This thesis describes NMR studies which probe weak interactions between amino acid side chains in folded peptide structures. Aromatic/aromatic interactions between facing phenylalanine residues have been probed in antiparallel β-sheets, while aromatic/proline interactions have been examined using cyclic peptide disulfides that occur in the venom of marine cone snails. Novel intramolecular hydrogen bonded structures in hybrid peptides containing backbone homologated residues, specifically γ-amino acids, are also described. Chapter 1 provides a brief background to the principles involved in the design of antiparallel β-sheet structures and an introduction to previous studies on aromatic/aromatic and aromatic/proline interactions in influencing peptide conformations. A summary of the NMR methods used is also presented. Chapter 2 discusses the structural characterisation of a designed 14 residue, three stranded β-sheet peptide, Boc-LFVDP-PLFVADP-PLFV-OMe (LFV14). The results described in this Chapter support the presence of multiple conformational states about the χ1 (Cα-Cβ) torsional degree of freedom for the interacting aromatic pairs in solution. Chapter 3 presents the structural characterisation of a designed 19 residue three stranded hybrid β-sheet peptide, Boc-LVβFVDPGLβFVVLDPGLVLβFVV-OMe (BBH19). β-amino acid residues (β-phenylalanine, βPhe) were incorporated at facing positions on antiparallel β-sheets. The BBH19 structure provides an example of interaction between the N and C-terminal strands in a three stranded structure with an α/β hybrid backbone. Chapter 4 focuses on studies of the conformations of the contryphan In936 (GCVDLYPWC*) from Conus inscriptus and the related peptide Lo959 (GCPDWDPWC*) from Conus loroissi. Both peptides possess a macrocyclic 23 membered ring, with multiple accessible conformational states. Chapter 5 describes conformational analysis of a novel 20 membered cyclic peptide disulfide, CIWPWC (Vi804), from Conus virgo. NMR structures were calculated for Vi804 and an analog peptide, CIDWPWC, DW3-Vi804. Chapter 6 explores the solution conformation of hybrid sequences containing α and γ residues. Oligopeptides of the type (αγ)n and (αγγ)n have been studied in solution by NMR methods. Chapter 7 provides a summary of the results described in this thesis and highlights the major conclusions.

Cette thèse se place dans le cadre de l'étude in silico, c'est-à-dire assistée par ordinateur, des liens qui unissent la séquence d'une protéine à la (ou aux) structure(s) tri-dimensionnelle(s) qu'elle adopte. Le décryptage de ces liens présente de nombreuses applications dans divers domaines et constitue sans doute l'une des problématiques les plus fascinantes de la recherche en biologie moléculaire.<p><p>Le premier aspect de notre travail concerne le développement de potentiels statistiques dérivés de bases de données de protéines dont les structures sont connues. Ces potentiels présentent plusieurs avantages: ils peuvent être aisément adaptés à des représentations structurales simplifiées, et permettent de définir un nombre limité de fonctions énergétiques qui incarnent l'ensemble complexe d'interactions gouvernant la structure et la stabilité des protéines, et qui incluent également certaines contributions entropiques. Cependant, leur signification physique reste assez nébuleuse, car l'impact des diverses hypothèses nécessaires à leur dérivation est loin d'être clairement établi. Nous nous sommes attachés à l'étude de certaines limitations des ces potentiels: leur dépendance en la taille des protéines incluses dans la base de données, la non-additivité des termes de potentiels, et l'importance souvent négligée de l'environnement protéique spécifique ressenti par chaque résidu. Nous avons ainsi mis en évidence que l'influence de la taille des protéines de la base de données sur les potentiels de distance entre résidus est spécifique à chaque paire d'acides aminés, peut être relativement importante, et résulte essentiellement de la répartition inhomogène des résidus hydrophobes et hydrophiles entre le coeur et la surface des protéines. Ces résultats ont guidé la mise au point de fonctions correctives qui permettent de tenir compte de cette influence lors de la dérivation des potentiels. Par ailleurs, la définition d'une procédure générale de dérivation de potentiels et de termes de couplage a rendu possible la création d'une fonction énergétique qui tient compte simultanément de plusieurs descripteurs de séquence et de structure (la nature des résidus, leurs conformations, leurs accessibilités au solvant, ainsi que les distances qui les séparent dans l'espace et le long de la séquence). Cette fonction énergétique présente des performances nettement améliorées par rapport aux potentiels originaux, et par rapport à d'autres potentiels décrits dans la littérature.<p><p>Le deuxième aspect de notre travail concerne l'application de programmes basés sur des potentiels statistiques à l'étude de protéines qui adoptent des structures alternatives. La permutation de domaines est un phénomène qui affecte diverses protéines et qui implique la génération d'un oligomère suite à l'échange de fragments structuraux entre monomères identiques. Nos résultats suggèrent que la présence de "faiblesses structurales", c'est-à-dire de régions qui ne sont pas optimales vis-à-vis de la stabilité de la structure native ou qui présentent une préférence marquée pour une conformation non-native en absence d'interactions tertiaires, est intimement liée aux mécanismes de permutation. Nous avons également mis en évidence l'importance des interactions de type cation-{pi}, qui sont fréquemment observées dans certaines zones clés de la permutation. Finalement, nous avons sélectionné un ensemble de mutations susceptibles de modifier sensiblement la propension de diverses protéines à permuter. L'étude expérimentale de ces mutations devrait permettre de valider, ou de raffiner, les hypothèses que nous avons proposées quant au rôle joué par les faiblesses structurales et les interactions de type cation-{pi}. Nous avons également analysé une autre protéine soumise à d'importants réarrangements conformationnels: l'{alpha}1-antitrypsine. Dans le cas de cette protéine, les modifications structurales sont indispensables à l'exécution de l'activité biologique normale, mais peuvent sous certaines conditions mener à la formation de polymères insolubles et au développement de maladies. Afin de contribuer à une meilleure compréhension des mécanismes responsables de la polymérisation, nous avons cherché à concevoir rationnellement des protéines mutantes qui présentent une propension à polymériser contrôlée. Des tests expérimentaux ont été réalisés par le groupe australien du Professeur S.P. Bottomley, et ont permis de valider nos prédictions de manière assez remarquable.<p><p><p><p>The work presented in this thesis concerns the computational study of the relationships between the sequence of a protein and its three-dimensional structure(s). The unravelling of these relationships has many applications in different domains and is probably one of the most fascinating issues in molecular biology.<p><p>The first part of our work is devoted to the development of statistical potentials derived from databases of known protein structures. These potentials allow to define a limited number of energetic functions embodying the complex ensemble of interactions that rule protein folding and stability (including some entropic contributions), and can be easily adapted to simplified representations of protein structures. However, their physical meaning remains unclear since several hypotheses and approximations are necessary, whose impact is far from clearly understood. We studied some of the limitations of these potentials: their dependence on the size of the proteins included in the database, the non-additivity of the different potential terms, and the importance of the specific environment of each residue. Our results show that residue-based distance potentials are affected by the size of the database proteins, and that this effect can be quite strong, is residue-specific, and seems to result mostly from the inhomogeneous partition of hydrophobic and hydrophilic residues between the surface and the core of proteins. On the basis of these observations, we defined a set of corrective functions in order to take protein size into account while deriving the potentials. On the other hand, we developed a general procedure of derivation of potentials and coupling terms and consequently created an energetic function describing the correlations between several sequence and structure descriptors (the nature of each residue, the conformation of its main chain, its solvent accessibility, and the distances that separate it from other residues, in space and along the sequence). This energetic function presents a strongly improved predictive power, in comparison with the original potentials and with other potentials described in the literature.<p><p>The second part describes the application of different programs, based on statistical potentials, to the study of proteins that adopt alternative structures. Domain swapping involves the exchange of a structural element between identical proteins, and leads to the generation of an oligomeric unit. We showed that the presence of “structural weaknesses”, regions that are not optimal with respect to the folding mechanisms or to the stability of the native structure, seems to be intimately linked with the swapping mechanisms. In addition, cation-{pi} interactions were frequently detected in some key locations and might also play an important role. Finally, we designed a set of mutations that are likely to affect the swapping propensities of different proteins. The experimental study of these mutations should allow to validate, or refine, our hypotheses concerning the importance of structural weaknesses and cation-{pi} interactions. We also analysed another protein that undergoes large conformational changes: {alpha}1-antitrypsin. In this case, the structural modifications are necessary to the proper execution of the biological activity. However, under certain circumstances, they lead to the formation of insoluble polymers and the development of diseases. With the aim of reaching a better understanding of the mechanisms that are responsible for this polymerisation, we tried to design mutant proteins that display a controlled polymerisation propensity. An experimental study of these mutants was conducted by the group of Prof. S.P. Bottomley, and remarkably confirmed our predictions.<p> / Doctorat en sciences appliquées / info:eu-repo/semantics/nonPublished

Indiana University-Purdue University Indianapolis (IUPUI) / RNA polymerase II (RNAPII) is regulated by multiple modifications to the C-terminal domain (CTD) of the largest subunit, Rpb1. This study has focused on the relationship between hyperphosphorylation of the CTD and RNAPII turnover and proteolytic degradation as well as post-translational modifications of the globular core of RNAPII. Following tandem affinity purification, western blot analysis showed that MG132 treated RTR1 ERG6 deletion yeast cells have accumulation of total RNAPII and in particular, the hyperphosphorylated form of the protein complex. In addition, proteomic studies using MuDPIT have revealed increased interaction between proteins of the ubiquitin-proteasome degradation system in the mutant MG132 treated yeast cells as well as potential ubiquitin and phosphorylation sites in RNAPII subunits, Rpb6 and Rpb1, respectively. A novel Rpb1 phosphorylation site, T1471-P, is located in the linker region between the CTD and globular domain of Rpb1 and will be the focus of future studies to determine biological significance of this post-translational modification.

Indiana University-Purdue University Indianapolis (IUPUI) / Traditional treatment strategy development for diseases involves the identification of target proteins related to disease states, and the interference of these proteins with drug molecules. Computational drug discovery and virtual screening from thousands of chemical compounds have accelerated this process. The thesis presents a comprehensive framework of computational drug discovery using system biology approaches. The thesis mainly consists of two parts: disease biomarker identification and disease treatment discoveries. The first part of the thesis focuses on the research in biomarker identification for human diseases in the post-genomic era with an emphasis in system biology approaches such as using the protein interaction networks. There are two major types of biomarkers: Diagnostic Biomarker is expected to detect a given type of disease in an individual with both high sensitivity and specificity; Predictive Biomarker serves to predict drug response before treatment is started. Both are essential before we even start seeking any treatment for the patients. In this part, we first studied how the coverage of the disease genes, the protein interaction quality, and gene ranking strategies can affect the identification of disease genes. Second, we addressed the challenge of constructing a central database to collect the system level data such as protein interaction, pathway, etc. Finally, we built case studies for biomarker identification for using dabetes as a case study. The second part of the thesis mainly addresses how to find treatments after disease identification. It specifically focuses on computational drug repositioning due to its low lost, few translational issues and other benefits. First, we described how to implement literature mining approaches to build the disease-protein-drug connectivity map and demonstrated its superior performances compared to other existing applications. Second, we presented a valuable drug-protein directionality database which filled the research gap of lacking alternatives for the experimental CMAP in computational drug discovery field. We also extended the correlation based ranking algorithms by including the underlying topology among proteins. Finally, we demonstrated how to study drug repositioning beyond genomic level and from one dimension to two dimensions with clinical side effect as prediction features.

A alteração do tráfego mitocondrial em neurônios leva ao aumento do estresse oxidativo, privação de energia, deficiência da comunicação intercelular e neurodegeneração. Há evidências de que essas alterações de tráfego antecedem a morte neuronal associada à agregação proteica. Portanto, conhecer a relação entre a mobilidade mitocondrial e a formação de agregados proteicos pode ser um passo importante para o melhor entendimento dos mecanismos da neurodegeneração. Com isso, o objetivo do presente estudo é analisar a mobilidade das mitocôndrias em culturas de células do hipocampo, substância negra e locus coeruleus expostas a rotenona e MPTP, como agentes neurodegenerativos, e à rapamicina como ativador da autofagia. Um outro objetivo do estudo é avaliar o papel do cálcio (através do emprego de EGTA e ionomicina) no modelo experimental. Os resultados mostraram aumento da mobilidade mitocondrial no hipocampo e diminuição na substância negra, já no locus coeruleus houve aumento seguido de diminuição da mobilidade mitocondrial dependendo da concentração de rotenona. O emprego do EGTA e ionomicina mostra que a ação da rotenona sobre o tráfego mitocondrial envolve o cálcio, mas não se relaciona com uma possível alteração da integridade mitocondrial, já que não foi observada alteração no potencial de membrana mitocondrial. Foram também realizados experimentos a fim de avaliar a mobilidade mitocondrial em modelo utilizando rapamicina para ativar a autofagia e MPTP como indutor da neurodegeneração em culturas de células, onde foi observado aumento da mobilidade no hipocampo e no locus coeruleus quando exposto a rapamicina e aumento da mobilidade mitocondrial em cultura de células do hipocampo exposto a MPTP já no locus coeruleus houve uma diminuição significativa da mobilidade mitocondrial. Os resultados permitem concluir que o tráfego mitocondrial está alterado antes da agregação proteica podendo contribuir com a neurodegeneração / Altered mitochondrial traffic in neurons can lead to increased oxidative stress, energy deprivation, impaired intercellular communication and neurodegeneration. There are evidences mitochondria disturbing precedes neuronal death associated with protein aggregation. Therefore, the study of mitochondrial traffic and protein aggregation can be an important step towards a better understanding of the mechanisms of neurodegeneration. Thus, the aim of this study is to analyze mitochondria mobility in cultured cells of the hippocampus, substantia nigra and locus coeruleus exposed to rotenone and MPTP, as neurodegeneration-promoting agents, and rapamycin to activate autophagy. The other objective of the study was to analyze the role of calcium (through EGTA and ionomycin) in the experimental model. The results showed increased and decreased mobility mitochondrial in cells from hippocampus and substantia nigra, respectively, while the locus coeruleus cell culture has increased followed by decreased mitochondrial mobility depending upon rotenone concentration. The use of EGTA and ionomycin showed that alteration of mitochondrial traffic is associated with calcium, however it is not related with changes in mitochondrial membrane potential. Additional experiments were also conducted to assess mitochondrial mobility in a model using rapamycin to activate autophagy and MPTP to induce neurodegeneration in cell cultures. The results of these experiments showed increased mitochondrial mobility in the hippocampus and locus coeruleus when exposed to rapamycin; while MPTP also increased mitochondria mobility in hippocampal cell cultures, but decreased it in locus coeruleus. Results suggest that mitochondrial traffic is altered before protein aggregation, which may contribute to neurodegeneration

O gene PKHD1, mutado na doença renal policística autossômica recessiva, apresenta um padrão de splicing complexo associado a múltiplos transcritos alternativos. Neste trabalho estudamos o perfil de expressão de seu produto, poliductina. Análises por western blot revelaram produtos putativos de membrana de > 440 kDa e ~230 kDa, e de ~140 kDa em frações solúveis de rim, fígado e pâncreas. Estudos imunoistoquímicos mostraram marcação em ductos coletores renais e porção ascendente espessa da alça de Henle, em epitélios ductais biliar e pancreático e, no período embrionário, em broto ureteral, ductos biliar e pancreático e glândula salivar. Análises por imunofluorescência e microscopia imunoeletrônica sugerem que poliductina se localize em cílio apical primário, membrana apical e citoplasma de células do ducto coletor. Nossos resultados indicam que PKHD1 codifica isoformas de membrana e solúveis / PKHD1, the gene mutated in autosomal recessive polycystic kidney disease, presents a complex splicing pattern, associated with multiple alternative transcripts. In this work we have studied the expression profile of its product, polyductin. Western blot analysis revealed putative membrane products of > 440 kDa and 230 kDa, and of ~140 kDa in soluble fractions in kidney, liver and pancreas. Immunohistochemistry studies showed staining in renal collecting duct and thick ascending limb of Henle, in biliary and pancreatic ductal epithelia and, in the embryonic period, in ureteric bud, biliary and pancreatic ducts and salivary gland. Immunofluorescence and immunoelectron microscopy studies suggest that polyductin localizes to primary apical cilium, apical membrane and cytoplasm of collecting duct cells. Our data indicate that PKHD1 codifies membrane and soluble isoforms

O gene PKHD1, mutado na doença renal policística autossômica recessiva, apresenta um padrão de splicing complexo associado a múltiplos transcritos alternativos. Neste trabalho estudamos o perfil de expressão de seu produto, poliductina. Análises por western blot revelaram produtos putativos de membrana de > 440 kDa e ~230 kDa, e de ~140 kDa em frações solúveis de rim, fígado e pâncreas. Estudos imunoistoquímicos mostraram marcação em ductos coletores renais e porção ascendente espessa da alça de Henle, em epitélios ductais biliar e pancreático e, no período embrionário, em broto ureteral, ductos biliar e pancreático e glândula salivar. Análises por imunofluorescência e microscopia imunoeletrônica sugerem que poliductina se localize em cílio apical primário, membrana apical e citoplasma de células do ducto coletor. Nossos resultados indicam que PKHD1 codifica isoformas de membrana e solúveis / PKHD1, the gene mutated in autosomal recessive polycystic kidney disease, presents a complex splicing pattern, associated with multiple alternative transcripts. In this work we have studied the expression profile of its product, polyductin. Western blot analysis revealed putative membrane products of > 440 kDa and 230 kDa, and of ~140 kDa in soluble fractions in kidney, liver and pancreas. Immunohistochemistry studies showed staining in renal collecting duct and thick ascending limb of Henle, in biliary and pancreatic ductal epithelia and, in the embryonic period, in ureteric bud, biliary and pancreatic ducts and salivary gland. Immunofluorescence and immunoelectron microscopy studies suggest that polyductin localizes to primary apical cilium, apical membrane and cytoplasm of collecting duct cells. Our data indicate that PKHD1 codifies membrane and soluble isoforms

A alteração do tráfego mitocondrial em neurônios leva ao aumento do estresse oxidativo, privação de energia, deficiência da comunicação intercelular e neurodegeneração. Há evidências de que essas alterações de tráfego antecedem a morte neuronal associada à agregação proteica. Portanto, conhecer a relação entre a mobilidade mitocondrial e a formação de agregados proteicos pode ser um passo importante para o melhor entendimento dos mecanismos da neurodegeneração. Com isso, o objetivo do presente estudo é analisar a mobilidade das mitocôndrias em culturas de células do hipocampo, substância negra e locus coeruleus expostas a rotenona e MPTP, como agentes neurodegenerativos, e à rapamicina como ativador da autofagia. Um outro objetivo do estudo é avaliar o papel do cálcio (através do emprego de EGTA e ionomicina) no modelo experimental. Os resultados mostraram aumento da mobilidade mitocondrial no hipocampo e diminuição na substância negra, já no locus coeruleus houve aumento seguido de diminuição da mobilidade mitocondrial dependendo da concentração de rotenona. O emprego do EGTA e ionomicina mostra que a ação da rotenona sobre o tráfego mitocondrial envolve o cálcio, mas não se relaciona com uma possível alteração da integridade mitocondrial, já que não foi observada alteração no potencial de membrana mitocondrial. Foram também realizados experimentos a fim de avaliar a mobilidade mitocondrial em modelo utilizando rapamicina para ativar a autofagia e MPTP como indutor da neurodegeneração em culturas de células, onde foi observado aumento da mobilidade no hipocampo e no locus coeruleus quando exposto a rapamicina e aumento da mobilidade mitocondrial em cultura de células do hipocampo exposto a MPTP já no locus coeruleus houve uma diminuição significativa da mobilidade mitocondrial. Os resultados permitem concluir que o tráfego mitocondrial está alterado antes da agregação proteica podendo contribuir com a neurodegeneração / Altered mitochondrial traffic in neurons can lead to increased oxidative stress, energy deprivation, impaired intercellular communication and neurodegeneration. There are evidences mitochondria disturbing precedes neuronal death associated with protein aggregation. Therefore, the study of mitochondrial traffic and protein aggregation can be an important step towards a better understanding of the mechanisms of neurodegeneration. Thus, the aim of this study is to analyze mitochondria mobility in cultured cells of the hippocampus, substantia nigra and locus coeruleus exposed to rotenone and MPTP, as neurodegeneration-promoting agents, and rapamycin to activate autophagy. The other objective of the study was to analyze the role of calcium (through EGTA and ionomycin) in the experimental model. The results showed increased and decreased mobility mitochondrial in cells from hippocampus and substantia nigra, respectively, while the locus coeruleus cell culture has increased followed by decreased mitochondrial mobility depending upon rotenone concentration. The use of EGTA and ionomycin showed that alteration of mitochondrial traffic is associated with calcium, however it is not related with changes in mitochondrial membrane potential. Additional experiments were also conducted to assess mitochondrial mobility in a model using rapamycin to activate autophagy and MPTP to induce neurodegeneration in cell cultures. The results of these experiments showed increased mitochondrial mobility in the hippocampus and locus coeruleus when exposed to rapamycin; while MPTP also increased mitochondria mobility in hippocampal cell cultures, but decreased it in locus coeruleus. Results suggest that mitochondrial traffic is altered before protein aggregation, which may contribute to neurodegeneration

Indiana University-Purdue University Indianapolis (IUPUI) / Intrinsically disordered proteins are proteins characterized by lack of stable tertiary structures under physiological conditions. Evidence shows that disordered proteins are not only highly involved in protein interactions, but also have the capability to associate with more than one partner. Short disordered protein fragments, called “molecular recognition features” (MoRFs), were hypothesized to facilitate the binding diversity of highly-connected proteins termed “hubs”. MoRFs often couple folding with binding while forming interaction complexes. Two protein disorder mechanisms were proposed to facilitate multiple partner binding and enable hub proteins to bind to multiple partners: 1. One region of disorder could bind to many different partners (one-to-many binding), so the hub protein itself uses disorder for multiple partner binding; and 2. Many different regions of disorder could bind to a single partner (many-to-one binding), so the hub protein is structured but binds to many disordered partners via interaction with disorder. Thousands of MoRF-partner protein complexes were collected from Protein Data Bank in this study, including 321 one-to-many binding examples and 514 many-to-one binding examples. The conformational flexibility of MoRFs was observed at atomic resolution to help the MoRFs to adapt themselves to various binding surfaces of partners or to enable different MoRFs with non-identical sequences to associate with one specific binding pocket. Strikingly, in one-to-many binding, post-translational modification, alternative splicing and partner topology were revealed to play key roles for partner selection of these fuzzy complexes. On the other hand, three distinct binding profiles were identified in the collected many-to-one dataset: similar, intersecting and independent. For the similar binding profile, the distinct MoRFs interact with almost identical binding sites on the same partner. The MoRFs can also interact with a partially the same but partially different binding site, giving the intersecting binding profile. Finally, the MoRFs can interact with completely different binding sites, thus giving the independent binding profile. In conclusion, we suggest that protein disorder with post-translational modifications and alternative splicing are all working together to rewire the protein interaction networks.

Indiana University-Purdue University Indianapolis (IUPUI) / Carbon nanomaterials are widely produced and used in industry, medicine and scientific research. To examine the impact of exposure to nanoparticles on human health, the human airway epithelial cell line, Calu-3, was used to evaluate changes in the cellular proteome that could account for alterations in cellular function of airway epithelia after 24 h exposure to 10 μg/mL and 100 ng/mL of two common carbon nanoparticles, singleand multi-wall carbon nanotubes (SWCNT, MWCNT). After exposure to the nanoparticles, label-free quantitative mass spectrometry (LFQMS) was used to study differential protein expression. Ingenuity Pathway Analysis (IPA) was used to conduct a bioinformatics analysis of proteins identified by LFQMS. Interestingly, after exposure to a high concentration (10 μg/mL; 0.4 μg/cm2) of MWCNT or SWCNT, only 8 and 13 proteins, respectively, exhibited changes in abundance. In contrast, the abundance of hundreds of proteins was altered in response to a low concentration (100 ng/mL; 4 ng/cm2) of either CNT. Of the 281 and 282 proteins that were significantly altered in response to MWCNT or SWCNT, respectively, 231 proteins were the same. Bioinformatic analyses found that the proteins common to both kinds of nanotubes are associated with the cellular functions of cell death and survival, cell-to-cell signaling and interaction, cellular assembly and organization, cellular growth and proliferation, infectious disease, molecular transport and protein synthesis. The decrease in expression of the majority proteins suggests a general stress response to protect cells. The STRING database was used to analyze the various functional protein networks. Interestingly, some proteins like cadherin 1 (CDH1), signal transducer and activator of transcription 1 (STAT1), junction plakoglobin (JUP), and apoptosis-associated speck-like protein containing a CARD (PYCARD), appear in several functional categories and tend to be in the center of the networks. This central positioning suggests they may play important roles in multiple cellular functions and activities that are altered in response to carbon nanotube exposure. To examine the effect of nanotubes on the plasma membrane, we investigated the interaction of short purified MWCNT with model lipid membranes using a planar bilayer workstation. Bilayer lipid membranes were synthesized using neutral 1, 2-diphytanoylsn-glycero-3-phosphocholine (DPhPC) in 1 M KCl. The ion channel model protein, Gramicidin A (gA), was incorporated into the bilayers and used to measure the effect of MWCNT on ion transport. The opening and closing of ion channels, amplitude of current, and open probability and lifetime of ion channels were measured and analyzed by Clampfit. The presence of an intermediate concentration of MWCNT (2 μg/ml) could be related to a statistically significant decrease of the open probability and lifetime of gA channels. The proteomic studies revealed changes in response to CNT exposure. An analysis of the changes using multiple databases revealed alterations in pathways, which were consistent with the physiological changes that were observed in cultured cells exposed to very low concentrations of CNT. The physiological changes included the break down of the barrier function and the inhibition of the mucocillary clearance, both of which could increase the risk of CNT’s toxicity to human health. The biophysical studies indicate MWCNTs have an effect on single channel kinetics of Gramicidin A model cation channel. These changes are consistent with the inhibitory effect of nanoparticles on hormone stimulated transepithelial ion flux, but additional experiments will be necessary to substantiate this correlation.