brownian fluctuations of an atomic force microscope (AFM) have provided a new window onto their internal dynamics in an underlying conformational landscape. Here we develop and apply these methods to examine the internal friction of unfolded polypeptide chains at high stretch. The results reveal a power law dependence of internal friction with tension (exponent 1.3 +/- 0.5) and a relaxation time approximately independent of force. To explain these results we develop a frictional worm-like chain (FWLC) model based on the Rayleigh dissipation function of a stiff chain with dynamical resistance to local bending. We analyse the dissipation rate integrated over the chain length by its Fourier components to calculate an effective tension-dependent friction constant for the end-to-end vector of the chain. The result is an internal friction that increases as a power law with tension with an exponent 3/2, consistent with experiment. Extracting the intrinsic bending friction constant of the chain it is found to be approximately 7 orders of magnitude greater than expected from solvent friction alone; a possible explanation we offer is that the underlying energy landscape for bending amino acids and/or peptide bond is rough, consistent with recent results on both proteins and polysaccharides.

Chemical probing of single cancer cells with gold nanoaggregates by surface-enhanced Raman scattering.

By using near-infrared surface-enhanced Raman scattering (SERS) with 60 nm gold nanoparticles (Au-NPs) to probe the chemical composition inside single human osteosarcoma cells we have shown that the SERS intensity may increase by a factor of 3-6 times in different parts of the cells depending on the density of gold nanoaggregates within the probed volume after the cell is dehydrated. The cellular points of low-density gold nanoaggregates exhibit more significant increase of SERS signal levels, the cellular macrochemicals such as nucleic acids show conformational changes, and new components can be probed after the cell is completely dried. A comparative study between viable and apoptotic cells indicates that most of the Au-NPs that enter the living cell reside in the cytoplasm and around the nucleus, whereas glyoxal-induced apoptotic cells show relatively uniform distribution of Au-NPs and, interestingly, the presence of DNA fragments is detected throughout the cell, including the cell surface.

Single-molecule fluorescence resonance energy transfer assays reveal heterogeneous folding ensembles in a simple RNA stem-loop.

We have examined the folding ensembles present in solution for a series of RNA oligonucleotides that encompass the replicase translational operator stem-loop of the RNA bacteriophage MS2. Single-molecule (SM) fluorescence assays suggest that these RNAs exist in solution as ensembles of differentially base-paired/base-stacked states at equilibrium. There are two distinct ensembles for the wild-type sequence, implying the existence of a significant free energy barrier between "folded" and "unfolded" ensembles. Experiments with sequence variants are consistent with an unfolding mechanism in which interruptions to base-paired duplexes, in this example by the single-stranded loop and a single-base bulge in the base-paired stem, as well as the free ends, act as nucleation points for unfolding. The switch between folded and unfolded ensembles is consistent with a transition that occurs when all base-pairing and/or base-stacking interactions that would orientate the legs of the RNA stem are broken. Strikingly, a U-to-C replacement of a residue in the loop, which creates a high-affinity form of the operator for coat protein binding, results in dramatically different (un)folding behaviour, revealing distinct subpopulations that are either stabilised or destabilised with respect to the wild-type sequence. This result suggests additional reasons for selection against the C-variant stem-loop in vivo and provides an explanation for the increased affinity.

Common variation in the C-terminal region of the fibrinogen beta-chain: effects on fibrin structure, fibrinolysis and clot rigidity.

Fibrinogen BbetaArg448Lys is a common polymorphism, positioned within the carboxyl terminus of the Bbeta-chain of the molecule. Studies suggest that it is associated with severity of coronary artery disease and development of stroke. The effects of the amino acid substitution on clot structure remains controversial, and the aim of this study was to investigate the effect(s) of this polymorphism on fibrin clot structure using recombinant techniques. Permeation, turbidity, and scanning electron microscopy showed that recombinant Lys448 fibrin had a significantly more compact structure, with thin fibers and small pores, compared with Arg448. Clot stiffness, measured by means of a novel method using magnetic tweezers, was significantly higher for the Lys448 compared with the Arg448 variant. Clots made from recombinant protein variants had similar lysis rates outside the plasma environment, but when added to fibrinogen-depleted plasma, the fibrinolysis rates for Lys448 were significantly slower compared with Arg448. This study demonstrates for the first time that clots made from recombinant BbetaLys448 fibrinogen are characterized by thin fibers and small pores, show increased stiffness, and appear more resistant to fibrinolysis. Fibrinogen BbetaArg448Lys is a primary example of common genetic variation with a significant phenotypic effect at the molecular level.

Visualization of detergent solubilization of membranes: implications for the isolation of rafts.

Although different detergents can give rise to detergent-resistant membranes of different composition, it is unclear whether this represents domain heterogeneity in the original membrane. We compared the mechanism of action of five detergents on supported lipid bilayers composed of equimolar sphingomyelin, cholesterol, and dioleoylphosphatidylcholine imaged by atomic force microscopy, and on raft and nonraft marker proteins in live cells imaged by confocal microscopy. There was a marked correlation between the detergent solubilization of the cell membrane and that of the supported lipid bilayers. In both systems Triton X-100 and CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) distinguished between the nonraft liquid-disordered (l(d)) and raft liquid ordered (l(o)) lipid phases by selectively solubilizing the l(d) phase. A higher concentration of Lubrol was required, and not all the l(d) phase was solubilized. The solubilization by Brij 96 occurred by a two-stage mechanism that initially resulted in the solubilization of some l(d) phase and then progressed to the solubilization of both l(d) and l(o) phases simultaneously. Octyl glucoside simultaneously solubilized both l(o) and l(d) phases. These data show that the mechanism of membrane solubilization is unique to an individual detergent. Our observations have significant implications for using different detergents to isolate membrane rafts from biological systems.

Sphingomyelin chain length influences the distribution of GPI-anchored proteins in rafts in supported lipid bilayers.

Glycosyl-phosphatidylinositol (GPI)-anchored proteins are enriched in cholesterol- and sphingolipid-rich lipid rafts within the membrane. Rafts are known to have roles in cellular organization and function, but little is understood about the factors controlling the distribution of proteins in rafts. We have used atomic force microscopy to directly visualize proteins in supported lipid bilayers composed of equimolar sphingomyelin, dioleoyl-sn-glycero-3-phosphocholine and cholesterol. The transmembrane anchored angiotensin converting enzyme (TM-ACE) was excluded from the liquid ordered raft domains. Replacement of the transmembrane and cytoplasmic domains of TM-ACE with a GPI anchor (GPI-ACE) promoted the association of the protein with rafts in the bilayers formed with brain sphingomyelin (mainly C18:0). Association with the rafts did not occur if the shorter chain egg sphingomyelin (mainly C16:0) was used. The distribution of GPI-anchored proteins in supported lipid bilayers was investigated further using membrane dipeptidase (MDP) whose GPI anchor contains distearoyl phosphatidylinositol. MDP was also excluded from rafts when egg sphingomyelin was used but associated with raft domains formed using brain sphingomyelin. The effect of sphingomyelin chain length on the distribution of GPI-anchored proteins in rafts was verified using synthetic palmitoyl or stearoyl sphingomyelin. Both GPI-ACE and MDP only associated with the longer chain stearoyl sphingomyelin rafts. These data obtained using supported lipid bilayers provide the first direct evidence that the nature of the membrane-anchoring domain influences the association of a protein with lipid rafts and that acyl chain length hydrophobic mismatch influences the distribution of GPI-anchored proteins in rafts.

Probing intrinsic and extrinsic components in single osteosarcoma cells by near-infrared surface-enhanced Raman scattering.

We report on the capabilities of near-infrared surface-enhanced Raman scattering (SERS) using gold nanoparticles to obtain detailed chemical information with high spatial resolution from within single cancer cells, living or fixed. Colloidal gold particles, 60 nm in size, were introduced into live human osteosarcoma cells by endocytosis by adding them to the growth medium. Rapid SERS mapping of cells indicated that not only could rich vibrational spectra be obtained from intrinsic cellular constituents both in the cytoplasm and nucleus and but also the distribution of extrinsic molecules introduced into the cells, in this case, rhodamine 6G could be characterized, suggesting that the intracellular distribution of chemotherapeutic agents could potentially be measured by this technique. We show that the SERS signal intensity from the cellular components increases and more spectral detail is acquired from dried cells when compared with hydrated cells in buffer. The data also show spectral fluctuations, mainly in intensity but also in peak position, which are dependent upon the intensity of the excitation light and are probably due to diffusion of molecules on the surface of the gold nanoparticles. A detailed understanding of the origins of these effects is still not complete, but the ability to acquire very sensitive SERS inside living cancer cells indicates the potential of this technique as a useful tool in biomedicine.

The effect of protein complexation on the mechanical stability of Im9.

Force mode microscopy can be used to examine the effect of mechanical manipulation on the noncovalent interactions that stabilize proteins and their complexes. Here we describe the effect of complexation by the high affinity protein ligand E9 on the mechanical resistance of the simple four-helical protein, Im9. When concatenated into a construct of alternating I27 domains, Im9 unfolded below the thermal noise limit of the instrument ( approximately 20 pN). Complexation of E9 had little effect on the mechanical resistance of Im9 (unfolding force approximately 30 pN) despite the high avidity of this complex (K(d) approximately 10 fM).

Entropy and barrier-controlled fluctuations determine conformational viscoelasticity of single biomolecules.

Biological macromolecules have complex and nontrivial energy landscapes, endowing them with a unique conformational adaptability and diversity in function. Hence, understanding the processes of elasticity and dissipation at the nanoscale is important to molecular biology and emerging fields such as nanotechnology. Here we analyze single molecule fluctuations in an atomic force microscope, using a generic model of biopolymer viscoelasticity that includes local "internal" conformational dissipation. Comparing two biopolymers, dextran and cellulose (polysaccharides with and without local bistable transitions), demonstrates that signatures of simple conformational change are minima in both the elastic and internal friction constants around a characteristic force. A novel analysis of dynamics on a bistable energy landscape provides a simple explanation: an elasticity driven by the entropy, and friction by a barrier-controlled hopping time of populations between states, which is surprisingly distinct to the well-known relaxation time. This nonequilibrium microscopic analysis thus provides a means of quantifying new dynamical features of the energy landscape of the glucopyranose ring, revealing an unexpected underlying roughness and information on the shape of the barrier of the chair-boat transition in dextran. The results presented herein provide a basis toward probing the viscoelasticity of macromolecular conformational transitions on more complex energy landscapes, such as during protein folding.

Viscoelastic properties of single poly(ethylene glycol) molecules.

The viscoelastic properties of single poly(ethylene glycol) (PEG) molecules were measured by analysis of thermally and magnetically driven oscillations of an atomic force microscope (AFM) cantilever/molecule system. The molecular and monomer stiffness and friction of the PEG polymer were derived using a simple harmonic oscillator (SHO) model. Excellent agreement between the values of these two parameters obtained by the two approaches indicates the validity of the SHO model under the experimental regimes and the excellent reproducibility of the techniques. A sharp minimum in the monomeric friction is seen at around 180 pN applied force which we propose is due to a force induced change in the shape of the energy landscape describing the conformational transition of PEG from a helical to a planar state, which in turn affects the timescale of the transition and therefore modifies the measured internal friction. A knowledge of the viscoelastic response of PEG monomers is particularly important since PEG is widely used as a linker molecule for tethering groups of interest to the AFM tip in force spectroscopy experiments, and we show here that care must be exercised because of the force-dependent viscoelastic properties of these linkers.

Urea-induced unfolding of the immunity protein Im9 monitored by spFRET.

We have studied the urea-induced unfolding of the E colicin immunity protein Im9 using diffusion single-pair fluorescence resonance energy transfer. Detailed examination of the proximity ratio of the native and denatured molecules over a wide range of urea concentrations suggests that the conformational properties of both species are denaturant-dependent. Whereas native molecules become gradually more expanded as urea concentration increases, denatured molecules show a dramatic dependence of the relationship between proximity ratio and denaturant concentration, consistent with substantial compaction of the denatured ensemble at low denaturant concentrations. Analysis of the widths of the proximity ratio distributions for each state suggests that whereas the native state ensemble is relatively narrow and homogeneous, the denatured state may possess heterogeneity in mildly denaturing conditions.

Evidence for direct amelogenin-target cell interactions using dynamic force spectroscopy.

Increasing evidence suggests that amelogenin, long held to be a structural protein of developing enamel matrix, may also have cell signaling functions. However, a mechanism for amelogenin cell signaling has yet to be described. The aim of the present study was to use dynamic chemical force spectroscopy to measure amelogenin interactions with possible target cells. Full-length amelogenin (rM179) was covalently attached to silicon nitride AFM tips. Synthetic RGD peptides and unmodified AFM tips were used as controls. Amelogenin-RGD cell binding force measurements were carried out using human periodontal ligament fibroblasts (HPDF) from primary explants and a commercially available osteoblast-like human sarcoma cell line as the targets. Results indicated a linear logarithmic dependence between loading rate and unbinding force for amelogenin-RGD target cells across the range of loading rates used. For RGD controls, binding events measured at 5.5 nN s-1 force loading rate resulted in a mean force of 60 pN. Values for amelogenin-fibroblast and amelogenin-osteoblast-like cell unbinding forces, measured at similar loading rates, were 50 and 55 pN, respectively. These data suggest that amelogenin interacts with potential target cells with forces characteristic of specific ligand-receptor binding, suggesting a direct effect for amelogenin at target cell membranes.