The transition between the closed and open conformations of the Sec61 complex permits nascent protein insertion into the translocation channel. A critical event in this structural transition is the opening of the lateral translocon gate that is formed by four transmembrane (TM) spans (TM2, TM3, TM7 and TM8 in Sec61p) to expose the signal sequence-binding (SSB) site. To gain mechanistic insight into lateral gate opening, mutations were introduced into a lumenal loop (L7) that connects TM7 and TM8. The sec61 L7 mutants were found to have defects in both the posttranslational and cotranslational translocation pathways due to a kinetic delay in channel gating. The translocation defect caused by L7 mutations could be suppressed by the prl class of sec61 alleles that reduce the fidelity of signal sequence recognition. The prl mutants are proposed to act by destabilizing the closed conformation of the translocation channel. Our results indicate that the equilibrium between the open and closed conformations of the protein translocation channel maintains a balance between translocation activity and signal sequence recognition fidelity. In the opening of the translocation channel, both the lateral and lumenal gate must open in a coordinated fashion for efficient protein translocation to occur. The lumenal gate is composed of a short helix of the loop preceding the second TM span, referred to as the plug helix, and six hydrophobic pore ring residues which form the constriction ring in the center of the channel. We identified three lateral gate polar residues and three hydrophobic residues from the plug domain that affect channel gating. Mutagenesis of the lateral gate polar cluster residues yields either a gain of function (prl phenotype) or a loss of function (translocation defect) phenotype. The combination of polar cluster mutations with each other or with plug domain mutations which cause a prl phenotype resulted in the mutually suppressive or additive phenotypes in double mutant strains. Cooperation between these residues is made possible through a structural link which connects the two translocation channel gates at their interface. The structural link provides a mechanism for the channel to coordinate the movement of multiple domains in the channel gating conformational change. Translocation assays demonstrated that this mechanism of gating regulation is particularly important for efficient protein translocation of substrates using the posttranslational translocation pathway. Our results indicate that residues from the plug and lateral gate domain form a regulatory cluster of residues responsible for efficient translocation channel gating.

Understanding the conformational and dynamic changes of biomacromolecular complexes in different states, such as the membrane protein photoreceptor-transducer complex NpSRII/NpHtrII, is a key step to gaining insight into the functional mechanism of these important classes of protein complexes, since ~30 % of the human proteome are membrane proteins, yet they are largely underrepresented in terms of structural information with <1 % of all structures in the protein data bank. Hence for the development of methods suitable to study the conformation and dynamics of such complexes there is a strong demand and a vast potential field of applications. Here we combined method development at the interface between biomolecular simulations and model-based analysis of EPR- and fluorescence spectroscopic data with application studies using state-of-the-art spectroscopic techniques in conjunction with site-directed spin- or fluorescence labeling. In an initial benchmark study on the rigid globular protein complex Rpo4/7, we compared experimental inter fluorescence label distances or spin label distance distributions to a variety of predicted inter label distances based on molecular dynamics simulations, Monte Carlo sampling and a discrete rotamer library analysis. We found that while for the molecular dynamics simulations with explicit solvent considerable sampling challenges have to be overcome to reproduce the experimentally observed inter label distance distributions, the Monte Carlo sampling performed well when compared to the experimental data and was computationally less demanding. Significantly more efficient and equally accurate for our examples was the so-called rotamer library analysis available for the spin labels since it relies on a pre-calculated set of rotational isomers. In general, predictions for the mean distances were in agreement within the error margins while distribution shapes were more challenging to reproduce. Overall this study shows a positive evaluation for the assessed tools and the developed simulation protocols as well as their potential applications. Using the combination of EPR and fluorescence spectroscopy for distance determination we studied the structural influence of RNA binding on Rpo4/7, and showed that the protein complex stays conformationally rigid and thereby serves as a guiding rail for the nascent RNA chain that leaves the RNA polymerase along the Rpo4/7 RNA binding interface. To enhance the interpretation of experimentally determined changes of conformation and dynamics in protein complexes and to discuss the observed changes in terms of structural information, we built models of the two transcription factors TFE and the Spt4/5 complex, as well as of Argonaute, a 713 amino acid four-domain protein nuclease from Methanocaldococcus jannaschii. These structural models not only allowed a more accurate planning of fluorescence or EPR labeling experiments, but also the models enabled the discussion of the experimental data in structural terms. Based on such an initial structure further computational analysis techniques may be applied to identify putative structural changes or dynamic modes. This was shown for the histidine transporter HisQMP2, where we combined normal mode analysis to model protein flexibility with the rotamer library analysis to screen for possible conformational changes in comparison to experimental inter spin distance data. The most prominent agreement with one mode led to a working hypothesis of a conformational change and provides the basis for validation in future experiments. Due to the inherent synergy effects, we applied a combined experimental and simulation approach for the EPR-based distance determination in the globular DNA-binding protein LexA to probe conformation and dynamics of the N-terminal DNA-binding domains with respect to the C-terminal domains within the LexA homodimer. While the C-terminal dimerization domains exhibit a well-defined conformation that proved to be independent of DNA-binding, large-scale changes in conformation and dynamics were detected for the N-terminal domains. They were only found in a defined conformation when bound to DNA while in its absence a large rotational freedom of the entire N-terminal domains contributed to the conformational ensemble. Combined with a biochemical characterization of the autocatalytic cleavage of LexA, our data explains how LexA induces the SOS response after DNA damage or under latent antibiotic stress. We further studied the membrane photoreceptor-transducer complex NpSRII/NpHtrII that governs the light-dependent swimming behavior in Natronomonas pharaonis by a two-component signaling system. This system comprises extraordinary features of sensitivity, signal amplification, integration and transducer cooperativity, yet the molecular details of these features are poorly understood, as is signal propagation itself. By combining time-resolved cw EPR spectroscopy of NpSRII/NpHtrII variants spin labeled in the HAMP1 domain with time-resolved optical absorbance spectroscopy to report on the receptor signaling state, we found a tight kinetic coupling of receptor and transducer during the relaxation back to the ground state and hence a prolonged activation period, that with ~500 - ~700 ms is sufficiently long to cause phosphorylation bursts of the cognate kinase CheA. This explains signal amplification already on the level of the NpSRII/NpHtrII dimers. We further determined the transient difference spectra from the time-resolved EPR data that show local differences in dynamics and steric restrictions upon light-activation. Comparing these experimentally observed differences to predictions confirms the assumed two-state structural model and shows this transition between the two states for a single HAMP domain in a light-dependent manner. Additionally, our approach integrates a dynamic view into the model, since the two states are shown to exhibit different local dynamics in a fashion described previously as a competing model for signaling by dynamic differences based on biochemical studies. Here we show unification of the two models into one congruent description encompassing a transition between the two previously suggested states by concerted structural and dynamic changes. In an independent analysis using all-atom and coarse grained molecular dynamics of the NpSRII/NpHtrII complex in the minimal unit that can exert kinase control, the trimer of receptor-transducer dimers, we revealed a distinct dynamical pattern encoded in the primary sequence of the coiled-coil heptad-repeats. Upon receptor activation, these segments alter their dynamics in a concerted fashion with regions such as HAMP1 and the adaptation region becoming more compact, while HAMP2 and the tip become more dynamic, leading to dynamic and to limited structural changes at the CheA-kinase binding sites. Together with an extensive validation against experimental data, these findings suggest the altered dynamics as the mechanism for signal propagation along the extended coiled-coil structure of NpHtrII. This working model, that explains the current body of experimental data, allows for further refinement by all-atom molecular dynamics and provides a basis to devise future experiments for validation. The presented studies outline the versatile methodology of combined experimental and simulation approaches to analyze the conformation and dynamics of biomacromolecules including membrane protein complexes.

No description available.

Mass spectrometry has enjoyed enormous popularity over the years for studying biological systems. The theme of this dissertation was to develop and use mass spectrometry based tools to solve five biologically oriented problems associated with protein architecture and extend the utility of these tools to study protein polymer conjugation. The first problem involved elucidating the false negatives of how proteins with few basic residues, forms highly charged ions in electrospray ionization mass spectrometry (ESI MS). This study showed that the unfolding of polypeptide chains in solution leads to the emergence of highly charged protein ions in ESI MS mass spectra, even if the polypeptide chains lack a sufficient number of basic sites. In the second problem, a new technique was developed that can monitor small-scale conformational transitions that triggers protein activity and inactivity using porcine pepsin as a model protein. This work allowed us to revise a commonly accepted scenario of pepsin inactivation and denaturation. The physiological relevance of an enzyme-substrate complex was probed in our third problem. We observed by ESI MS that pepsin forms a facile complex with a substrate protein, N-lobe transferrin under mildly acidic pH. The observed complex could either be a true enzyme-substrate complex or may likely results from an electrostatically driven association. Our investigation suggested that the enzyme binds nonspecifically to substrate proteins under mild acidic pH conditions. The fourth problem dealt with the investigation of conformational heterogeneity of natively unstructured proteins using a combination of spectroscopic techniques and ESI MS as tools. It was observed that four different conformations of alpha-synuclein coexist in equilibrium. One of these conformations appeared to be tightly folded. Conclusions regarding the nature of these states were made by correlating the abundance evolution of the conformers as a function of pH with earlier spectroscopic measurements. The final problem was aimed at monitoring conformational transitions in polypeptide and polymer segments of PEGylated proteins using PEGylated ubiquitin as a model system. This studies suggested that for a PEGylated protein, polypeptides maintain their folded conformation to a greater extent whiles the polymer segments are bound freely to the protein.

Proteins are among the most versatile organic macromolecules essential for living systems and present in almost all biological processes. Cancer is associated with mutations that either enhance or disrupt the conformation of proteins. These mutations have been shown to accumulate in specific regions of a proteins three dimensional structure. In this thesis, the aim is to find connections that secondary structure elements make and explore them using a self-organizing map (SOM). The detection of these connections is done by first mapping the three-dimensional structure onto a novice type of distance matrix that also incorporates chemical information, and then deploying a density-based clustering algorithm. The connections found are mapped onto the SOM and later analyzed in order to see if highly mutated connections are more common among certain SOM-nodes. This was tested with an ANOVA that indicated that there are indeed mutational asymmetries among the nodes. By further analyzing the map it could also be stated that certain nodes were to a large extent activated by connections from genes associated with cancer. / Proteiner tillhör några av de mest mångsidiga organiska makromolekylerna, och är direkt nödvändiga för alla levande system och biologiska processer. Cancer orsakas av mutationer som antingen förstärker eller stör strukturen hos proteinet. Dessa mutationer tenderar att att samlas i specifika områden av proteinets tredimensionella struktur. I den här rapporten är målet att hitta kopplingar som sekundärstrukturselement skapar, och utforska dem med hjälp av en självorganiserande karta. Dessa kopplingar finnes genom att först skapa en tvådimensionell representation av proteinets tredimensionella struktur, och sedan använda en densitetsbaserad klustringsalgoritm. De funna kopplingarna mappas till de olika neuronerna i kartan och analyseras sedan för att se om kopplingar med hög mutationsnivå är mer vanliga hos vissa neuron. För att undersöka detta användes ett ANOVA-test som visade att så var fallet. Genom att ytterligare studera kartan upptäcktes fynd som indikerade att vissa neuron i högre utsträckning var aktiverade av kopplingar som härstammar från gener vi vet är associerade med cancer.

Wong, Siu Lun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 129-141). / Abstracts in English and Chinese. / Abstract --- p.i / Abstract (Chinese version) --- p.iii / Acknowledgements --- p.iv / List of Abbreviations --- p.v / List of Tables --- p.vii / List of Figures --- p.viii / Chapter 1. --- INTRODUCTION / Chapter 1.1 --- Neurodegenerative disorders - a brief overview --- p.1 / Chapter 1.2 --- Polyglutamine diseases --- p.2 / Chapter 1.3 --- Microscopically visible polyglutamine protein aggregates and its relation to toxicity --- p.7 / Chapter 1.4 --- Polyglutamine protein conformers and their relation to toxicity --- p.10 / Chapter 1.5 --- Modeling polyglutamine diseases in Drosophila / Chapter 1.5.1 --- GAL4/UAS spatial transgene expression system in Drosophila --- p.14 / Chapter 1.5.2 --- Temporal control of GAL4/UAS transgene expression system in Drosophila --- p.16 / Chapter 1.5.3 --- Drosophila as a model to study human pathologies --- p.19 / Chapter 1.5.4 --- Drosophila as a model to study polyglutamine diseases --- p.21 / Chapter 1.6 --- Aims of study --- p.26 / Chapter 2. --- MATERIALS AND METHODS / Chapter 2.1 --- Drosophila culture and manipulation / Chapter 2.1.1 --- Drosophila culture --- p.27 / Chapter 2.1.2 --- Phenotypic examination of adult external eye degeneration --- p.27 / Chapter 2.1.3 --- Pseudopupil assay of adult retinal degeneration and observation of green fluorescent protein in adult eyes --- p.28 / Chapter 2.2 --- Semi-quantitative Reverse Transcription-Polymerase Chain Reaction / Chapter 2.2.1 --- RNA extraction from adult Drosophila heads --- p.30 / Chapter 2.2.2 --- DNase treatment of extracted RNA --- p.31 / Chapter 2.2.3 --- Reverse transcription-Polymerase Chain Reaction (RT-PCR) --- p.31 / Chapter 2.2.4 --- Agarose gel electrophoresis --- p.33 / Chapter 2.3 --- Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) / Chapter 2.3.1 --- Protein extraction from adult Drosophila heads --- p.33 / Chapter 2.3.2 --- Preparation of SDS-polyacrylamide gel and electrophoresis --- p.34 / Chapter 2.3.3 --- Western blotting --- p.35 / Chapter 2.3.4 --- Immunodetection --- p.36 / Chapter 2.4 --- Immunoprecipitation --- p.38 / Chapter 2.5 --- Filter retardation assay --- p.39 / Chapter 2.6 --- Isolation and solubilization of SDS-insoluble protein --- p.40 / Chapter 2.7 --- Sucrose gradient sedimentation --- p.41 / Chapter 2.8 --- Preparation of Drosophila tissues for immunofluorescence analysis / Chapter 2.8.1 --- Dissection and immunostaining of Drosophila larval imaginal eye discs --- p.42 / Chapter 2.8.2 --- Cryosectioning and immunostaining of adult Drosophila heads --- p.44 / Chapter 2.9 --- Atomic force microscopy --- p.47 / Chapter 2.10 --- Reagents and buffers / Chapter 2.10.1 --- Reagents for Drosophila culture --- p.48 / Chapter 2.10.2 --- Reagents for RT-PCR --- p.52 / Chapter 2.10.3 --- Reagents for SDS-PAGE --- p.54 / Chapter 2.10.4 --- Reagents for immunoprecipitation --- p.57 / Chapter 2.10.5 --- Reagents for filter retardation assay --- p.57 / Chapter 2.10.6 --- Reagents for isolation and solubilization of SDS-insoluble protein --- p.58 / Chapter 2.10.7 --- Reagents for sucrose gradient sedimentation --- p.58 / Chapter 2.10.8 --- Reagents for immunofluorescence --- p.59 / Chapter 3. --- RESULTS / Chapter 3.1 --- Establishment of an inducible transgenic Drosophila model of polyglutamine diseases / Chapter 3.1.1 --- Introduction --- p.60 / Chapter 3.1.2 --- Results / Chapter 3.1.2.1 --- GAL80ts-mediated inducible expression of expanded polyglutamine protein in Drosophila / Chapter 3.1.2.1.1 --- GAL80ts controls GAL4/UAS-mediated polyQ protein expression --- p.61 / Chapter 3.1.2.1.2 --- Inducible expression of SDS-soluble expanded polyglutamine protein --- p.64 / Chapter 3.1.2.1.3 --- Inducible expression of expanded polyglutamine protein accumulates gradually in form of SDS-insoluble protein --- p.66 / Chapter 3.1.2.1.4 --- Inducible expression of expanded polyglutamine protein results in progressive accumulation of microscopically visible aggregates --- p.68 / Chapter 3.1.2.2 --- Inducible expression of expanded polyglutamine protein causes late-onset progressive neuronal degeneration in Drosophila / Chapter 3.1.2.2.1 --- Inducible expression of expanded polyglutamine protein leads to late-onset progressive deterioration of photoreceptor neurons --- p.68 / Chapter 3.1.2.2.2 --- Inducible expression of expanded polyglutamine protein neither causes external eye degenerative phenotype nor disrupts gross retinal morphology despite deterioration of photoreceptor neurons --- p.72 / Chapter 3.1.2.3 --- Co-expression of caspase inhibitor P35 suppresses polyglutamine-induced neuronal degeneration --- p.72 / Chapter 3.1.2.4 --- Co-expression of molecular chaperone Hsp70 suppresses polyglutamine-induced neuronal degeneration --- p.74 / Chapter 3.1.2.5 --- Inducible expression of expanded polyglutamine protein results in biphasic expression of molecular chaperone Hsp70 in Drosophila --- p.76 / Chapter 3.1.3 --- Discussion --- p.76 / Chapter 3.2 --- Involvement of microscopically visible polyglutamine aggregates in neurodegeneration / Chapter 3.2.1 --- Introduction --- p.83 / Chapter 3.2.2 --- Results / Chapter 3.2.2.1 --- Effect of Hsc70-K71S on microscopically visible polyglutamine aggregates and neuronal degeneration / Chapter 3.2.2.1.1 --- Co-expression of Hsc70-K71S reduces the level of microscopically visible polyglutamine aggregates --- p.83 / Chapter 3.2.2.1.2 --- Co-expression of Hsc70-K71S does not alter polyglutamine transgene expression --- p.84 / Chapter 3.2.2.1.3 --- Co-expression of Hsc70-K71S does not modify polyglutamine-induced neuronal degeneration --- p.87 / Chapter 3.2.2.2 --- Microscopically visible polyglutamine aggregates do not correlate with neuronal degeneration --- p.90 / Chapter 3.2.3 --- Discussion --- p.93 / Chapter 3.3 --- Detection of small SDS-insoluble expanded polyglutamine protein species and its association with neurodegeneration / Chapter 3.3.1 --- Introduction --- p.97 / Chapter 3.3.2 --- Results / Chapter 3.3.2.1 --- Accumulation of SDS-soluble expanded polyglutamine protein does not correlate with neuronal degeneration --- p.98 / Chapter 3.3.2.2 --- Identification of small SDS-insoluble expanded polyglutamine protein species / Chapter 3.3.2.2.1 --- Accumulation of total SDS-insoluble expanded polyglutamine protein positively correlates with progressive neuronal degeneration --- p.99 / Chapter 3.3.2.2.2 --- Accumulation of large SDS-insoluble expanded polyglutamine protein does not correlate with neuronal degeneration --- p.99 / Chapter 3.3.2.2.3 --- Accumulation of small SDS-insoluble expanded polyglutamine protein correlates with neuronal degeneration --- p.104 / Chapter 3.3.3 --- Discussion --- p.107 / Chapter 3.4 --- Biophysical characterization of small SDS-insoluble expanded polyglutamine protein species / Chapter 3.4.1 --- Introduction --- p.109 / Chapter 3.4.2 --- Results / Chapter 3.4.2.1 --- Separation of expanded polyglutamine protein species by sucrose gradient sedimentation --- p.110 / Chapter 3.4.2.2 --- Morphological studies of small SDS-insoluble expanded polyglutamine protein species by atomic force microscopy --- p.112 / Chapter 3.4.3 --- Discussion --- p.118 / Chapter 4. --- GENERAL DISCUSSION --- p.124 / Chapter 5. --- CONCLUSION --- p.127 / Chapter 6. --- REFERENCES --- p.129

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 5 uppsatser.

How proteins undergo conformational changes to bind a ligand is one of the most fundamental questions of protein biology. MD simulations provide a useful computational tool for studying the theoretical movements of protein in solution on nanosecond timescales. The results of these simulations can be used to guide experimental design. By correlating the theoretical models with the results of experimental studies, we can obtain a significant amount of information about protein dynamics. This study represents the application of both computational and traditional experimental techniques to study protein dynamics in HIV-1 protease. The results provide a novel mechanism for the conformational changes in proteins and address the role of residues outside the active site in protein dynamics. Additionally, these results are applied to the complex role of non-active site mutations in the development of drug resistance. Chapter II examines an invariant Thr80 at the apex of the P1 loop of HIV-1, HIV-2, and simian immunodeficiency virus protease. Sequence variability associated with human immunodeficiency virus type 1 (HIV-1) is useful for inferring structural and/or functional constraints at specific residues within the viral protease. Positions that are invariant even in the presence of drug selection define critically important residues for protease function. Three protease variants (T80V, T80N, and T80S) were examined for changes in structure, dynamics, enzymatic activity, affinity for protease inhibitors, and viral infectivity. While all three variants were structurally similar to the wild type, only T80S was functionally similar. T80V significantly decreased the ability of the enzyme to cleave a peptide substrate but maintained infectivity, while T80N abolished both activity and viral infectivity. Additionally, T80N decreased the conformational flexibility of the flap region, as observed by simulations of molecular dynamics. Taken together, these data indicate that HIV-1 protease functions best when residue 80 is a small polar residue and that mutations to other amino acids significantly impair enzyme function, possibly by affecting the flexibility of the flap domain. Chapter III focuses on residues within the hydrophobic core of each monomer in HIV-1 protease. Many hydrophobic residues located in the core of this dimeric enzyme frequently mutate in patients undergoing protease inhibitor therapy. The mechanism by which these mutations aid the development of drug resistance is not well understood. Using MD simulations, this study suggests that the hydrophobic residues outside the active site facilitate the conformational change that occurs in HIV-1 protease upon binding substrates and inhibitors. In these simulations, the core of each monomer significantly rearranges to assist in the expansion of the active site as hydrophobic core residues slide by each other, exchanging one hydrophobic contact for another. Such hydrophobic sliding may represent a general mechanism by which proteins undergo conformational changes. Mutation of these hydrophobic core residues would alter the packing of the hydrophobic core. Thus, these residues could facilitate drug resistance in HIV-1 protease by altering dynamic properties of HIV-1 protease preferentially affecting the relative affinity for inhibitors versus substrates. Chapter IV concentrates on a residue in the flap region, Ile54, which is significantly correlated with the development of drug resistance. A series of patient sequences containing the mutation I54A were evaluated for the most frequently occurring co-mutations. I54A was found to occur with mutations that were previously correlated with I54V mutations, including L10I, G48V, and V82A. Based on the results of this evaluation, the binding properties of five variant proteases were investigated: MDI54V, MDRI54A, I54V, I54A, and G48V. MDRI54V and MDRI54Aeach contained the mutations L10I, G48V, and V82A, and either I54V or I54A, respectively. The other variants contained only the mutation indicated. Mutations at Ile54 were able to significantly impact the thermodynamics of binding to saquinavir, amprenavir, and the recently approved darunavir. The magnitude of this impact depended on the presence or absence of other drug resistance mutations, including another mutation in the flap region, G48V. Therefore, while residues 48 and 54 are not in contact with each other, mutations at both sites had a cooperative effect that varies between inhibitors. The results demonstrate that residues outside the active site of HIV-1 protease are clearly important to enzyme function, possibly through their role in the dynamic properties of protease. Mutations outside the active site of protease that are known to cause drug resistance could alter the conformational flexibility of protease. While the role of protein dynamics in molecular recognition is still not fully understood, the results of this study indicate that altering the dynamic properties of a protein affects its ability to recognize ligands. Therefore, to design better inhibitors we will have to develop a more thorough understanding of protein dynamics.

Rotavirus is responsible for the death of many children annually, and current vaccines have lower efficiency in developing countries. A reverse translated consensus gene sequence of the rotavirus VP6 cloned into a pET-28a(+) plasmid was used to transform BL21 and KRX Escherichia coli cells. Optimal expression of soluble protein was induced in KRX cells by adding 0.05% L-rhamnose and 0.0001 M IPTG, with an incubation temperature of 25ºC for 6 h. VP6 was purified by combining anion exchange chromatography followed by affinity chromatography. Far-UV circular dichroism and intrinsic fluorescence were used as probes to assess the native structure of VP6 and structural in the presence of a denaturant, high sodium chloride concentrations and varying temperatures. The 0.2 M sodium chloride had an impact on the VP6’s tertiary structure and also influenced the proteins conformational changes as detected during thermal unfolding to 90ºC. Although treatment with 3 M urea showed tertiary structural changes no secondary structural loss occurred due to the presence of a denaturant. / Life Sciences / M. Sc. (Life Sciences)

Les biocapteurs sont des moyens d’analyse en plein essor à la fois rapides, sélectifs et peu coûteux applicables à des domaines extrêmement variés (environnement, santé, agroalimentaire,…). Dans ce type d’outil, un élément sensible de nature biologique (anticorps, enzyme, microorganisme, ADN…) doté d’un pouvoir de reconnaissance pour un analyte ou un groupe d’analytes est associé à un transducteur pouvant être de type électrochimique, optique ou thermique. Dans ce travail, nous nous sommes intéressés au développement de différents biocapteurs se basant sur l'immobilisation d'enzymes ou de bactéries sur des microélectrodes en vue d’une détection électrochimique. Nous avons montré les potentialités d’application de deux biocapteurs conductimétriques à base de protéinase K ou de protéinase K et de pronase à la détection des modifications de conformation de la myoglobine et de l’albumine de sérum bovin au cours de leur relargage à partir de microsphères de poly (ε-caprolactone). Nous avons également mis au point un biocapteur conductimétrique à base de catalase et d’alcool oxydase pour une détection rapide et sensible des alcools ainsi que deux biocapteurs à catalase pour la détection impédimétrique et conductimétrique du cyanure et l’étude des interactions catalase-cyanure. Nous avons enfin élaboré des biocapteurs bactériens à base de Pseudomonas putida F1 pour la détection du trichloroéthylène dans les eaux souterraines. Pour cela, une voie originale d’immobilisation des cellules, basée sur la fonctionnalisation du transducteur à l’aide de couches autoassemblées et d’anticorps, ainsi que l’utilisation de nanotubes de carbone, a été explorée / The development of biosensors is an expanding research area. Indeed, biosensors are rapid, selective and cost-effective analytical tools which find applications in various fields (environment, health, food,…). They are constituted of a sensitive biological element (antibody, enzyme, microorganism, DNA…), which can selectively recognize one analyte or a group of analytes, associated to an electrochemical, optical or thermal transducer. In this work, we developed different biosensors based on enzymes or bacteria immobilised onto microelectrodes in view of electrochemical detection. First, we demonstrated the potentialities of two conductometric biosensors based on proteinase K or proteinase K and pronase for the detection of myoglobin and bovine serum albumine conformation changes during their release from poly (ε-caprolactone) microspheres. Then, we elaborated a bi-enzymatic conductometric biosensor with catalase and alcohol oxidase as sensing elements, for a rapid and sensitive detection of alcohols. Catalase impedimetric and conductometric biosensors were also developed for cyanide detection and used for the study of catalase-cyanide interactions. Finally, we prepared Pseudomonas putida F1 whole cell biosensors for the determination of trichloroethylene in groundwaters. For that, an original route, including the functionalisation of the transducer with a self-assembled-monolayer and antibodies, and the use of single-wall carbon nanotubes, was investigated for cell immobilisation