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Robin S Dothager

Publications and source records attributed to Robin S Dothager.

9 recordsLinked to original sources
PubMed2007

Characterizing protein folding transition States using Psi-analysis.

We discuss the implementation of Psi-analysis for the structural characterization of protein folding transition states. In Psi-analysis, engineered bi-histidine metal ion binding sites are introduced at surface positions to stabilize secondary and tertiary structures. The addition of metal ions stabilizes the interaction between the two known histidines in a continuous fashion. Measuring the ratio of transition state stabilization to that of the native state provides information about the presence of the metal binding site in the transition state. Psi-Analysis uses noninvasive surface mutations and does not require specialized equipment, so it can be readily applied to characterize the folding of many proteins. As a result, this method can provide a wealth of high-resolution quantitative data for comparison with theoretical folding simulations. Additionally, investigations of other biological processes also may utilize metal binding sites and Psi-analysis to detect conformational events during catalysis, assembly, and function.

Binding Sites↗
PubMed2006

The compound 13-D selectively induces apoptosis in white blood cancers versus other cancer cell types.

As general cytotoxins are still the backbone of anticancer chemotherapy, the identification of selective inducers of cell death in defined cancer types and subtypes is one of the major goals of modern oncology research. Thus, compounds identified with such selectivity have utility as probes of cancer-type-specific biological pathways, and optimized versions have potential in targeted anticancer therapy. Described herein is the discovery that compound 13-D selectively induces apoptotic cell death in white blood cancer cell lines but not in other cancer cell lines. Further experiments indicate that this selectivity is not simply due to selective cell permeability. The compound localizes to both the nucleus and cytoplasm and arrests cells in the prophase/prometaphase of the cell cycle, and there is a very sharp dependence of activity on compound structure, with the trans-alpha,beta-unsaturated amide of 13-D being critical for inducing cell death. The macromolecular target of 13-D could be involved in white blood cell-specific oncogenic pathways.

Antineoplastic Agents↗
PubMed2005

Synthesis and identification of small molecules that potently induce apoptosis in melanoma cells through G1 cell cycle arrest.

Late-stage malignant melanoma is a cancer that is refractory to current chemotherapeutic treatments. The average survival time for patients with such a diagnosis is 6 months. In general, the vast majority of anticancer drugs operate through induction of cell cycle arrest and cell death in either the DNA synthesis (S) or mitosis (M) phase of the cell cycle. Unfortunately, the same mechanisms that melanocytes possess to protect cells from DNA damage often confer resistance to drugs that derive their toxicity from S or M phase arrest. Described herein is the synthesis of a combinatorial library of potential proapoptotic agents and the subsequent identification of a class of small molecules (triphenylmethylamides, TPMAs) that arrest the growth of melanoma cells in the G1 phase of the cell cycle. Several of these TPMAs are quite potent inducers of apoptotic death in melanoma cell lines (IC(50) approximately 0.5 muM), and importantly, some TPMAs are comparatively nontoxic to normal cells isolated from the bone marrow of healthy donors. Furthermore, the TPMAs were found to dramatically reduce the level of active nuclear factor kappa-B (NFkappaB) in the cell; NFkappaB is known to be constitutively active in melanoma, and this activity is critical for the proliferation of melanoma cells and their evasion of apoptosis. Compounds that reduce the level of NFkappaB and arrest cells in the G1 phase of the cell cycle can provide insights into the biology of melanoma and may be effective antimelanoma agents.

Amides↗
PubMed2004

Differences in the folding transition state of ubiquitin indicated by phi and psi analyses.

We compare the folding transition state (TS) of ubiquitin previously identified by using psi analysis to that determined by using analysis. Both methods attempt to identify interactions and their relative populations at the rate-limiting step for folding. The TS ensemble derived from psi analysis has an extensive native-like chain topology, with a four-stranded beta-sheet network and a portion of the major helix. According to analysis, however, the TS is much smaller and more polarized, with only a local helix/hairpin motif. We find that structured regions can have values far from unity, the canonical value for such sites, because of structural relaxation of the TS. Consequently, these sites may be incorrectly interpreted as contributing little to the structure of the TS. These results stress the need for caution when interpreting and drawing conclusions from analysis alone and highlight the need for more specific tools for examining the structure and energetics of the TS ensemble.

Computer Simulation↗
PubMed2004

Random-coil behavior and the dimensions of chemically unfolded proteins.

Spectroscopic studies have identified a number of proteins that appear to retain significant residual structure under even strongly denaturing conditions. Intrinsic viscosity, hydrodynamic radii, and small-angle x-ray scattering studies, in contrast, indicate that the dimensions of most chemically denatured proteins scale with polypeptide length by means of the power-law relationship expected for random-coil behavior. Here we further explore this discrepancy by expanding the length range of characterized denatured-state radii of gyration (R(G)) and by reexamining proteins that reportedly do not fit the expected dimensional scaling. We find that only 2 of 28 crosslink-free, prosthetic-group-free, chemically denatured polypeptides deviate significantly from a power-law relationship with polymer length. The R(G) of the remaining 26 polypeptides, which range from 16 to 549 residues, are well fitted (r(2) = 0.988) by a power-law relationship with a best-fit exponent, 0.598 +/- 0.028, coinciding closely with the 0.588 predicted for an excluded volume random coil. Therefore, it appears that the mean dimensions of the large majority of chemically denatured proteins are effectively indistinguishable from the mean dimensions of a random-coil ensemble.

Guanidine↗
PubMed2004

Early collapse is not an obligate step in protein folding.

The dimensions and secondary structure content of two proteins which fold in a two-state manner are measured within milliseconds of denaturant dilution using synchrotron-based, stopped-flow small-angle X-ray scattering and far-UV circular dichroism spectroscopy. Even upon a jump to strongly native conditions, neither ubiquitin nor common-type acylphosphatase contract prior to the major folding event. Circular dichroism and fluorescence indicate that negligible amounts of secondary and tertiary structures form in the burst phase. Thus, for these two denatured states, collapse and secondary structure formation are not energetically downhill processes even under aqueous, low-denaturant conditions. In addition, water appears to be as good a solvent as that with high concentrations of denaturant, when considering the over-all dimensions of the denatured state. However, the removal of denaturant does subtly alter the distribution of backbone dihedral phi,psi angles, most likely resulting in a shift from the polyproline II region to the helical region of the Ramachandran map. We consider the thermodynamic origins of these behaviors along with implications for folding mechanisms and computer simulations thereof.

Acid Anhydride Hydrolases↗
PubMed2004

Discerning the structure and energy of multiple transition states in protein folding using psi-analysis.

We quantify the degree to which folding occurs along a complex landscape with structurally distinct pathways using psi-analysis in combination with a protein engineering method that identifies native, non-covalent polypeptide interactions and their relative populations at the rate-limiting step. By probing the proximity of two specific partners, this method is extremely well-suited for comparison to theoretical simulations. Using ubiquitin as a model system, we detect individual pathways with site-resolved resolution, demonstrating that the protein folds through a native-like transition state ensemble with a common nucleus that contains heterogeneous features on its periphery. The consensus transition state topology has part of the major helix docked against four properly aligned beta-strands. However, structural heterogeneity exists in the transition state ensemble, wherein peripheral regions are differentially populated according to their relative stability. Pathway diversity reflects the variable order of formation of these peripheral elements, which radiate outward from the common nucleus. These results, which show only moderate agreement with traditional mutational phi-analysis, provide an extraordinarily detailed and quantitative description of protein folding.

Amino Acid Sequence↗