Single molecule protein patterning using hole mask colloidal lithography.

The ability to manipulate single protein molecules on a surface is useful for interfacing biology with many types of devices in optics, catalysis, bioengineering, and biosensing. Control of distance, orientation, and activity at the single molecule level will allow for the production of on-chip devices with increased biological activity. Cost effective methodologies for single molecule protein patterning with tunable pattern density and scalable coverage area remain a challenge. Herein, Hole Mask Colloidal Lithography is presented as a bench-top colloidal lithography technique that enables a glass coverslip to be patterned with functional streptavidin protein onto patches from 15-200 nm in diameter with variable pitch. Atomic force microscopy (AFM) was used to characterize the size of the patterned features on the glass surface. Additionally, single-molecule fluorescence microscopy was used to demonstrate the tunable pattern density, measure binding controls, and confirm patterned single molecules of functional streptavidin.

Single plasmonic nanoparticle tracking studies of solid supported bilayers with ganglioside lipids.

Single-particle tracking experiments were carried out with gold nanoparticle-labeled solid supported lipid bilayers (SLBs) containing increasing concentrations of ganglioside (GM(1)). The negatively charged nanoparticles electrostatically associate with a small percentage of positively charged lipids (ethyl phosphatidylcholine) in the bilayers. The samples containing no GM(1) show random diffusion in 92% of the particles examined with a diffusion constant of 4.3(±4.5) × 10(-9) cm(2)/s. In contrast, samples containing 14% GM(1) showed a mixture of particles displaying both random and confined diffusion, with the majority of particles, 62%, showing confined diffusion. Control experiments support the notion that the nanoparticles are not associating with the GM(1) moieties but instead most likely confined to regions in between the GM(1) clusters. Analysis of the root-mean-squared displacement plots for all of the data reveals decreasing trends in the confined diffusion constant and diameter of the confining region versus increasing GM(1) concentration. In addition, a linearly decreasing trend is observed for the percentage of randomly diffusing particles versus GM(1) concentration, which offers a simple, direct way to measure the percolation threshold for this system, which has not previously been measured. The percolation threshold is found to be 22% GM(1) and the confining diameter at the percolation threshold only ∼50 nm.

Advances in localized surface plasmon resonance spectroscopy biosensing.

In recent years, localized surface plasmon resonance (LSPR) spectroscopy advancements have made it a sensitive, flexible tool for probing biological interactions. Here, we describe the basic principles of this nanoparticle-based sensing technique, the ways nanoparticles can be tailored to optimize sensing, and examples of novel LSPR spectroscopy applications. These include detecting small molecules via protein conformational changes and resonance LSPR spectroscopy, as well as coupling LSPR with mass spectrometry to identify bound analytes. The last few sections highlight the advantages of single nanoparticle LSPR, in that it lowers limits of detection, allows multiplexing on the nanometer scale, and enables free diffusion of sensors in solution. The cases discussed herein illustrate creative ways that LSPR spectroscopy has been improved to achieve new sensing capabilities.

Methyl groups of trimethylamine N-oxide orient away from hydrophobic interfaces.

The molecular orientation of trimethylamine N-oxide (TMAO), a powerful protein stabilizer, was explored at aqueous/hydrophobic interfaces using vibrational sum frequency spectroscopy (VSFS). The systems studied included the octadecyltrichlorosilane (OTS)/water interface, which represents an aqueous solution in direct contact with a hydrophobic medium. Surprisingly, the measurements revealed that the methyl groups of TMAO pointed into the aqueous phase and away from the OTS. This orientation may arise from the more hydrophilic nature of methyl groups attached to a strongly electron-withdrawing atom such as a quaternary nitrogen. Additional studies were performed at the air/water interface. This interface showed a high degree of TMAO alignment, but the dangling OH from water was present even at 5 M TAMO. Moreover, the addition of this osmolyte modestly increased the surface tension of the interface. This meant that this species was somewhat depleted at the interface compared to the bulk solution. These findings may have implications for the stabilizing effect of TMAO on proteins. Specifically, the strong hydration required for the methyl groups as well as the oxide moiety should be responsible for the osmolyte's depletion from hydrophobic/aqueous interfaces. Such depletion effects should help stabilize proteins in their folded and native conformations on entropic grounds, although orientational effects may play an additional role.

Hydrogen bonding of beta-turn structure is stabilized in D(2)O.

The lower critical solution temperature (LCST) of elastin-like polypeptides (ELPs) was investigated as a function of ELP chain length and guest residue chemistry. These measurements were made in both D(2)O and H(2)O. Differences in the LCST values with heavy and light water were correlated with secondary structure formation of the polypeptide chains. Such structural information was obtained by circular dichroism and infrared measurements. Additional thermodynamic data were obtained by differential scanning calorimetry. It was found that there is a greater change in the LCST value between H(2)O and D(2)O for those polypeptides which form the greatest amount of beta-turn/beta-aggregate structure. Moreover, these same molecules were the least hydrophobic ELPs. Therefore, hydrogen bonding rather than hydrophobicity was the key factor in the stabilization of the collapsed state of ELPs in D(2)O compared with H(2)O.

Investigating the hydrogen-bonding model of urea denaturation.

The direct binding mechanism for urea-based denaturation of proteins was tested with a thermoresponsive polymer, poly(N-isopropylacrylamide) (PNIPAM). Thermodynamic measurements of the polymer's hydrophobic collapse were complemented by Fourier transform infrared (FTIR) spectroscopy, Stokes radius measurements, and methylated urea experiments. It was found that the lower critical solution temperature (LCST) of PNIPAM decreased as urea was added to the solution. Therefore, urea actually facilitated the hydrophobic collapse of the macromolecule. Moreover, these thermodynamic measurements were strongly correlated with amide I band data which indicated that the decrease in the LCST was coupled to the direct hydrogen bonding of urea to the amide moieties of the polymer. In addition, the hydrogen bonding was found to be highly cooperative, which is consistent with a cross-linking (bivalent binding) mechanism. Cross-linking was confirmed by Stokes radius measurements below the polymer's LCST using gel filtration chromatography. Finally, phase transition measurements with methylurea, dimethylurea, and tetramethylurea indicated that these substituted compounds caused the LCST of PNIPAM to rise with increasing methyl group content. No evidence could be found for the direct binding of any of these methylated ureas to the polymer amide moieties by FTIR. These results are inconsistent with a direct hydrogen-bonding mechanism for the urea-induced denaturation of proteins.

Characterization of alkaline transitions in ferricytochrome c using carbon-deuterium infrared probes.

The alkaline-induced structural transitions of ferricytochrome c have been studied intensively as a model for how changes in metal ligation contribute to protein function and folding. Previous studies have demonstrated that multiple non-native species accumulate with increasing pH. Here, we used a combination of experiments and simulations to provide a high-resolution view of the changes associated with increasing alkaline conditions. Alkaline-induced transitions were characterized under equilibrium conditions by following changes in the IR absorptions of carbon-deuterium chromophores incorporated at Leu68, Lys72, Lys73, Lys79, and Met80. The data suggest that at least four intermediates are formed as the pH is increased prior to complete unfolding of the protein. The first alkaline transition observed appears to be driven by a single deprotonation and occurs with a midpoint of pH 8.8, but surprisingly, the intermediate formed does not appear to be one of the well-characterized lysine misligates. At higher pH, second and third deprotonations, with a combined apparent midpoint pH of 10.2, induce transitions to Lys73- or Lys79-misligated species. Interestingly, the lysine misligates appear to undergo iron reduction by the coordinated amine. A transition from the lysine misligates to another intermediate, likely a hydroxide-misligated species, is associated with a fourth deprotonation and a midpoint of pH 10.7. Finally, the protein loses tertiary structure with a fifth deprotonation that occurs with a midpoint of pH 12.7. Native topology-based models with enforced misligation are employed to help understand the structures of the observed intermediates.

Effects of Hofmeister anions on the phase transition temperature of elastin-like polypeptides.

The modulation of the lower critical solution temperature (LCST) of two elastin-like polypeptides (ELPs) was investigated in the presence of 11 sodium salts that span the Hofmeister series for anions. It was found that the hydrophobic collapse/aggregation of these ELPs generally followed the series. Specifically, kosmotropic anions decreased the LCST by polarizing interfacial water molecules involved in hydrating amide groups on the ELPs. On the other hand, chaotropic anions lowered the LCST through a surface tension effect. Additionally, chaotropic anions showed salting-in properties at low salt concentrations that were related to the saturation binding of anions with the biopolymers. These overall mechanistic effects were similar to those previously found for the hydrophobic collapse and aggregation of poly(N-isopropylacrylamide), PNIPAM. There is, however, a crucial difference between PNIPAM and ELPs. Namely, PNIPAM undergoes a two-step collapse process as a function of temperature in the presence of sufficient concentrations of kosmotropic salts. By contrast, ELPs undergo collapse in a single step in all cases studied herein. This suggests that the removal of water molecules from around the amide moieties triggers the removal of hydrophobic hydration waters in a highly coupled process. There are also some key differences between the LCST behavior of the two ELPs. Specifically, the more hydrophilic ELP V5A2G(3)-120 construct displays collapse/aggregation behavior that is consistent with a higher concentration of anions partitioning to polymer/aqueous interface as compared to the more hydrophobic ELP V(5)-120. It was also found that larger anions could bind with ELP V5A2G(3)-120 more readily in comparison with ELP V(5)-120. These latter results were interpreted in terms of relative binding site accessibility of the anion for the ELP.

Effects of Hofmeister Anions on the LCST of PNIPAM as a Function of Molecular Weight.

The effect of a series of sodium salts on the lower critical solution temperature (LCST) of poly(N-isopropylacrylamide), PNIPAM, was investigated as a function of molecular weight and polymer concentration with a temperature gradient microfluidic device under a dark-field microscope. In solutions containing sufficient concentrations of kosmotropic anions, the phase transition of PNIPAM was resolved into two separate steps for higher molecular weight samples. The first step of this two step transition was found to be sensitive to the polymer's molecular weight and solution concentration, while the second step was not. Moreover, the binding of chaotropic anions to the polymer was also influenced by molecular weight. Both sets of results could be explained by the formation of intramolecular and intermolecular hydrogen-bonding between polymer chains. By contrast, the hydrophobic hydration of the isopropyl moieties and polymer backbone was found to be unaffected by either the polymer's molecular weight or solution concentration.

Direct and high resolution characterization of cytochrome c equilibrium folding.

Protein folding has emerged as a central problem in biophysics, and the equilibrium folding mechanism of cytochrome c (cyt c) has served as a model system. Unfortunately, the detailed characterization of both the folding process and of any intermediate that might be populated has been limited by the low structural and/or temporal resolution of the available techniques. Here, we report the use of a recently developed technique to study folding that is based on the site-selective incorporation of carbon-deuterium (C-D) bonds and their characterization by IR spectroscopy. Specifically, we synthesize and characterize the protein with deuterated residues spread throughout four structural motifs: (d3)Leu68 in the 60's helix, (d8)Lys72 and (d8)Lys73 in the 70's helix, (d8)Lys79, (d3)Met80, and (d3)Ala83 in the D-loop, and (d3)Leu94, (d3)Leu98, and (d3)Ala101 in the C-terminal helix. The data reveal correlated behavior of the residues within each structural motif, as well as between the residues of the 60's and C-terminal helices and between residues of the 70's helix and D-loop. Residues of the 70's helix and the D-loop are more stable than those within the 60's and C-terminal helices, although the former are more sensitive to added denaturant. The data also suggest that the hydrophobicity of the heme cofactor plays a central role in folding. These results contrast with those from previous H/D exchange studies and suggest that the low denaturant fluctuations observed in the H/D exchange experiments are not similar to those through which the protein actually unfolds. The inherently fast time scale of IR also allows us to characterize the folding intermediate, long thought to be present, but which has proven difficult to characterize by other techniques.

Redox-coupled dynamics and folding in cytochrome c.

Cytochrome c functions as an electron carrier in the mitochondrial electron-transport chain using the Fe(II)-Fe(III) redox couple of a covalently attached heme prosthetic group, and it has served as a paradigm for both biological redox activity and protein folding. On the basis of a wide variety of biophysical techniques, it has been suggested that the protein is more flexible in the oxidized state than in the reduced state, which has led to speculation that it is the dynamics of the protein that has been evolved to control the cofactor's redox properties. To test this hypothesis, we incorporated carbon-deuterium bonds throughout cytochrome c and characterized their absorption frequencies and line widths using IR spectroscopy. The absorption frequencies of several residues on the proximal side of the heme show redox-dependent changes, but none show changes in line width, implying that the flexibility of the oxidized and reduced proteins is not different. However, the spectra demonstrate that folded protein is in equilibrium with a surprisingly large amount of locally unfolded protein, which increases with oxidation for residues localized to the proximal side of the heme. The data suggest that while the oxidized protein is not more flexible than the reduced protein, it is more locally unfolded. Local unfolding of cytochrome c might be one mechanism whereby the protein evolved to control electron transfer.

Efforts toward developing direct probes of protein dynamics.

We report the first IR characterization of a single C-D bond within a protein, methyl-d1 Met80 of horse heart cytochrome c. A comparison was made to methyl-d1/d3 methionine as well as methyl-d3 Met80. We found that for methyl-d1 and the asymmetric stretches of methyl-d3, line widths/line shapes are dominated by inhomogeneous broadening, whereas the symmetric stretch of methyl-d3 has a significant homogeneous component. Vibrational energy relaxation calculations found that a significantly stronger Fermi resonance exists for the symmetric stretch than for the asymmetric stretches, thereby suggesting that a difference in intramolecular vibrational relaxation (IVR) causes the observed line width/line shape difference between the symmetric and asymmetric stretches.

A high-resolution probe of protein folding.

Protein folding is a central problem in the biological sciences. To generate residue-specific information on the equilibrium folding of cytochrome c, we have semisynthesized the protein with specifically deuterated residues. The C-D bonds may be easily visualized in an otherwise transparent region of the IR spectra, even at high protein and denaturant concentrations. Plotted as a function of added guanidine hydrochloride denaturant, the absorption intensities reveal that the protein undergoes a conformational change at the protein-based ligand, Met80, which is then followed by a more global unfolding at 2.3 M denaturant. Deuteration and characterization of other residues in cytochrome c, or other protein of interest, should provide complete views of folding with residue specific detail that is capable of resolving even the most rapidly interconverting intermediates.