Here, we analyse the RanGTPase system and its coupling to receptor-mediated nuclear transport. Our simulations predict nuclear RanGTP levels in HeLa cells to be very sensitive towards the cellular energy charge and to exceed the cytoplasmic concentration »1000-fold. The steepness of the RanGTP gradient appears limited by both the cytoplasmic RanGAP concentration and the imperfect retention of nuclear RanGTP by nuclear pore complexes (NPCs), but not by the nucleotide exchange activity of RCC1. Neither RanBP1 nor the NPC localization of RanGAP has a signi®cant direct impact on the RanGTP gradient. NTF2-mediated import of Ran appears to be the bottleneck for maximal capacity of Ran-driven nuclear transport. We show that unidirectional nuclear transport can be faithfully simulated without the assumption of a vectorial NPC passage; transport receptors only need to reversibly cross NPCs and switch their af®nity for cargo in response to the RanGTP gradient. A signi®cant RanGTP gradient after nuclear envelope (NE) breakdown can apparently exist only in large cytoplasm. This indicates that RanGTP gradients can provide positional information for mitotic spindle and NE assembly in early embryonic cells, but hardly any in small somatic cells.

GTPase-activating proteins (GAPs) increase the rate of GTP hydrolysis on guanine nucleotide-binding proteins by many orders of magnitude. Studies with Ras and Rho have elucidated the mechanism of GAP action by showing that their catalytic machinery is both stabilized by GAP binding and complemented by the insertion of a so-called 'arginine finger' into the phosphate-binding pocket. This has been proposed as a universal mechanism for GAP-mediated GTP hydrolysis. Ran is a nuclear Ras-related protein that regulates both transport between the nucleus and cytoplasm during interphase, and formation of the mitotic spindle and/or nuclear envelope in dividing cells. Ran-GTP is hydrolysed by the combined action of Ran-binding proteins (RanBPs) and RanGAP. Here we present the three-dimensional structure of a Ran-RanBP1-RanGAP ternary complex in the ground state and in a transition-state mimic. The structure and biochemical experiments show that RanGAP does not act through an arginine finger, that the basic machinery for fast GTP hydrolysis is provided exclusively by Ran and that correct positioning of the catalytic glutamine is essential for catalysis.

The contributions of backbone NH group dynamics to the conformational heat capacity of the B1 domain of Streptococcal protein G have been estimated from the temperature dependence of 15 N NMR-derived order parameters. Longitudinal~R 1 ! and transverse~R 2 ! relaxation rates, transverse cross-relaxation rates~h xy !, and steady state $ 1 H%-15 N nuclear Overhauser effects were measured at temperatures of 0, 10, 20, 30, 40, and 50 8C for 89-100% of the backbone secondary amide nitrogen nuclei in the B1 domain. The ratio R 2 0h xy was used to identify nuclei for which conformational exchange makes a significant contribution to R 2 . Relaxation data were fit to the extended model-free dynamics formalism, incorporating an axially symmetric molecular rotational diffusion tensor. The temperature dependence of the order parameter~S 2 ! was used to calculate the contribution of each NH group to conformational heat capacity~C p ! and a characteristic temperature~T *!, representing the density of conformational energy states accessible to each NH group. The heat capacities of the secondary structure regions of the B1 domain are significantly higher than those of comparable regions of other proteins, whereas the heat capacities of less structured regions are similar to those in other proteins. The higher local heat capacities are estimated to contribute up to ;0.8 kJ0mol K to the total heat capacity of the B1 domain, without which the denaturation temperature would be ;9 8C lower~78 8C rather than 87 8C!. Thus, variation of backbone conformational heat capacity of native proteins may be a novel mechanism that contributes to high temperature stabilization of proteins.Keywords: B1 domain; entropy; heat capacity; NMR relaxation; order parameter; protein dynamics; protein stability Most globular proteins are marginally stable because the factors that favor formation of the native state, primarily desolvation of hydrophobic groups and formation of intramolecular hydrogen bonds and salt bridges, are almost equally balanced against those that favor denaturation, primarily the higher conformational entropy of the unfolded protein chain relative to that of the native state~Creigh-ton, 1993; Fersht, 1999!. At high temperatures, the balance between these factors is altered such that many proteins exhibit reversible thermal denaturation with a characteristic midpoint, the melting temperature~T m !. The free energy of unfolding of a proteiñ DG NϪU ! depends upon the changes in enthalpy, entropy, and heat capacity that occur upon unfolding and the temperature~T ! according to the equation~Creighton, 1993; Fersht, 1999!:in which DH 0 and DS 0 are the enthalpy and entropy changes, respectively, at a reference temperature T 0 and DC p,NϪU is the change in heat capacity at constant pressure, which is assumed to be invariant with temperature. Equation 1 indicates that native proteins may be stabilized by an increase in DH 0 or by a decrease in either DS 0 or DC p,NϪU . Thus, one possible strategy for a protein to achieve high thermal ...

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Purpose: Accurate testing of HER2 is centrally important for breast cancer therapy and prognosis. Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) are current standard testing methods. As a potential alternative for assessment of HER2, we explored quantitative real-time reverse transcription-PCR (RT-PCR), a fast and inexpensive method yielding quantitative results insensitive to interobserver variability and amenable to standardized scoring. Experimental Design: We assessed HER2 status at the DNA, mRNA, and protein levels with FISH, quantitative RT-PCR, and IHC in 136 tumor samples from 85 breast cancer patients. Expression of GRB7, MLN64, and p21, genes coregulated with HER2, was also quantified with quantitative RT-PCR and correlated with the overall survival (OS) and disease-free survival (DFS) individually and in combination with HER2. Results: Twenty-nine percent and 19% of the patients scored HER2 positive with IHC and quantitative RT-PCR, respectively. In 18 of 19 cases, HER2 statuses in tumors and lymph node metastases were identical. HER2 status significantly correlated with DFS when determined by IHC (P < 0.01), quantitative RT-PCR (P < 0.003), but not with FISH (P = 0.09). The combination of HER2 with MLN64, but not with GRB7 or p21, enhanced the prognostic power for the DFS (P < 0.00005) and OS (P < 0.0008).Conclusions: Quantitative RT-PCR seems to be clinically as useful in the assessment of HER2 status as IHC and FISH, yielding comparable correlations of HER2 status with the OS and DFS. Thus, quantitative RT-PCR analysis of HER2 or HER2 plus MLN64 is a promising complement or alternative to current methods for HER2 testing, particularly in laboratories lacking FISH or IHC technology.