Nucleocytoplasmic transport: a thermodynamic mechanism

The nuclear pore supports molecular communication between cytoplasm and nucleus in eukaryotic cells. Selective transport of proteins is mediated by soluble receptors, whose regulation by the small GTPase Ran leads to cargo accumulation in, or depletion from the nucleus, i.e., nuclear import or nuclear export. We consider the operation of this transport system by a combined analytical and experimental approach. Provocative predictions of a simple model were tested using cell-free nuclei reconstituted in Xenopus egg extract, a system well suited to quantitative studies. We found that accumulation capacity is limited, so that introduction of one import cargo leads to egress of another. Clearly, the pore per se does not determine transport directionality. Moreover, different cargo reach a similar ratio of nuclear to cytoplasmic concentration in steady-state. The model shows that this ratio should in fact be independent of the receptor-cargo affinity, though kinetics may be strongly influenced. Numerical conservation of the system components highlights a conflict between the observations and the popular concept of transport cycles. We suggest that chemical partitioning provides a framework to understand the capacity to generate concentration gradients by equilibration of the receptor-cargo intermediary.Comment: in press at HFSP Journal, vol 3 16 text pages, 1 table, 4 figures, plus Supplementary Material include

Structural effects of phosphorylation and β-O-GlcNAcylation on α-helices and structural effects of phosphorylation and R406W on tau395-411

The dynamic interplay between phosphorylation and beta-O-GlcNAcylation (OGlcNAc) of serine and threonine plays critical roles in numerous intracellular processes. Changes in phosphorylation and OGlcNAcylation are linked to Alzheimer's disease, diabetes, and cancer. We have conducted a systematic study on a model alpha-helix to determine the structural effects of phosphorylation and OGlcNAcylation of serine and threonine residues, on the N-terminus, C-terminus, and internal positions (Ac-XKAAXAKAAXAKAAGY-NH2, Ac-YGAKAAAAKAAAAKAX-NH 2). We found that both phosphorylation and OGlcNAcylation on the N-terminus increase alpha-helix stability, with phosphorylation exhibiting a greater increase in alpha-helix stability than OGlcNAcylation. These stabilizing effects were found to be greater for threonine than serine, and for the dianionic phosphate over the monoanionic phosphate. In contrast, both phosphorylation and OGlcNAcylation reduced helix stability on internal and C-terminal positions relative to serine or threonine. These effects are not simply electrostatic interactions; we observe a unique cyclization of serine and threonine residues due to an intra-residue phosphate-amide hydrogen bond. Furthermore this interaction is greater for threonine than serine and for the dianionic phosphate over the monoanionic phosphate. On the N-terminus NMR data are consistent with an alpha-helix capping mechanism in which the phosphate hydrogen bonds to its own amide, organizing and nucleating the first turn of the alpha-helix through an induced n to pi star interaction between the i-1 carbonyl and the i (phosphorylated) carbonyl. On internal and C-terminal positions, alpha-helix destabilization is due to this phosphate amide intra-residue hydrogen bond disrupting the backbone hydrogen bonding network. Interestingly, the overall effects of phosphorylation and OGlcNAcylation on an alpha-helix are analogous to the effects observed for proline, with the effects of phosphothreonine greater than the effects of proline on the alpha-helix at all positions. To further understand the effects of serine GlcNAcylation on alpha-helices, we sought to explore possible side-chain interactions with neighboring residues, such as hydrophobic, CH/pi, or boronic acid/diol conjugates. A series of Baldwin model alpha-helices (Ac-AKAAAAKAAAAKAAGY-NH2) were designed to explore i+2, i+3, i+4, and i+7 interactions utilizing 4-iodo-phenylalanine (4-iodo-Phe) or 4-B(OH)2-phenylalanine (4-B(OH)2-Phe) as interacting residues. Although no interaction was found to exist between GlcNAc and either 4-iodo-Phe or 4-B(OH)2-Phe, an alpha-helix stabilizing interaction was found involving lysine and boronic acid in a relative i / i+4 relationship. The interaction between lysine and boronic acid exhibited an increase in alpha-helix stability as the concentration of KF was increased, yet showed no increase in alpha-helix stability when NaCl was used rather than KF. This observation is consistent with fluoride ions playing a crucial role in the alpha-helix stabilizing interaction, as well as a mechanism involving more than a simple electrostatic model. Phosphorylation is known to affect protein structure when there is no defined secondary structure such as an alpha-helix. Many examples exist where phosphorylation sites have been identified in natively disordered proteins, such as the microtubule binding protein tau. Hyperphosphorylation of tau is associated with numerous neurodegenerative diseases, most notably Alzheimer's disease (AD). Within tau, phosphorylation of Ser404 has shown to occur within patients with AD. Furthermore, elevated rates of AD onset have been observed due to a mutation of Arg 406 to Trp406. Given the roles of both Ser404 phosphorylation and R406W mutation in the onset of AD and other neurodegenerative disorders, I sought to determine the local structural effects of both phosphorylation and R406W mutation within tau. Due to the lack of organized secondary structure, one way to study regions of disordered proteins is to study small peptide fragments. Tetra-peptides of the sequence TSPX, representing residues 403-406 of tau, were synthesized, containing either or both phosphorylated Thr403 and Ser404 residues, containing either the native arginine or tryptophan mutation. We have found that phosphorylation of Ser404 and R406W mutation both independently and dependently lead to a higher population of cis amide bonds. To validate the data obtained from tetra-peptides, larger tau395-411 peptides with synthesized with both unmodified and phosphorylated Ser404 and either Arg406 or Trp406. The data herein are consistent with both phosphorylation of Ser404 and R406W mutation leading to a higher population of cis amide bonds within tau. This increase in cis-Pro amide bond population may be directly correlated to the onset of AD

On the failure of water to freeze from its surface

We show that water in equilibrium is not expected to freeze from its surface inward, contrary to the expectation of elementary theories. We do this in two ways; by utilizing a recent calculation of the interfacial energy of the ice/water interface, and by an explicit calculation of the surface free energy of a water/vapor interface which incorporates a film of ice

Cyclopean vs. Dominant Eye in Gaze-Interface-Tracking

User-centered design questions in gaze interfaces have been explored in multitude empirical investigations. Interestingly, the question of what eye should be the input device has never been studied. We compared tracking accuracy between the “cyclopean” (i.e., midpoint between eyes) dominant and non-dominant eye. In two experiments, participants performed tracking tasks. In Experiment 1, participants did not use a crosshair. Results showed that mean distance from target was smaller with cyclopean than with dominant or non-dominant eyes. In Experiment 2 participants controlled a crosshair with their cyclopean, dominant and non-dominant eye intermittently and had to align the crosshair with the target. Overall tracking accuracy was highest with cyclopean eye, yet similar between cyclopean and dominant eye in the second half of the experiment. From a theoretical viewpoint, our findings correspond with the cyclopean eye theory of egocentric direction and lend support to the hemispheric laterality approach of eye dominance. From a practical viewpoint, we show that what eye to use as input should be a design consideration in gaze interfaces

Design of a Redox-Sensitive Supramolecular Protein Assembly System Operating in Live Cells

A fusion construct between Citrine (a YFP variant) and human ferritin (H-chain) was recently shown to form supramolecular assemblies of micrometer size when expressed in mammalian cells. The assembly process is driven by weak hydrophobic interactions leading to dimerization of YFP. Protein assembly could be suppressed at the gene level by mutation in the primary sequence of the construct. In this work, we describe the engineering of a self-assembly interface sensitive to redox state in the cell. Key hydrophobic residues of YFP were mutated systematically to cysteines. Supramolecular assembly of the Citrine–ferritin construct was in some cases preserved by formation of disulfide bonds in place of hydrophobic interactions. In others cases, assembly was abolished, resulting in a diffuse distribution of the expressed protein. A specific variant that remained diffuse under normally reducing intracellular conditions was found to self-assemble rapidly upon exposure to a thiol-specific oxidizing reagent