Protein folding barriers can be so low that a substantial protein population diffusing in the transition state region can be detected. The very fast kinetic phase contributed by transition state transit is the molecular phase. We detect the molecular phase of the beta-sheet protein FiP35 from 60 to 83 degrees C by T-jump relaxation experiments. The molecular phase actually slows down slightly with increasing temperature. Thus the friction that controls the prefactor in Kramers' transition state model does not scale with solvent viscosity. Instead, we postulate that an increase in the energy landscape roughness as the hydrophobic effect strengthens with increasing temperature explains the slowing of the molecular phase. We measured that the duration tau(m) of the molecular phase depends slightly on the size of the T-jump, in agreement with this explanation. The tau(m) measured here provides the best current estimate for the transit time from folded to unfolded state of a single protein molecule. We confirm this by directly comparing relaxation and single molecule signals computed by using Langevin trajectory models on a realistic FiP35 free energy surface.

Coupling between the amide linkages in a peptide or protein is the key physical property that gives vibrational spectra and circular dichroism sensitivity to secondary structures. By use of (13)C isotopic labeling on individual and pairs of amide C═O groups, the amide I band for selected residues was effectively isolated in designed hexa- and octapeptides having dominant 3(10)-helical conformations. The resultant frequency and intensity responses were measured with IR absorption, vibrational circular dichroism (VCD), and Raman spectroscopies and simulated with density functional theory (DFT) based computations. Band fitting the spectral components and correlating the results to the computed coupling between selected labeled positions were used to determine coupling constant signs and to estimate their magnitudes for specific sequences. The observed frequency and intensity patterns, and their variation between IR and VCD with label position in the sequence, follow the theoretical predictions to a large degree, but are complicated by end effects that alter the local force field (FF) for some residues in these short peptides. These FF variations were overestimated in the theoretical models which may be evidence of structural variations not included in the model. By analyzing the simulations with different coupling models, the coupling constants were determined to lie in a range (positive) +3-5 cm(-1) for sequential residues (i,i+1) and with (negative) -3 cm(-1) as an upper bound for alternate ones (i,i+2). The sequential amide coupling for 3(10)-helices is weaker than for α-helices but has the same sign and is larger than and oppositely signed as compared to 3(1)-, or poly-(Pro)(n) type-II, helices.

We have developed a rapid, reproducible, easy to execute, surface enhanced Raman scattering (SERS) method for detection of low volumes and total amounts of biological antigens using an analyte capture system derived from methods commonly used in Western blotting. Our method is a "half-sandwich" assay with an antigen detection scheme that employs a nitrocellulose (NC) membrane with 200 nm pore size to capture subnanograms of analyte and concentrate them in a small area from applied volumes as low as one microliter. The SERS probes used for detection consist of gold-silica nanoshells modified with a two-component mixed monolayer system. One component consists of a poly(ethylene glycol) (PEG)-modified Raman-active chromophore bound to the gold surface which allows for SERS detection and imparts particle stability. The second component uses (ortho-pyridyl) disulfide-PEG-succinimidyl ester to couple the recognition antibody to the particle surface. By controlling the reaction time and concentration of thiols, a mixed monolayer is prepared on the nanoshell surface with the ability to recognize low concentrations of analyte and generate reproducible SERS signals. Using this strategy, we have achieved SERS signals that are proportional to antigen present on the membrane allowing detection of total antigen amounts as low as 1.25 ng for some cases. The performance of this new SERS bioassay has been tested with a variety of potential antigens, demonstrating the potential for multiplexed detection of analytes.

Unordered proteins, unfolded peptides, and several "random coil" models have been shown to have local conformations similar to that of polyproline II (PPII). Inter-residue coupling of selected residues in a series of related peptides having predominantly PPII conformations were measured using IR, VCD, and Raman spectra of selected variants that were doubly C(1)-labeled with (13)C on the amide C═O. The characteristics of the (13)C═O component of the IR, VCD, and Raman amide I' bands and their sensitivity to the local structure of the peptide are compared to predictions based on DFT level calculations for related structures and used to estimate coupling interactions between pairs of C═O groups along the backbone of this helical structure. In the PPII case, the coupling is relatively weak, due to the extended structure, yet by combining IR, Raman, and VCD observations with results of DFT level model calculations, we have determined bounds for experimental interaction constants for this structure. Correlation of properties for PPII structures with those of "random coils" can be done by comparing Pro(n) and Pro-rich sequences with Lys-rich sequences. The experimental band shifts and implied couplings reflect the computed results in both cases. Thermal unfolding of these peptides appears to be multistate, with monotonic spectral changes but little evidence of a cooperative (sigmoidal) transition. For the Lys-rich series, a transition from PPII to α-helix structure was induced by TFE addition, and the spectra were fit to an equilibrium model. These spectral changes show a large variation in (13)C═O coupling that occurs with a local conformational change from PPII- to α-helical, which is both well-fit by our theoretical results and offers a new site-specific method of assigning local PPII/disordered vs α-helical (or other) structure.

Effects of thermal annealing on the emission properties of type-II InAs/GaAsSb quantum dots Appl. Phys. Lett. 94, 053101 (2009); 10.1063/1.3062979Photoluminescence studies of type-II self-assembled In 0.55 Al 0.45 As/Al 0.5 Ga 0.5 As quantum dots grown on (311)A GaAs substrate