Feng Gai

Helix formation via conformation diffusion search

The helix-coil transition kinetics of an α-helical peptide were investigated by time-resolved infrared spectroscopy coupled with laser-induced temperature-jump initiation method. Specific isotope labeling of the amide carbonyl groups with 13C at selected residues was used to obtain site-specific information. The relaxation kinetics following a temperature jump, obtained by probing the amide I′ band of the peptide backbone, exhibit nonexponential behavior and are sensitive to both initial and final temperatures. These data are consistent with a conformation diffusion process on the folding energy landscape, in accord with a recent molecular dynamics simulation study.

Ultrafast folding of α3D: A de novo designed three-helix bundle protein

Here, we describe the folding/unfolding kinetics of α3D, a small designed three-helix bundle. Both IR temperature jump and ultrafast fluorescence mixing methods reveal a single-exponential process consistent with a minimal folding time of 3.2 ± 1.2 μs (at ≈50°C), indicating that a protein can fold on the 1- to 5-μs time scale. Furthermore, the single-exponential nature of the relaxation indicates that the prefactor for transition state (TS)-folding models is probably ≥1 (μs)–1 for a protein of this size and topology. Molecular dynamics simulations and IR spectroscopy provide a molecular rationale for the rapid, single-exponential folding of this protein. α3D shows a significant bias toward local helical structure in the thermally denatured state. The molecular dynamics-simulated TS ensemble is highly heterogeneous and dynamic, allowing access to the TS via multiple pathways.

Microscopic insights into the protein-stabilizing effect of trimethylamine N-oxide (TMAO)

Significance The ability of trimethylamine N-oxide (TMAO) to counteract osmotic stress along with the deleterious effects of denaturants such as urea is fascinating and has inspired many studies. To further our understanding of how TMAO acts to stabilize folded proteins, we carry out an infrared experiment designed to probe the microscopic details of this action explicitly from the perspective of the solute molecule. Our results reveal that the protein-stabilizing effect of TMAO originates from two contributions: One is entropic and the other is enthalpic in nature. Thus, this study provides not only microscopic details underlying the stabilizing action of TMAO, but also a method that can be used to study the stability-perturbing effect of other cosolvents. Although it is widely known that trimethylamine N-oxide (TMAO), an osmolyte used by nature, stabilizes the folded state of proteins, the underlying mechanism of action is not entirely understood. To gain further insight into this important biological phenomenon, we use the C≡N stretching vibration of an unnatural amino acid, p-cyano-phenylalanine, to directly probe how TMAO affects the hydration and conformational dynamics of a model peptide and a small protein. By assessing how the lineshape and spectral diffusion properties of this vibration change with cosolvent conditions, we are able to show that TMAO achieves its protein-stabilizing ability through the combination of (at least) two mechanisms: (i) It decreases the hydrogen bonding ability of water and hence the stability of the unfolded state, and (ii) it acts as a molecular crowder, as suggested by a recent computational study, that can increase the stability of the folded state via the excluded volume effect.

Cholesterol-dependent phase separation in cell-derived giant plasma membrane vesicles

Cell-derived GPMVs (giant plasma-membrane vesicles) enable investigation of lipid phase separation in a system with appropriate biological complexity under physiological conditions, and in the present study were used to investigate the cholesterol-dependence of domain formation and stability. The cholesterol level is directly related to the abundance of the liquid-ordered phase fraction, which is the majority phase in vesicles from untreated cells. Miscibility transition temperature depends on cholesterol and correlates strongly with the presence of detergent-insoluble membrane in cell lysates. Fluorescence correlation spectroscopy reveals two distinct diffusing populations in phase-separated cell membrane-derived vesicles whose diffusivities correspond well to diffusivities in both model systems and live cells. The results of the present study extend previous observations in purified lipid systems to the complex environment of the plasma membrane and provide insight into the effect of cholesterol on lipid phase separation and abundance.

Divalent cation-induced cluster formation by polyphosphoinositides in model membranes

Polyphosphoinositides (PPIs) and in particular phosphatidylinositol-(4,5)-bisphosphate (PI4,5P2), control many cellular events and bind with variable levels of specificity to hundreds of intracellular proteins in vitro. The much more restricted targeting of proteins to PPIs in cell membranes is thought to result in part from the formation of spatially distinct PIP2 pools, but the mechanisms that cause formation and maintenance of PIP2 clusters are still under debate. The hypothesis that PIP2 forms submicrometer-sized clusters in the membrane by electrostatic interactions with intracellular divalent cations is tested here using lipid monolayer and bilayer model membranes. Competitive binding between Ca(2+) and Mg(2+) to PIP2 is quantified by surface pressure measurements and analyzed by a Langmuir competitive adsorption model. The physical chemical differences among three PIP2 isomers are also investigated. Addition of Ca(2+) but not Mg(2+), Zn(2+), or polyamines to PIP2-containing monolayers induces surface pressure drops coincident with the formation of PIP2 clusters visualized by fluorescence, atomic force, and electron microscopy. Studies of bilayer membranes using steady-state probe-partitioning fluorescence resonance energy transfer (SP-FRET) and fluorescence correlation spectroscopy (FCS) also reveal divalent metal ion (Me(2+))-induced cluster formation or diffusion retardation, which follows the trend: Ca(2+) ≫ Mg(2+) > Zn(2+), and polyamines have minimal effects. These results suggest that divalent metal ions have substantial effects on PIP2 lateral organization at physiological concentrations, and local fluxes in their cytoplasmic levels can contribute to regulating protein-PIP2 interactions.

Effect of Macromolecular Crowding on Protein Folding Dynamics at the Secondary Structure Level

Macromolecular crowding is one of the key characteristics of the cellular environment and is therefore intimately coupled to the process of protein folding in vivo. While previous studies have provided invaluable insight into the effect of crowding on the stability and folding rate of protein tertiary structures, very little is known about how crowding affects protein folding dynamics at the secondary structure level. In this study, we examined the thermal stability and folding-unfolding kinetics of three small folding motifs (i.e., a 34-residue alpha-helix, a 34-residue cross-linked helix-turn-helix, and a 16-residue beta-hairpin) in the presence of two commonly used crowding agents, Dextran 70 (200 g/L) and Ficoll 70 (200 g/L). We found that these polymers do not induce any appreciable changes in the folding kinetics of the two helical peptides, which is somewhat surprising as the helix-coil transition kinetics have been shown to depend on viscosity. Also to our surprise and in contrast to what has been observed for larger proteins, we found that crowding leads to an appreciable decrease in the folding rate of the shortest beta-hairpin peptide, indicating that besides the excluded volume effect, other factors also need to be considered when evaluating the net effect of crowding on protein folding kinetics. A model considering both the static and the dynamic effects arising from the presence of the crowding agent is proposed to rationalize these results.

Effect of dehydration on the aggregation kinetics of two amyloid peptides.

It is well-known that water plays a crucial role in the folding, dynamics, and function of proteins. Here we provide further evidence showing that the aggregation kinetics of peptides also depend strongly on their hydration status. Using reverse micelles as a tool to modulate the accessible number of water molecules and infrared spectroscopy and transmission electron microscopy as means to monitor aggregate formation, we show that the rate of aggregation of two amyloid forming peptides increases significantly under conditions where limited hydration of the peptide molecule is expected to occur. These results not only are in accord with recent computer simulations indicating that the expulsion of interfacial water molecules is a key event in the dimerization/oligmerization of amyloid beta (Abeta) peptides but also have implications for amyloid formation in vivo where molecular crowding is expected to influence the solvation status of proteins.

Site-Specific Infrared Probes of Proteins

Infrared spectroscopy has played an instrumental role in the study of a wide variety of biological questions. However, in many cases, it is impossible or difficult to rely on the intrinsic vibrational modes of biological molecules of interest, such as proteins, to reveal structural and environmental information in a site-specific manner. To overcome this limitation, investigators have dedicated many recent efforts to the development and application of various extrinsic vibrational probes that can be incorporated into biological molecules and used to site-specifically interrogate their structural or environmental properties. In this review, we highlight recent advancements in this rapidly growing research area.

Achieving Secondary Structural Resolution in Kinetic Measurements of Protein Folding: A Case Study of the Folding Mechanism of Trp-cage

Protein folding kinetics are often measured by monitoring the change of a single spectroscopic signal, such as the fluorescence of an intrinsic fluorophore or the absorbance at a single frequency within an electronic or vibrational band of the protein backbone. While such an experimental strategy is easy to implement, the use of a single spectroscopic signal can leave important folding events undetected and overlooked. Herein, we demonstrate, using the mini-protein Trp-cage as an example, that the structural resolution of protein folding kinetics can be significantly improved when a multi-probe and multi-frequency approach is used, thus allowing a more complete understanding of the folding mechanism.

Probing the folding transition state structure of the villin headpiece subdomain via side chain and backbone mutagenesis.

Backbone-backbone hydrogen bonds are a common feature of native protein structures, yet their thermodynamic and kinetic influence on folding has long been debated. This is reflected by the disparity between current protein folding models, which place hydrogen bond formation at different stages along the folding trajectory. For example, previous studies have suggested that the denatured state of the villin headpiece subdomain contains a residual helical structure that may provide a bias toward the folded state by confining the conformational search associated with its folding. Although helical hydrogen bonds clearly stabilize the folded state, here we show, using an amide-to-ester mutation strategy, that the formation of backbone hydrogen bonds within helices is not rate-limiting in the folding of the subdomain, thereby suggesting that such hydrogen bonds are unlikely to be formed en route from the denatured to the transition state. On the other hand, elimination of hydrogen bonds within the turn region elicits a slower folding rate, consistent with the hypothesis that these residues are involved in the formation of a folding nucleus. While illustrating a potentially conserved aspect of helix-turn-helix folding, our results further underscore the inherent importance of turns in protein supersecondary structure formation.