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.

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.

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.

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.

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.

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.

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.

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.

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.

Urea orientation at protein surfaces.

We have exploited the unique ability of vibrational sum frequency spectroscopy (VSFS) to investigate interfacial urea molecules at protein surfaces. Experiments were carried out at the bovine serum albumin/water interface. The absolute orientation of interfacial urea could be followed directly by VSFS.

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.

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.

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.

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.