Framed: The Interior Woman Artist-Observer in Modernity.

Beginning in fin-de-siécle London and closing with the aged modernism of New York in the 1930s, my project examines the spatial and social possibilities engendered through modernist versions of a pervasive figure in Western literature and visual art: the woman at the window. Using architectural and visual theory in dialogue with cultural history and literary narrative, I examine the window—a classic vantage for both the artist and the domestic woman—as a site/object/image that modernist writers use to negotiate tensions and form creative integrations of the aesthetic and the domestic and to define women’s evolving relationships to private and public spaces. Walter Benjamin’s Arcades Project, with its fixation on spatial interpenetration, provides the theoretical basis for this study, and the literary writers explored include Amy Levy, Edith Wharton, E. M. Forster, Virginia Woolf, Djuna Barnes, and Nathanael West. I aim to provide a case study in the continual feminist project of reworking traditional roles and spaces and to trouble the street-centrist grounds of value on which many modernist critics think it means to write of modernity and of women’s place and experience in that period.Ph.D.English Language & LiteratureUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/64689/1/smithrg_1.pd

Protein folding in the cell: challenges and progress.

It is hard to imagine a more extreme contrast than that between the dilute solutions used for in vitro studies of protein folding and the crowded, compartmentalized, sticky, spatially inhomogeneous interior of a cell. This review highlights recent research exploring protein folding in the cell with a focus on issues that are generally not relevant to in vitro studies of protein folding, such as macromolecular crowding, hindered diffusion, cotranslational folding, molecular chaperones, and evolutionary pressures. The technical obstacles that must be overcome to characterize protein folding in the cell are driving methodological advances, and we draw attention to several examples, such as fluorescence imaging of folding in cells and genetic screens for in-cell stability. DOI 10.1016DOI 10. /j.sbi.2010 Introduction Chris Anfinsen launched the field of protein folding by showing that ribonuclease (specifically, bovine pancreatic ribonuclease A) could refold to an active enzyme after reductive denaturation. Naturally, ribonuclease became emblematic of the fundamental tenet of protein folding -that the primary sequence of a protein specifies an energy landscape and a successful route to the native state at the global energy minimum. Yet ribonuclease folds in vivo during a complex journey through the secretory pathway of the cell. Notably, in its biological folding process, ribonuclease confronts milieux that are densely crowded with macromolecules; it samples the microenvironments of the ribosome tunnel, the translocon, and the ER lumen; it has the opportunity to fold from its N-terminus to C-terminus; and it is not left on its own, but instead is accompanied by lumenal chaperones that facilitate its folding and post-translational modification. As this journey abundantly illustrates, protein folding in the cell confronts many issues that are nonexistent in high dilution refolding experiments. It is thus not surprising that an increasing research effort is being applied to issues and processes involved in cellular protein folding. This review presents research in the expanding area of protein folding in the cell. We mainly confine our discussion to publications on cellular protein folding that have appeared in the last two years and to areas that have not been recently reviewed. We first describe a number of issues, such as macromolecular crowding and cotranslational folding, that arise when considering folding in the cell but are absent in vitro ( Macromolecular crowding A striking difference between most in vitro folding experiments and the cellular environment is the high concentration of macromolecules, which severely limits the cellular volume accessible to a polypeptide chain. Like many issues related to folding in the cell, determining the effects of crowding on folding presents major technical challenges to both computational and experimental studies; moreover, crowding is generally accompanied by other effects including altered diffusion and weak interactions. Macromolecular crowding also affects the viscosity of the cellular environment and solvent viscosity has been invoked as an important factor in determining folding rates and mechanisms (e.g., [9]). In a recent study, Dhar et al. used computational and experimental approaches to study the effects of a model crowder, Ficoll, on activity and folding of phosphoglycerate kinase (PGK), a 412-aa protein with two domains connected by a flexible hinge www.sciencedirect.com Current Opinion in Structural Biology 2011, 21:32-41 decreased the inter-domain separation. Also, the relaxation rate from temperature-jump unfolding experiments showed a maximum at 100 g/L Ficoll. The authors interpreted this result as arising from opposing effects of crowding and viscosity Hindered mobility and sticky neighbors Several recent studies show that the cellular environment affects macromolecular motion and provide insight into how. For example, two recent papers have used fluorescence recovery after photobleaching to monitor translational diffusion of GFP constructs in E. coli cells Vectorial synthesis and roles of mRNA and ribosomes in folding Newly synthesized polypeptide chains emerge from the ribosome vectorially, allowing their N-terminal portions to sample conformational space before the chain is completely synthesized. Additionally, the earliest environments encountered by a nascent chain are the ribosome tunnel and ribosome-associated chaperones. There have been excellent recent reviews on issues related to cotranslational folding including one in this issue A single domain stabilized by many long-range contacts is not expected to fold until the entire chain is complete, and recent studies of ribosome-bound nascent chains (RNCs) have confirmed this expectation for an SH3 domain by NMR [20] and GFP by observing chromophore maturation [21 ]. In fact, the NMR studies on SH3 RNCs reveal little or no compaction until the entire chain has exited the ribosomal tunnel [20]. By comparison, RNCs of the larger GFP may populate a more compact state before full translation [21 ]. The interrelationship of translation rate and folding has been discussed in a number of recent reviews [22][23][24][25]. Intuitively, slowing translation might allow more time for proper folding, and indeed in a recent study mutant ribosomes with reduced translation rates increased the soluble expression of eukaryotic proteins in E. coli [26 ]. The messenger RNA sequence can also affect the translation rate either through the use of rare codons [22,25,[27][28][29] or by RNA folding [29,30]. Either of these factors may be changed by synonymous mutations, where mRNA sequences are altered without affecting the encoded amino acid. Increasing the translation rate of the multidomain E. coli protein SufI by synonymously exchanging rare codon clusters for common codons was found to decrease cotranslational folding and the production of mature, folded protein [31 ]. Intriguingly, the mutation that leads to the deletion of F508 (DF508) in the cystic fibrosis transmembrane conductance regulator (CFTR), the most common mutation linked to cystic fibrosis, also changes the preceding codon for Ile507 [32 ]. Alteration of the local mRNA structure in the mutant retards translation and presumably impairs folding, increasing cotranslational ubiquitination and leading to protein degradation [32 ,33]. Restoration of the original Ile507 codon in the DF508 background significantly increases the amount of mature CFTR in the plasma membrane, demonstrating the potential impact of synonymous mutations on in vivo protein folding and maturation [32 ,33]. As described in recent reviews Molecular chaperones remodel the in vivo folding energy landscape The ability of molecular chaperones to interact with nascent or incompletely folded chains so as to favor successful folding and disfavor aggregation is well established. Yet the impact of chaperones on the folding mechanisms and stabilities of their clients is less clear, despite expanding literature on the functions and substrate-omes of several chaperones (e.g., Consider the case of arguably the best-studied chaperone, the E. coli chaperonin GroEL and its partner GroES. On Spatial organization, membranes and compartmentalization Cellular interiors are highly anisotropic with elaborate and physiologically critical architectures. This subcellular organization plays a major role in folding at several levels. For example, the native structures of membrane proteins are tuned to the diverse microenvironments and twodimensional character of membranes. Additionally, membranes are barriers, requiring proteins made on one side but destined to perform their functions extracytoplasmically to be translocated across the membrane, in most cases unfolded. For secretory proteins, passage through cellular compartments is highly choreographed with an assembly line of modifying enzymes, chaperones and transport mechanisms. Compartmentalization also opens up the possibility of chemical gradients, for example pH or oxidizing potential. Taken together, the spatial organization and compartmentalization of the cellular environment enable folding to occur in temporally optimized steps. Bacterial proteins destined for the periplasm or the cell exterior can be secreted across membranes either folded (e.g., by the Tat system) or unfolded (e.g., by the Sec system) Proteins that translocate through the Sec channel in bacteria and eukaryotes are greeted by an array of chaperones and modifying enzymes that alter the folding energy landscape. In addition, they move from an ATPrich, reducing environment to one that is ATP-poor and oxidizing as they enter either the bacterial periplasm, mitochondrial intermembrane space (IMS) or the eukaryotic ER. We refer interested readers to recent reviews of protein folding in the ER Exposure of nascent chains to the oxidizing environment of the periplasm or ER lumen enables step-wise disulfide bond formation, fixing the topology of secretory proteins. Not surprisingly, the timing and specificity of disulfide bond formation is integral to their in vivo folding, and this issue has been widely studied in eukaryotic proteins Many proteins that fold in the ER lumen are large with complicated topologies including multiple domains, disulfides, and glyosylation sites, which play roles in specialized in vivo folding mechanisms. A recent study on the influenza membrane glycoprotein neuraminidase Promising new methods for the study of in vivo protein folding The Holy Grail in studies of protein folding in the cell is to directly observe a protein of interest (POI) in intact cells and to characterize its folding, both thermodynamically and kinetically, in situ. Not surprisingly, this has proven exceedingly difficult. Several years ago, Ghaemmaghami and Oas took advantage of E. coli's urea tolerance to perform in-cell urea titrations of the l repressor headpiece utilizing a novel hydrogen exchange/ mass spectrometry method to assess stability Exciting recent work from the Gruebele lab combines temperature-jump perturbation methods with fast relaxation imaging (FReI) to interrogate the in vivo folding landscape of a POI, here a temperature-sensitive variant of phosphoglycerate kinase (tsPGK) In-cell NMR, recently reviewed by Pielak et al. [70] and by Ito and Selenko [71], is a potentially powerful approach to study proteins in vivo and to gain insight into their stability and folding mechanisms. Unfortunately, many folded proteins fail to show measurable NMR spectra in the cellular environment, most likely because of hindered rotational diffusion. Despite the inherent obstacles, the Shirakawa group has had impressive success applying NMR to small proteins in eukaryotic cells Clever use of split reporters, in which folding of the POI is coupled to successful binding and folding of two pieces of 36 Folding and Binding Figure 2 Monitoring protein folding kinetics in a living cell using fast relaxation imaging (FReI) These approaches to in-cell stability use a single flanking reporter and are clearly powerful as selections for enhanced stability. However, they are end-point assays based on the proteolytic lability of the fusion construct when the POI is unstable and therefore cannot readily yield an estimate of folding free energy. A related in vivo screen flanks the POI between a DNA-binding domain and a transcriptional activation domai

Evolution of Male-Killer Suppression in a Natural Population

Male-killing bacteria are widespread in arthropods, and can profoundly alter the reproductive biology of their host species. Here we detail the first case of complete suppression of a male killer. The nymphalid butterfly Hypolimnas bolina is infected with a strain of the bacterium Wolbachia, wBol1, which kills male host embryos in Polynesian populations, but does not do so in many areas of Southeast Asia, where both males and female adults are naturally infected, and wBol1-infected females produce a 1:1 sex ratio. We demonstrate that absence of male killing by wBol1 is associated with dominant zygotic suppression of the action of the male killer. Simulations demonstrate host suppressors of male-killer action can spread very rapidly, and historical data indicating the presence of male killing in Southeast Asia in the very recent past suggests suppressor spread has been a very recent occurrence. Thus, male killer/host interactions are much more dynamic than previously recognised, with rapid and dramatic loss of the phenotype. Our results also indicate that suppression can render male killers completely quiescent, leading to the conclusion that some species that do not currently express a male killer may have done so in the past, and thus that more species have had their biology affected by these parasites than previously believed

Time-resolved optical methods for the study of protein folding and conformation.

Studies of protein folding are necessary in order to understand how a one dimensional chain of amino acids becomes a functional three-dimensional protein. An understanding of the physical basis of this process can lead to the ability to predict protein structures from amino acid sequences and to predict how the environment affects protein folding. Laser pump-probe spectroscopy can monitor protein folding in real time on time scales from nanoseconds to seconds. This spectroscopy may be used to excite molecules and probe their conformationally sensitive excited states, transient absorption for example, or to change solution characteristics such as pH and temperature in order to trigger protein conformational changes. Comparisons were made between the tryptophan triplet state lifetimes determined by phosphorescence and by transient absorption for alkaline phosphatase and phosphoglycerate kinase. Differences found in lifetimes reported by the two techniques were due to the transient absorption of non-luminescent species. Thus, transient absorption may be used to study protein conformation and folding in cases where the phosphorescence signal is unmeasurable; additionally it provides a window unto tryptophan photochemistry. Experiments on early events in protein folding or unfolding allow direct measurements of the time scales and mechanisms of collapse from the random coil state or of the breakdown of the native structure. In these experiments, a laser pulse was used to abruptly change the solution pH or temperature in order to initiate protein folding or unfolding. pH jumps of 2 pH units were achieved. However, the ultra-violet absorption of the pH jumping dyes obscures intrinsic protein fluorescence and absorption; therefore, peptides or proteins used for such experiments must be labeled with extrinsic probes. Such probes must be able to reveal protein conformational changes without interfering in the protein folding process itself. Temperature jump experiments allow studies of protein unfolding due to thermal denaturation as well as protein folding from cold denatured states. Steady state measurements of the cold denaturation of apomyoglobin at various pHs and heat denaturation of ribonuclease T1 were conducted, a temperature jump apparatus was constructed and temperature jumps of 15-20\sp\circC were demonstrated.Ph.D.Biological SciencesBiophysicsUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/129992/2/9711968.pd

Protein Folding in the Cell: Challenges and Progress

It is hard to imagine a more extreme contrast than that between the dilute solutions used for in vitro studies of protein folding and the crowded, compartmentalized, sticky, spatially inhomogeneous interior of a cell. This review highlights recent research exploring protein folding in the cell with a focus on issues that are generally not relevant to in vitro studies of protein folding, such as macromolecular crowding, hindered diffusion, co-translational folding, molecular chaperones, and evolutionary pressures. The technical obstacles that must be overcome to characterize protein folding in the cell are driving methodological advances, and we draw attention to several examples, such as fluorescence imaging of folding in cells and genetic screens for incell stability

Phosphatidylcholine Cation—Tyrosine π Complexes: Motifs for Membrane Binding by a Bacterial Phospholipase C

Phosphatidylinositol-specific phospholipase C (PI-PLC) enzymes are a virulence factor in many Gram-positive organisms. The specific activity of the Bacillus thuringiensis PI-PLC is significantly increased by adding phosphatidylcholine (PC) to vesicles composed of the substrate phosphatidylinositol, in part because the inclusion of PC reduces the apparent Kd for the vesicle binding by as much as 1000-fold when comparing PC-rich vesicles to PI vesicles. This review summarizes (i) the experimental work that localized a site on BtPI-PLC where PC is bound as a PC choline cation—Tyr-π complex and (ii) the computational work (including all-atom molecular dynamics simulations) that refined the original complex and found a second persistent PC cation—Tyr-π complex. Both complexes are critical for vesicle binding. These results have led to a model for PC functioning as an allosteric effector of the enzyme by altering the protein dynamics and stabilizing an ‘open’ active site conformation.publishedVersio

Fluorescence Correlation Spectroscopy of Phosphatidylinositolspecific Phospholipase C Monitors the Interplay of Substrate and Activator Lipid Binding

Phosphatidylinositol-specific phospholipase C (PI-PLC) enzymes simultaneously interact with the substrate, PI, and with non-substrate lipids such as phosphatidylcholine (PC). For Bacillus thuringiensis PI-PLC these interactions are synergistic with maximal catalytic activity observed at low to moderate mole fractions of PC (XPC) and maximal binding occurring at low mole fractions of anionic lipids. It has been proposed that residues in α helix B help modulate membrane binding and that dimerization on the membrane surface both increases affinity for PC and activates PI-PLC yielding the observed PI/PC synergy. Vesicle binding and activity measurements using a variety of PI-PLC mutants support many aspects of this model and reveal that while single mutations can disrupt anionic lipid binding and the anionic lipid/PC synergy, the residues important for PC binding are less localized. Interestingly, at high XPC mutations can both decrease membrane affinity and increase activity, supporting a model where reductions in wildtype activity at XPC\u3e0.6 results from both dilution of the substrate and tight membrane binding of PI-PLC limiting enzyme hopping or scooting to the next substrate molecule. These results provide a direct analysis of vesicle binding and catalytic activity and shed light on how occupation of the activator site enhances enzymatic activity