Non-native Forms Research Articles

Chaperone rings in protein folding and degradation.

Chaperone rings play a vital role in the opposing ATP-mediated processes of folding and degradation of many cellular proteins, but the mechanisms by which they assist these life and death actions are only beginning to be understood. Ring structures present an advantage to both processes, providing for compartmentalization of the substrate protein inside a central cavity in which multivalent, potentially cooperative interactions can take place between the substrate and a high local concentration of binding sites, while access of other proteins to the cavity is restricted sterically. Such restriction prevents outside interference that could lead to nonproductive fates of the substrate protein while it is present in non-native form, such as aggregation. At the step of recognition, chaperone rings recognize different motifs in their substrates, exposed hydrophobicity in the case of protein-folding chaperonins, and specific "tag" sequences in at least some cases of the proteolytic chaperones. For both folding and proteolytic complexes, ATP directs conformational changes in the chaperone rings that govern release of the bound polypeptide. In the case of chaperonins, ATP enables a released protein to pursue the native state in a sequestered hydrophilic folding chamber, and, in the case of the proteases, the released polypeptide is translocated into a degradation chamber. These divergent fates are at least partly governed by very different cooperating components that associate with the chaperone rings: that is, cochaperonin rings on one hand and proteolytic ring assemblies on the other. Here we review the structures and mechanisms of the two types of chaperone ring system.

Antibodies to the Nonnative Forms of d-Glyceraldehyde-3-Phosphate Dehydrogenase: Identification, Purification, and Influence on the Renaturation of the Enzyme

Monoclonal antibodies of two clones reacting with the nonnative forms of d-glyceraldehyde-3-phosphate dehydrogenase, EC 1.2.1.12 (GAPDH), were obtained. Antibodies of clone 6C5 belonged to IgG1 subtype; antibodies of clone 6G7 belonged to IgM type. The interaction of antibodies of both clones with the immobilized and soluble enzyme was studied. The specificity of antibodies to the definite oligomeric forms was demonstrated on immobilized monomers, dimers, and tetramers of GAPDH. The affinity of antibodies to monomeric and dimeric forms of GAPDH, either active or not, was demonstrated. At the same time the antibodies did not react with the tetrameric enzyme. The binding of antibodies had no influence on the enzymatic activity. However, the addition of antibodies to the denatured enzyme blocked the spontaneous renaturation of GAPDH. The immobilized antibodies of both clones were successfully used for the purification of GAPDH solution from the denatured admixtures.

N-Ethylmaleimide-Modified Hsp70 Inhibits Protein Folding

Hsp70 molecular chaperones facilitate protein folding and translocation by binding to hydrophobic regions of nascent or unfolded proteins, thereby preventing their aggregation. N-Ethylmaleimide (NEM) inhibits the ATPase and protein translocation-stimulating activities of the yeast Hsp70 Ssa1p by modifying its three cysteine residues, which are located in its ATPase domain. NEM alters the conformation of Ssa1p and disrupts the coupling between its nucleotide- and polypeptide-binding domains. Ssa1p and the yeast DnaJ homolog Ydj1p constitute a protein folding machinery of the yeast cytosol. Using firefly luciferase as a model protein to study chaperone-dependent protein refolding, we have found that NEM also inhibits the protein folding activity of Ssa1p. Interestingly, the NEM-modified protein (NEM-Ssa1p) is a potent inhibitor of protein folding. NEM-Ssa1p can prevent the aggregation of luciferase and stimulate the ATPase activity of Ssa1p suggesting that it acts as an inhibitor by binding to nonnative forms of luciferase and by competing with them for the polypeptide binding site of Ssa1p. NEM-Ssa1p inhibits Ssa1p/Ydj1p-dependent protein refolding at different stages indicating that the chaperones bind and release nonnative forms of luciferase multiple times before folding is completed.

Global unfolding of a substrate protein by the Hsp100 chaperone ClpA.

The bacterial protein CIpA, a member of the Hsp100 chaperone family, forms hexameric rings that bind to the free ends of the double-ring serine protease ClpP. ClpA directs the ATP-dependent degradation of substrate proteins bearing specific sequences, much as the 19S ATPase 'cap' of eukaryotic proteasomes functions in the degradation of ubiquitinated proteins. In isolation, ClpA and its relative ClpX can mediate the disassembly of oligomeric proteins; another similar eukaryotic protein, Hsp104, can dissociate low-order aggregates. ClpA has been proposed to destabilize protein structure, allowing passage of proteolysis substrates through a central channel into the ClpP proteolytic cylinder. Here we test the action of ClpA on a stable monomeric protein, the green fluorescent protein GFP, onto which has been added an 11-amino-acid carboxy-terminal recognition peptide, which is responsible for recruiting truncated proteins to ClpAP for degradation. Fluorescence studies both with and without a 'trap' version of the chaperonin GroEL, which binds non-native forms of GFP, and hydrogen-exchange experiments directly demonstrate that ClpA can unfold stable, native proteins in the presence of ATP.

Molten globule versus variety of intermediates: influence of anions on pH-denatured apomyoglobin

The molten globule state was shown to be the third thermodynamic state of protein molecules in addition to their native and unfolded states. On the other hand, it was reported that optical and hydrodynamic properties of pH-denatured apomyoglobin depend on the nature of anions added to the protein solution. This observation was used to conclude that there are many ‘partly folded’ intermediates between the native and unfolded states rather than one distinct molten globule state. However, little is known on the structures of pH-denatured apomyoglobin in the presence of different anions. Two tyrosine residues in horse apomyoglobin have been successively modified by the reaction with tetranitromethane. This approach was employed to measure the distances between tryptophans and modified tyrosines in different states of apomyoglobin by the method of direct energy transfer. Experimental data show that the distance between the middle of the A-helix and the beginning of the G-helix and/or the end of the H-helix in ‘anion-induced’ states are very close to those in the native holo- and apomyoglobins. This suggests that the AGH helical complex, being the most structured part of apomyoglobin in the molten globule state, exists also in pH-denatured apomyoglobin in the presence of different anions. Consequently, all non-native forms of apomyoglobin studied so far share the common important feature of its native structure.

ELISA methods for the analysis of antibody responses induced in multiple sclerosis patients treated with recombinant interferon-β

Upon treatment with protein therapeutics, a subset of patients will typically develop antibodies against the drug. These anti-drug antibodies can be of concern because they have the potential to alter the drug's therapeutic activity. In the case of relapsing-remitting multiple sclerosis (RRMS) patients receiving recombinant interferon-β (IFN-β), those receiving BETASERON® (IFN-β-1b; E. coli expressed, non-glycosylated, des-Met-1, Cys17Ser recombinant IFN-β) have a higher incidence of IFN-β specific antibodies compared to those receiving AVONEX® (IFN-β-1a; mammalian cell-expressed, natural sequence, glycosylated recombinant IFN-β). The current study reports the development and characterization of ELISAs that detect distinct components of the anti-IFN-β response in patients' sera, and therefore can potentially be used to characterize the composition of the anti-IFN-β antibody response. ELISAs were developed using a constant detecting reagent but a variety of IFN-β-derived test antigens (e.g., native IFN-β, biotinylated IFN-β, IFN-β peptides) and capture methods. Assays were characterized using serum samples from a small number of patients treated with recombinant IFN-β (either BETASERON® or AVONEX®). Assays in which IFN-β was captured via a specific mAb, or in which biotinylated IFN-β was captured via streptavidin, detected serum antibodies that recognize IFN-β in its native structural state. In contrast, assays in which IFN-β was coated directly onto the assay plates detected antibodies that recognize forms of IFN-β possessing a folded structure distinct from the native structure. Certain epitopes present on native IFN-β were not represented in these assays in which the test antigen was directly coated on plastic. Antibodies specific for linear epitopes could be detected using linear peptides as test antigens; the locations of these epitopes were mapped by reference to the X-ray crystal structure of IFN-β-1a. Together, these data show that the mode of antigen presentation employed in IFN-β ELISAs determines which antibody specificities are detected, and can affect whether or not a given serum sample is identified as positive for anti-IFN-β antibodies. As a consequence, screening samples in a single ELISA format presenting IFN-β in a non-native form may lead to underestimation of the incidence of IFN-β treated MS patients that have generated antibodies specific to the native, active form of the drug.

Molten globules

What are they? Molten globules are partially folded proteins that have some characteristics of both folded (or ‘native’) and unfolded proteins. Like a native protein, the classic molten globule is compact and has native secondary structure, such as α-helices and β-sheets; like an unfolded protein, it lacks specific native tertiary interactions. When were they discovered? As early as the 1960s, there was evidence that under certain conditions protein molecules might have properties intermediate between the rigid native and fully unfolded states. The concept of such a protein as an intermediate in protein folding was suggested by the late Oleg Ptitsyn in the 1970s. A compact, partly folded protein was first observed experimentally in the early 1980s and the term molten globule was coined in 1983. How can I make one? Take a protein and dissolve it under mildly denaturing conditions, such as low pH, low concentrations of urea, elevated temperature or a lack of cofactors like metal ions or heme groups. But beware, only certain proteins form molten globules, the best-studied being β-lactoglobulin, carbonic anhydrase, apomyoglobin, cytochrome c and α-lactalbumin. How do I know if I have one? A variety of biophysical techniques, including circular dichroism, can be used to detect the hallmarks of a molten globule: lack of specific fixed tertiary contacts and the presence of native secondary structure. What do they look like? Who knows? The structural analysis of molten globules is difficult because they are flexible structures and consist of an interconverting ensemble of conformations, so it's hard to apply standard X-ray and NMR techniques. Advances in NMR, however, mean that some structural information is beginning to emerge for apomyoglobin and α-lactalbumin. Do they existin vivo? Possibly. Transient structures detected in studies of the folding of certain small proteins are similar to the stable molten globules that can be generated under mildly denaturing conditions. So, whether or not molten globules represent genuine in vivo folding intermediates, they could provide insights into the nature of transient folding intermediates that are, well, too transient to study in detail. When is a molten globule not a molten globule? The term molten globule has been used to describe a wide range of partly folded states of proteins, giving rise to a lively debate as to which of these conform to the original definition. The term ‘pre-molten globule’ has been used for an intermediate between the unfolded protein and the molten globule. Confusingly, some authors have also used the term pre-molten globule to describe a more structured state of the α-lactalbumin molten globule. The term ‘highly-ordered molten globule’ has been used to describe an intermediate between the molten globule and the native protein. Because they have stable tertiary contacts, it has been possible to determine the structure of the highly-ordered molten globules of apocytochrome b562 and interleukin-4 using NMR. But their tertiary contacts also mean that these systems might be better described as native proteins with regions of significant dynamic disorder. Will the real molten globules please stand up? Do they have any biological significance? The misfolding and aggregation of proteins is now known to be involved in several diseases, including cystic fibrosis and the amyloid diseases. A protein that fails to fold properly and gets trapped in a folding intermediate — such as the molten globule — could be mislocated in the cell, or aggregate and be converted to a non-native fibrillar form. C Redfield, Oxford Centre for Molecular Sciences, University of Oxford, South Parks Road, Oxford OX1 3QT, UK.

A conformational change in cytochrome c of apoptotic and necrotic cells is detected by monoclonal antibody binding and mimicked by association of the native antigen with synthetic phospholipid vesicles.

By flow cytometry, a conformational change in mouse cytochrome c (cyt c) of apoptotic and necrotic T hybridoma cells was detected using a monoclonal antibody (mAb) that recognizes the region around amino acid residue 44 on a non-native form of the protein. The conformational change in cyt c is an early event in apoptosis, which can be identified in pre-apoptotic cells that are negative for other indicators of apoptosis. Since the mAb did not bind fixed and permeabilized live cells and did not immunoprecipitate soluble cyt c extracted with detergent from dead cells, it appears to recognize cyt cbound in a detergent-sensitive complex to other cellular components. Coincidentally, the mAb was also shown by competitive enzyme-linked immunosorbent assay to bind cyt c associated with synthetic phosphatidic acid vesicles. This suggests that the conformational change of cyt c in dying cells could be due to its association with intracellular membranes that are, perhaps, altered in cell death. By immunofluorescent confocal microscopy, conformationally altered cyt c in post-apoptotic T hybridoma cells showed a punctate distribution, indicating that it remained associated with mitochondria. Furthermore, the heavy membrane fraction of post-apoptotic cells but not of live cells was functional in caspase activation. This suggests that membrane-bound cyt c is the relevant caspase coactivation factor in the T hybridoma cells.

Native Escherichia coli and murine dihydrofolate reductases contain late-folding non-native structures

We have examined the equilibrium and kinetic folding properties of two structurally homologous dihydrofolate reductases, Escherichia coli DHFR (EcDHFR) and murine DHFR (MuDHFR), as a function of temperature and ligand concentration. Conformational heterogeneity in native DHFR is well documented, and the results demonstrate that the non-native form(s) represents late intermediate(s) in the folding process. We have measured the concentrations of native and non-native forms and the rate constants for their interconversion over a temperature range of 3°C to 49°C, allowing characterization of the thermodynamic as well as the kinetic properties of the final folding step(s) relative to the overall folding reaction. Differences in ligand binding suggest that the intermediate structures for these two proteins may be different during refolding.

Two-Phase Induction of the Nonnative α-Helical Form of β-Lactoglobulin in the Presence of Trifluoroethanol

The trifluoroethanol-dependent induction of the nonnative α-helical form of β-lactoglubulin has been studied by circular dichroism spectroscopy. Data analysis is performed by factor analysis and multivariate curve resolution. An intermediate form in the induction of the α-helical form of the β-lactoglobulin has been identified at low TFE concentration. By application of an alternating least-squares algorithm, the CD spectrum corresponding to the intermediate form has been resolved. The deconvolution of this CD spectrum shows a secondary structure content more in agreement with the one predicted from the amino acid sequence than the secondary structure of the helical form obtained at higher TFE concentrations. The additional α-helical content of the form present at higher TFE concentrations could be due to nonspecific interaction of TFE with the polypeptide chain.

Electrostatic stabilization in methionine aminopeptidase from hyperthermophile Pyrococcus furiosus.

The thermostability of methionine aminopeptidase from a hyperthermophile P. furiosus (PfMAP) was extremely high: the denaturation temperature was 106.2 degreesC at pH 10.2. To explore the contribution of electrostatic interaction to the superior thermostability of PfMAP, the thermostability of PfMAP was examined by differential scanning calorimetry (DSC) in various salt concentrations in the acidic region far from the isoelectric point of PfMAP. (1) In 20 mM glycine buffer, the DSC curve of PfMAP exhibited a single peak. Transition temperatures (Tm) were lowered with decreasing pH from 4 to 3. The heat denaturation of PfMAP was not reversible. (2) Denaturation enthalpy (DeltaH) measured at different pHs linearly correlated with Tm up to 102 degreesC, suggesting that the denaturation heat capacity (DeltaCp) for PfMAP is constant up to 100 degreesC. DeltaCp was estimated to be 0.82 J K-1 g-1. (3) In the presence of 10-100 mM KCl at pH 3.2, two peaks appeared on the DSC curves. The first peak shifted to lower temperatures with increasing concentration of KCl and, oppositely, the second one to higher temperatures. It was found that the first and second peaks originated from the heat denaturation of the native form of PfMAP and the melting of the non-native associated form having molten globule-like structure, respectively, judged from the CD spectra and ultracentrifugation analyses. This indicates the following: first, the attractive electrostatic interaction is an important factor in stabilizing the native form of PfMAP; second, the presence of KCl stimulates the formation of the molten globule-like state of PfMAP and stabilizes it. (4) In a comparison of the sequence and crystal structure of PfMAP, which has been recently determined (1xgs.pdb), with those of MAP from Escherichia coli (EcMAP), it was predicted that the extra four short-range ion pairs less than 3 A involved in PfMAP are crucial candidates as determinants for the superior thermostability of PfMAP.

Ero1p: A Novel and Ubiquitous Protein with an Essential Role in Oxidative Protein Folding in the Endoplasmic Reticulum

The structure of many proteins entering the secretory pathway is dependent on stabilization by disulfide bonds. To support disulfide-linked folding, the endoplasmic reticulum (ER) must maintain a strongly oxidizing environment compared to the highly reduced environment of the cytosol. We report here the identification and characterization of Ero1p, a novel and essential ER-resident protein. Mutations in Ero1p cause extreme sensitivity to the reducing agent DTT, whereas overexpression confers DTT resistance. Strikingly, compromised Ero1p function results in ER retention of disulfide-stabilized proteins in a reduced, nonnative form, while not affecting structural maturation of a disulfide-free protein. We conclude that there exists a specific cellular redox machinery required for disulfide-linked protein folding in the ER and that Ero1p is an essential component of this machinery.

11] Construction of single-ring and two-ring hybrid versions of bacterial chaperonin GroEL

The chapter presents a study related to the construction of single-ring and two-ring hybrid versions of bacterial chaperonin GroEL. A single-ring version of the bacterial chaperonin, GroEL, has been designed using information from the crystal structure of the native double-ring assembly. This molecule, designated SR1, has proved to be functional as a folding chamber, inside which proteins, such as rhodanese and green fluorescent protein (GFP), bound to the ring in nonnative form, can reach native form on addition of GroES and ATP. In particular, the substrate protein folds to the native state within the central channel underneath GroES and remains within this space because the absence of a second GroEL ring prevents the normal ATP mediated signaling that releases GroES. SR1 has been designed by examination of the model of unliganded GroEL at 2.8 Å, derived from the orthorhombic crystal. This reveals a contact surface between the two rings that is composed of both electrostatic and hydrophobic interactions. Each subunit in the homotetradecameric complex forms contacts through the inferior aspect of its equatorial domain with that region of two neighboring subunits in the opposite ring. The essential double-ring nature of GroEL, with critical signaling occurring between its rings during the folding reaction, encouraged the study of double-ring complexes containing defects in only one ring.

Chaperonin-Mediated Folding in the Eukaryotic Cytosol Proceeds through Rounds of Release of Native and Nonnative Forms

The eukaryotic cytosolic chaperonin, CCT, plays an essential role in mediating ATP-dependent folding of actin and tubulin. There is debate about whether it mediates folding through a single round of association followed by release of native forms, or through cycles of binding and full release in which only a fraction of released molecules reaches native form in any cycle. We examine the fate of newly synthesized substrate proteins bound to CCT in reticulocyte lysate or intact Xenopus oocytes. When a chaperonin “trap,” able to bind but not release substrate protein, is introduced, production of the native state is strongly inhibited, associated with transfer to trap. While predominantly nonnative forms of actin, tubulin, and a newly identified substrate, Gα-transducin, are released from CCT, a small fraction reaches native form with each round of release, inaccessible to trap. This overall mechanism resembles that of the bacterial chaperonin, GroEL.

Relevance of the antibody response against human immunodeficiency virus type 1 envelope to vaccine design

Understanding the antibody response in HIV-1 infection is important to vaccine design. We have studied the antibody response to HIV-1 envelope at the molecular level and determined the characteristics of neutralizing and non-neutralizing antibodies. These antibodies were isolated from phage display libraries prepared from long-term seropositive asymptomatic individuals. The HIV-1 envelope is presented to the immune system in several antigenically distinct configurations: unprocessed gp160, gp120 and gp41 subunits and native envelope, each of which may be important in eliciting an antibody response in HIV-1 infection. The antibodies tested characteristically had poor affinities for native envelope as expressed on the surface of virions or infected cells, but had high affinities against non-native forms of HIV-1 envelope (viral debris). An exceptionally potent neutralizing antibody in contrast, bound native envelope with equivalent or somewhat higher affinity than this. This indicates that the antibody response in HIV-1 infection is principally elicited by viral debris rather than virions, and that these antibodies bind and neutralize viruses sub-optimally. Potential vaccines should be designed to elicit responses against native envelope.

GroEL-mediated protein folding.

I. Architecture of GroEL and GroES and the reaction pathway A. Architecture of the chaperonins B. Reaction pathway of GroEL-GroES-mediated folding II. Polypeptide binding A. A parallel network of chaperones binding polypeptides in vivo B. Polypeptide binding in vitro 1. Role of hydrophobicity in recognition 2. Homologous proteins with differing recognition-differences in primary structure versus effects on folding pathway 3. Conformations recognized by GroEL a. Refolding studies b. Binding of metastable intermediates c. Conformations while stably bound at GroEL 4. Binding constants and rates of association 5. Conformational changes in the substrate protein associated with binding by GroEL a. Observations b. Kinetic versus thermodynamic action of GroEL in mediating unfolding c. Crossing the energy landscape in the presence of GroEL III. ATP binding and hydrolysis-driving the reaction cycle IV. GroEL-GroES-polypeptide ternary complexes-the folding-active cis complex A. Cis and trans ternary complexes B. Symmetric complexes C. The folding-active intermediate of a chaperonin reaction-cis ternary complex D. The role of the cis space in the folding reaction E. Folding governed by a "timer" mechanism F. Release of nonnative polypeptides during the GroEL-GroES reaction G. Release of both native and nonnative forms under physiologic conditions H. A role for ATP binding, as well as hydrolysis, in the folding cycle V. Concluding remarks.

A strategy for identifying introduced provenances and translocations

Summary Native species of oak in Britain have been of great importance in our history for their many uses, and have thus been subject to management and planting by man for centuries. Following events such as the Enclosure Acts of the late eighteenth century and the Napoleonic wars, many oaks would have been planted. Translocations and introductions of foreign genotypes were greatly encouraged by early landscape gardeners such as Capability Brown. Britain must, therefore be a mosaic of native and non-native oaks. A major problem arises when we try to identify non-native trees. Due to their long life-cycle, oaks are of necessity both phenotypically plastic and genetically very variable and it has been virtually impossible to discriminate between native and non-native forms using traditional methods. The advent of new molecular genetic techniques however, now allows us to identify DNA markers that can distinguish between such forms. The geographic patterns for two chloroplast DNA markers will be presented. One clearly differentiates between oaks from eastern Europe versus western Europe and can be used to identify translocations of eastern European oaks into Britain and western Europe. The second identifies genotypes native to East Anglia and can be used to recognize translocations into and out of East Anglia.

Release of both native and non-native proteins from a cis-only GroEL ternary complex.

Protein folding by the double-ring chaperonin GroEL is initiated in cis ternary complexes, in which polypeptide is sequestered in the central channel of a GroEL ring, capped by the co-chaperonin GroES. The cis ternary complex is dissociated (half-life of approximately 15 s) by trans-sided ATP hydrolysis, which triggers release of GroES. For the substrate protein rhodanese, only approximately 15% of cis-localized molecules attain their native form before hydrolysis. A major question concerning the GroEL mechanism is whether both native and non-native forms are released from the cis complex. Here we address this question using a 'cis-only' mixed-ring GroEL complex that binds polypeptide and GroES on only one of its two rings. This complex mediates refolding of rhodanese but, as with wild-type GroEL, renaturation is quenched by addition of mutant GroEL 'traps', which bind but do not release polypeptide substrate. This indicates that non-native forms are released from the cis complex. Quenching of refolding by traps was also observed under physiological conditions, both in undiluted Xenopus oocyte extract and in intact oocytes. We conclude that release of non-native forms from GroEL in vivo allows a kinetic partitioning among various chaperones and proteolytic components, which determines both the conformation and lifetime of a protein.

Representing Truth and History in Native American Documentary: Indigenous Efforts to Counter Mass Media Stereotypes

Leuthold | Representing Truth and History in Native American Documentary Steven Leuthold School of Art and Design, Syracuse University Representing Truth and History in Native American Documentary: Indigenous Efforts to Counter Mass Media Stereotypes hy has documentary emerged as the most common genre of indigenous media? Though one attraction of documentary to native directors is its financial accessibility, the attraction of the documentary medium extends far beyond the shaping factor of economics. The place of documentaries in education, the relation of electronic media documentaries to traditionally oral cultures, and the desire to document disappearing cultural practices have influenced the adoption of the documentary genre by Native Americans. Natives hope that through electronic media they can present role models to young Indians and expose non-Indians to cultures they might not encounter otherwise. I talked to one man today; he's a teacher and some of his students have been in the public schools eight or nine years. They have yet to see a native teacher. And education is an area [that] many people have gone into because we need that, but the other part is that we know about white culture all over ... we watch white TV, we learn about white culture, but we never have that opportunity to explain our culture to our non-Indian audience. That's whatwe're looking for ... thatwe can try to find people that can put together movies. We may notbe able to do it right ... because we lack the funding; we lack the resources; we lack equipment, but we can put together images. We can put together stories and, for many Indian nations, our oral historywas the tradition. That is the same tradition here, whether radio, television or film. We have that ability. We have that power and knowledge to put things together and explain ourselves to manynon-Indian people.1 Many native directors desire to counteract popular culture's influence on Indian youth. Instead of learning about the meaning of symbols, colors, images, and myths through the traditional channels of tribal communication, Native American youths imitate movie portrayals of Indians, a case in which life imitates fiction. "In our tribe, we have certain customs now that actually have been transformed by film ... We're doing stuff out offilm now: face painting, for instance. Youths ... are painting their faces, not in the traditional waywe did that—by clan, by family, by color—but they're doing it out ofwhat they see in the damn movies."2 From the perspective of some native directors, documentary acts as a form of truth speaking, a way of accurately recording and presenting both history and contemporary lives in contrast to the fictive world portrayed in popular imagery. Another way to describe documentaries as true is that they are empirically verifiable records of reality. A documentarían, like a scientist, makes 30 I Film & History The American Frontier in Film | Special In-Depth Section the implicit claim that 'ifyou would have been there, you would have observed the same events or relationships that I did.' The assumption that documentary records the truth about native cultures , as seen from a native perspective, underlies its educational and cultural preservational capacity . Simply put, if viewers did not believe documentary accounts to be true, the educational and cultural function of the films and videos would be lessened or undermined altogether. In this paper I examine the assumption that native produced documentaries portray native lives and history in a way that is more real or 'truer to life' than non-native mass mediated forms.3 1 explore the reasons why natives may consider documentary a form of truth telling and consider this position in the light of recent critiques about the capacity of documentary to truly represent 'reality'. Though modern theorists have argued against a view of documentary as a true record of 'reality,' I will contend that the efficacy of documentary depends upon the assumption by viewers that what they are seeing is more real than what is produced in other media forms. Similarly, the attraction of documentary to native producers and directors is that it portrays native realities, in contrast to the distortion and stereotypes found in the mass media. A major...

Conformational Effects on Reversed-Phase Chromatography of Proteins with Particle Beam LC/FT-IR Spectrometry and Free Solution Capillary Electrophoresis

In this study, reversed-phase HPLC (RPC), particle beam FT-IR spectrometry, and capillary electrophoresis (CE) were employed to ascertain the dynamic conformational structure empirically of various forms of bovine ribonuclease A (RNase A) to investigate the interdependence of protein conformation and RPC separation. RNase A was analyzed in four conformational states: native, partially denatured, completely unfolded, and completely unfolded and disulfide-reduced (denatured). Each form was analyzed individually and produced similar multizoned RPC profiles composed of several low-response peaks before the primary peak. Particle beam FT-IR spectrometry results from the RPC fractions of the partially denatured and unfolded forms were identical to one another and exhibited a loss of ordered structure. The infrared spectra of the RNase A that was introduced into the HPLC in the denatured and reduced form showed an excess of β-sheet content, particularly the primary peak. We believe this to be a non-native form of RNase A. CE migration times of RNase A samples that were reduced in 2-mercaptoethanol and unfolded in increasing concentrations of urea increased with the degree of unfolding. Mobilities of RNase A RPC fractions were compared to those of the urea unfolded samples. The migration time of the primary RPC fraction of the denatured form showed an intermediate migration rate between that of the native, spherical geometry and denatured, open geometry forms. It is unclear whether this β-structure formed during column propagation or upon elution.