Yi Qin Gao

The Transcriptome and DNA Methylome Landscapes of Human Primordial Germ Cells

Germ cells are vital for transmitting genetic information from one generation to the next and for maintaining the continuation of species. Here, we analyze the transcriptome of human primordial germ cells (PGCs) from the migrating stage to the gonadal stage at single-cell and single-base resolutions. Human PGCs show unique transcription patterns involving the simultaneous expression of both pluripotency genes and germline-specific genes, with a subset of them displaying developmental-stage-specific features. Furthermore, we analyze the DNA methylome of human PGCs and find global demethylation of their genomes. Approximately 10 to 11 weeks after gestation, the PGCs are nearly devoid of any DNA methylation, with only 7.8% and 6.0% of the median methylation levels in male and female PGCs, respectively. Our work paves the way toward deciphering the complex epigenetic reprogramming of the germline with the aim of restoring totipotency in fertilized oocytes.

Probing Allostery through DNA

Allostery Across DNA Proteins, such as transcription factors and RNA polymerase, bind close to each other on DNA and their function is coordinated. Kim et al. (p. 816; see the Perspective by Crothers) report single-molecule experiments that show that the DNA binding affinity of a protein is significantly altered by a second protein bound nearby. The effect oscillates between stabilizing and destabilizing the binding with a periodicity equal to the helical pitch of DNA. Allosteric coupling between a transcriptional repressor and RNA polymerase modulated gene expression in living bacteria. Proteins bound to the same, but not overlapping, stretch of DNA modulate each others DNA binding affinity. [Also see Perspective by Crothers] Allostery is well documented for proteins but less recognized for DNA-protein interactions. Here, we report that specific binding of a protein on DNA is substantially stabilized or destabilized by another protein bound nearby. The ternary complexs free energy oscillates as a function of the separation between the two proteins with a periodicity of ~10 base pairs, the helical pitch of B-form DNA, and a decay length of ~15 base pairs. The binding affinity of a protein near a DNA hairpin is similarly dependent on their separation, which—together with molecular dynamics simulations—suggests that deformation of the double-helical structure is the origin of DNA allostery. The physiological relevance of this phenomenon is illustrated by its effect on gene expression in live bacteria and on a transcription factors affinity near nucleosomes.

A Structure-Based Model for the Synthesis and Hydrolysis of ATP by F1-ATPase

Many essential functions of living cells are performed by nanoscale protein motors. The best characterized of these is F(o)F1-ATP synthase, the smallest rotary motor. This rotary motor catalyzes the synthesis of ATP with high efficiency under conditions where the reactants (ADP, H2PO4(-)) and the product (ATP) are present in the cell at similar concentrations. We present a detailed structure-based kinetic model for the mechanism of action of F1-ATPase and demonstrate the role of different protein conformations for substrate binding during ATP synthesis and ATP hydrolysis. The model shows that the pathway for ATP hydrolysis is not simply the pathway for ATP synthesis in reverse. The findings of the model also explain why the cellular concentration of ATP does not inhibit ATP synthesis.

Effects of Urea, Tetramethyl Urea, and Trimethylamine N-Oxide on Aqueous Solution Structure and Solvation of Protein Backbones: A Molecular Dynamics Simulation Study

The effects of urea, tetramethyl urea (TMU), and trimethylamine N-oxide (TMAO) on the structure and dynamics of aqueous solutions are studied using molecular dynamics simulations. It was found that urea has little effects on the water-water hydrogen-bond length and angle distributions except that it induces a slight collapse of the second shell in the hydrogen-bonding network. TMU and TMAO both strengthen the individual hydrogen bonds and significantly slow the orientational relaxation of water, but have opposite effects on the second shell structure of the hydrogen-bonding network: TMU distorts while TMAO enhances the tetrahedral water structure. Furthermore, TMAO significantly weakens the interactions between the amide carbonyl group and the water molecules, while TMU and urea both strengthen these interactions, with the effect of urea being much less significant than that of TMU. These conclusions are supported by molecular dynamics simulations of three different systems: a model amide compound CH(3)-NH-CO-CH(3) (NMA), and two polypeptides, GB1 and ELP. Consistent with earlier studies, we also found that urea interacts strongly with the carbonyl group through direct hydrogen bonding. The simulations for the denaturation of the polypeptide GB1 in urea solutions showed that the breaking of its native hydrogen bonds follows a step-by-step process and each step is strongly coupled to the formation of water-carbonyl hydrogen bonds, and to a less extent to the urea-carbonyl hydrogen-bond formation. Our simulation results reveal the potential importance of the indirect effects of cosolvents in protein denaturation or structure protection, particularly through modifying of the water-amide interactions.

On the Structure of Water at the Aqueous/Air Interface

Vibrational sum frequency spectroscopy (VSFS) and molecular dynamics (MD) simulations were used in concert to investigate the molecular structure and hydrogen bonding of the air/water interface. MD simulations were performed with a variety of water models. The results indicated that only the upper most two layers of water molecules are ordered in this system. There is a strong preference to have the top layer arranged such that the OH moiety points upward into the air. This orientational preference arises from two factors that involve the maximization of the number of hydrogen bonds formed and the minimization of partial charge that is exposed. Specifically, the lone pairs from oxygen are less likely to face into the air compared with the OH moiety because this would expose more partial charge and, therefore, be unfavorable on enthalpic grounds. The two-layer interfacial water structure model implies that there should be four distinct types of OH stretches for this system. Namely, one directs upward and another points downward in each layer. Interestingly, VSFS experiments revealed the presence of four OH stretch region peaks at 3117, 3222, 3448, and 3696 cm(-1). The phases of the 3117 and 3696 cm(-1) resonances carried a positive sign, which indicates that these features arise from OH groups with protons facing upward toward the air. The other two resonances emanate from OH groups with protons facing downward toward the bulk aqueous solution. On the basis of this, we assign the 3117 cm(-1) peak to the OH moiety from a water molecule in the second layer, which is hydrogen bonded upward toward the top layer. On the other hand, the peak at 3222 cm(-1) should arise from water molecules in the top layer with the OH moiety facing downward to hydrogen bond to the second layer. The 3448 cm(-1) peak arises from hydrogen bonding between water molecules in the second layer and the more disordered water molecules of the bulk liquid. Finally, the peak at 3696 cm(-1) is assigned to the free OH moiety pointing upward in the top layer.

The missing link between thermodynamics and structure in F1-ATPase

F1Fo-ATP synthase is the enzyme responsible for most of the ATP synthesis in living systems. The catalytic domain F1 of the F1Fo complex, F1-ATPase, has the ability to hydrolyze ATP. A fundamental problem in the development of a detailed mechanism for this enzyme is that it has not been possible to determine experimentally the relation between the ligand binding affinities measured in solution and the different conformations of the catalytic β subunits (βTP, βDP, βE) observed in the crystal structures of the mitochondrial enzyme, MF1. Using free energy difference simulations for the hydrolysis reaction ATP+H2O → ADP+Pi in the βTP and βDP sites and unisite hydrolysis data, we are able to identify βTP as the “tight” (KD = 10−12 M, MF1) binding site for ATP and βDP as the “loose” site. An energy decomposition analysis demonstrates how certain residues, some of which have been shown to be important in catalysis, modulate the free energy of the hydrolysis reaction in the βTP and βDP sites, even though their structures are very similar. Combined with the recently published simulations of the rotation cycle of F1-ATPase, the present results make possible a consistent description of the binding change mechanism of F1-ATPase at an atomic level of detail.

A Simple Theory for the Hofmeister Series

In cells, biological molecules function in an aqueous solution. Electrolytes and other small molecules play important roles in keeping the osmotic pressure of the cellular environment as well as the structure formation and function of biomolecules. The observed empirical rules such as Hofmeister series are still waiting for molecular interpretations. In this Perspective, we will discuss a simple and self-consistent theory that takes into account the cooperative effects of cations and anions in affecting water/air surface tension, water activity, and the solubility of model compounds including polypeptides. Molecular dynamics simulations used to test these theoretical models will also be discussed.

On the theory of electron transfer reactions at semiconductor electrode'liquid interfaces

Electron transfer reaction rate constants at semiconductor/liquid interfaces are calculated using the Fermi Golden Rule and a tight-binding model for the semiconductors. The slab method and a z-transform method are employed in obtaining the electronic structures of semiconductors with surfaces and are compared. The maximum electron transfer rate constants at Si/viologen2+/+ and InP/Me2Fc+/0 interfaces are computed using the tight-binding type calculations for the solid and the extended-Huckel for the coupling to the redox agent at the interface. These results for the bulk states are compared with the experimentally measured values of Lewis and co-workers, and are in reasonable agreement, without adjusting parameters. In the case of InP/liquid interface, the unusual current vs applied potential behavior is additionally interpreted, in part, by the presence of surface states.

An integrate-over-temperature approach for enhanced sampling.

A simple method is introduced to achieve efficient random walking in the energy space in molecular dynamics simulations which thus enhances the sampling over a large energy range. The approach is closely related to multicanonical and replica exchange simulation methods in that it allows configurations of the system to be sampled in a wide energy range by making use of Boltzmann distribution functions at multiple temperatures. A biased potential is quickly generated using this method and is then used in accelerated molecular dynamics simulations.

A model for the cooperative free energy transduction and kinetics of ATP hydrolysis by F1-ATPase

Although the binding change mechanism of rotary catalysis by which F1-ATPase hydrolyzes ATP has been supported by equilibrium, kinetic, and structural observations, many questions concerning the function remain unanswered. Because of the importance of this enzyme, the search for a full understanding of its mechanism is a key problem in structural biology. Making use of the results of free energy simulations and experimental binding constant measurements, a model is developed for the free energy change during the hydrolysis cycle. This model makes possible the development of a kinetic scheme for ATP hydrolysis by F1-ATPase, in which the rate constants are associated with specific configurations of the β subunits. An essential new element is that the strong binding site for ADP,Pi is shown to be the βDP site, in contrast to the strong binding site for ATP, which is βTP. This result provides a rationale for the rotation of the γ subunit, which induces the cooperativity required for a tri-site binding change mechanism. The model explains a series of experimental data, including the ATP concentration dependence of the rate of hydrolysis and catalytic site occupation for both the Escherichia coli F1-ATPase (EcF1) and Thermophilic Bacillus PS3 F1-ATPase (TF1), which have different behavior.