Generation of Lamprey Monoclonal Antibodies (Lampribodies) Using the Phage Display System.

The variable lymphocyte receptors (VLRs) consist of leucine rich repeats (LRRs) and comprise the humoral antibodies produced by lampreys and hagfishes. The diversity of the molecules is generated by stepwise genomic rearrangements of LRR cassettes dispersed throughout the VLRB locus. Previously, target-specific monovalent VLRB antibodies were isolated from sea lamprey larvae after immunization with model antigens. Further, the cloned VLR cDNAs from activated lamprey leukocytes were transfected into human cell lines or yeast to select best binders. Here, we expand on the overall utility of the VLRB technology by introducing it into a filamentous phage display system. We first tested the efficacy of isolating phage into which known VLRB molecules were cloned after a series of dilutions. These experiments showed that targeted VLRB clones could easily be recovered even after extensive dilutions (1 to 109). We further utilized the system to isolate target-specific "lampribodies" from phage display libraries from immunized animals and observed an amplification of binders with relative high affinities by competitive binding. The lampribodies can be individually purified and ostensibly utilized for applications for which conventional monoclonal antibodies are employed

Single-molecule experiments in biological physics: methods and applications

I review single-molecule experiments (SME) in biological physics. Recent technological developments have provided the tools to design and build scientific instruments of high enough sensitivity and precision to manipulate and visualize individual molecules and measure microscopic forces. Using SME it is possible to: manipulate molecules one at a time and measure distributions describing molecular properties; characterize the kinetics of biomolecular reactions and; detect molecular intermediates. SME provide the additional information about thermodynamics and kinetics of biomolecular processes. This complements information obtained in traditional bulk assays. In SME it is also possible to measure small energies and detect large Brownian deviations in biomolecular reactions, thereby offering new methods and systems to scrutinize the basic foundations of statistical mechanics. This review is written at a very introductory level emphasizing the importance of SME to scientists interested in knowing the common playground of ideas and the interdisciplinary topics accessible by these techniques. The review discusses SME from an experimental perspective, first exposing the most common experimental methodologies and later presenting various molecular systems where such techniques have been applied. I briefly discuss experimental techniques such as atomic-force microscopy (AFM), laser optical tweezers (LOT), magnetic tweezers (MT), biomembrane force probe (BFP) and single-molecule fluorescence (SMF). I then present several applications of SME to the study of nucleic acids (DNA, RNA and DNA condensation), proteins (protein-protein interactions, protein folding and molecular motors). Finally, I discuss applications of SME to the study of the nonequilibrium thermodynamics of small systems and the experimental verification of fluctuation theorems. I conclude with a discussion of open questions and future perspectives.Comment: Latex, 60 pages, 12 figures, Topical Review for J. Phys. C (Cond. Matt

Mehr als Abstandsmessungen : die Weiterentwicklung von gepulster X-Band-Elektron-Elektron-Doppel-Resonanzspektroskopie

This thesis demonstrates the advancement of PELDOR spectroscopy beyond its original design of distance measurements in order to disentangle a maximum amount of information additionally encoded in the PELDOR data. In particular, the successful synthesis of novel polynitroxide radicals is described as well as the extraction of the relative orientation of spin labels, conformational flexibility and the separation of dipolar and exchange coupling via orientation selective PELDOR measurements in combination with PESIM based simulations. Moreover, the method of PELDOR "Spin Counting" was experimentally validated.In dieser Arbeit wird die Weiterentwicklung der X-Band PELDOR-Spektroskopie über Abstandsmessungen hinaus, mit dem Ziel ein Maximum an zusätzlicher Information aus den erhaltenen PELDOR-Spektren zu gewinnen, beschrieben. Insbesondere wird die erfolgreiche Synthese neuartiger Polynitroxidradikale aufgezeigt sowie die Extraktion der relativen Orientierung von Spinsonden, die Bestimmung der konformationellen Flexibilität und die Trennung von dipolarer und Austauschwechselwirkung. Zusätzlich wird die Methode des PELDOR "Spinzählens" experimentell validiert

Detecting Repetitions and Periodicities in Proteins by Tiling the Structural Space

The notion of energy landscapes provides conceptual tools for understanding the complexities of protein folding and function. Energy Landscape Theory indicates that it is much easier to find sequences that satisfy the "Principle of Minimal Frustration" when the folded structure is symmetric (Wolynes, P. G. Symmetry and the Energy Landscapes of Biomolecules. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 14249-14255). Similarly, repeats and structural mosaics may be fundamentally related to landscapes with multiple embedded funnels. Here we present analytical tools to detect and compare structural repetitions in protein molecules. By an exhaustive analysis of the distribution of structural repeats using a robust metric we define those portions of a protein molecule that best describe the overall structure as a tessellation of basic units. The patterns produced by such tessellations provide intuitive representations of the repeating regions and their association towards higher order arrangements. We find that some protein architectures can be described as nearly periodic, while in others clear separations between repetitions exist. Since the method is independent of amino acid sequence information we can identify structural units that can be encoded by a variety of distinct amino acid sequences

Biosignature storage in sulfate minerals- synthetic and natural investigations of the jarosite group minerals

The discovery of jarosite on Mars in 2004 generated increased interest in the properties of the mineral related to the search for life on other planets. Several studies indicate that the formation of jarosite can be linked to biological activity on Earth and biomolecules such as amino acids have been found associated with terrestrial jarosite samples. A series of natural and synthetic investigations using different jarosite end-members has been conducted and is presented in this dissertation to investigate the possibility that jarosite can store biosignatures. Natural samples were analyzed by x-ray diffraction, elemental carbon analysis and laser-desorption Fourier transform mass spectrometry (LD-FTMS) and were found to contain the amino acid glycine. Synthetic experiments were conducted in which the different end-members were synthesized in the presence of glycine as well as the amino acid alanine and the amino acid breakdown product methylamine. These samples were analyzed by x-ray diffraction, neutron diffraction, LD-FTMS and thermogravimetric analysis (TGA) techniques. Results of these experiments show that the detection of the biosignature and the effect that biomolecule has on the jarosite minerals is dependent on the end-member and indicate that the jarosite minerals are an excellent target for detecting potential signs of past life on other planets

Directing hMSCs fate through geometrical cues and mimetics peptides

The native microenvironment of mesenchymal stem cells (hMSCs)—the extracellular matrix (ECM), is a complex and heterogenous environment structured at different scales. The present study aims at mimicking the hierarchical microorganization of proteins or growth factors within the ECM using the photolithography technique. Polyethylene terephthalate substrates were used as a model material to geometrically defined regions of RGD + BMP‐2 or RDG + OGP mimetic peptides. These ECM‐derived ligands are under research for regulation of mesenchymal stem cells osteogenic differentiation in a synergic manner. The hMSCs osteogenic differentiation was significantly affected by the spatial distribution of dually grafted peptides on surfaces, and hMSCs cells reacted differently according to the shape and size of peptide micropatterns. Our study demonstrates the presence of a strong interplay between peptide geometric cues and stem cell differentiation toward the osteoblastic lineage. These tethered surfaces provide valuable tools to investigate stem cell fate mechanisms regulated by multiple ECM cues, thereby contributing to the design of new biomaterials and improving hMSCs differentiation cues