The use of the Knob-Socket model in the synthesis and expression of a novel protein, Star1.0

The knob-socket model is a code to describe how proteins interact to form tertiary structures. The basis of the knob-socket model includes a one amino acid residue knob from one piece of secondary structure packing into a three amino acid residue socket from another piece of secondary structure. Sockets can be described as free (disfavoring knob packing), or filled (favoring knob packing) and are given these designations depending on their three amino acid composition. A propensity library was developed and gives the frequency at which each socket is found to be either free or filled based on data from the PDB database. We aim to show that the knob socket model and the socket propensity library can be used to predict protein secondary structure, and therefore be used in protein design. This idea was used in the de novo design of the STAR1.0 protein. The STAR1.0 protein was designed with a unique five alpha-helical structure in the shape of a five pointed star. The sequence was developed using the alpha-helical propensity library and was further optimized for expression in E. coli. The STAR1.0 protein was expressed and purified, and then secondary structure was analyzed using circular dichroism spectroscopy. STAR1.0 is now being expressed on a large scale and purified in order to produce a high concentration of protein for x-ray crystallography, which will determine if the structure matches the theoretical five pointed star shape

The use of the Knob-Socket model in the synthesis and expression of a novel protein, Star1.0

The knob-socket model is a code to describe how proteins interact to form tertiary structures. The basis of the knob-socket model includes a one amino acid residue knob from one piece of secondary structure packing into a three amino acid residue socket from another piece of secondary structure. Sockets can be described as free (disfavoring knob packing), or filled (favoring knob packing) and are given these designations depending on their three amino acid composition. A propensity library was developed and gives the frequency at which each socket is found to be either free or filled based on data from the PDB database. We aim to show that the knob socket model and the socket propensity library can be used to predict protein secondary structure, and therefore be used in protein design. This idea was used in the de novo design of the STAR1.0 protein. The STAR1.0 protein was designed with a unique five alpha-helical structure in the shape of a five pointed star. The sequence was developed using the alpha-helical propensity library and was further optimized for expression in E. coli. The STAR1.0 protein was expressed and purified, and then secondary structure was analyzed using circular dichroism spectroscopy. STAR1.0 is now being expressed on a large scale and purified in order to produce a high concentration of protein for x-ray crystallography, which will determine if the structure matches the theoretical five pointed star shape

Application of the Knob Socket Model to predict changes in alpha helical structure

The Knob Socket (KS) model is a 4 amino acid motif that describes the way a protein will fold and pack its residues to form tertiary structure. The model includes a one amino acid residue knob from one secondary structure that packs into a three amino acid residue socket of another secondary structure. The α-helical sockets can be placed into three different categories: (1) free, unpacked and favoring intra-helical interactions, (2) filled, packed with a knob, and favoring inter-helical interactions, and (3) non, unpacked and disfavoring α-helical structure. Data within the Protein Data Bank was used to develop propensity libraries for each type of secondary structures. An α-helical propensity library was used to determine the relative frequency in which specific amino acid composition of sockets were free or filled. From this library and use of the KS model, a novel anti-parallel α-helical homodimer, KSα1.1, was designed. A single point mutation in the KSα1.1 sequence was incorporated in order to change the propensities of the surrounding six sockets and named a ‘hexagon.’ Values calculated from the difference between the total socket propensities for KSα1.1 and its corresponding mutated versions were used to predict changes in alpha helical content. Negative values corresponded to a predicted decrease in alpha helical content whereas positive values corresponded to a predicted increase in alpha helical content. Point mutations were made in the KSα1.1 sequence through the use of site-directed mutagenesis. To obtain high amounts of the desired mutated protein, plasmid vectors containing the specific point mutations in KSα1.1 sequence were transformed and expressed in E.coli. The transformed cells were induced for protein expression with Isopropyl β-D-1-thiogalactopyranoside (IPTG) and purified via column chromatography. The mutated versions of KSα1.1 were analyzed via circular dichroism (CD) spectroscopy to confirm predictions made using the KS model and propensity libraries. Deconvolutions were used to analyze the CD graphs and determine the percent content of alpha helix, beta sheet, and random coil structures. Mutant KSα1.1 proteins were compared to wild-type KSα1.1 protein in order to analyze changes in higher ordered protein packing and alpha helical structure

Application of the Knob Socket Model to predict changes in alpha helical structure

The Knob Socket (KS) model is a 4 amino acid motif that describes the way a protein will fold and pack its residues to form tertiary structure. The model includes a one amino acid residue knob from one secondary structure that packs into a three amino acid residue socket of another secondary structure. The α-helical sockets can be placed into three different categories: (1) free, unpacked and favoring intra-helical interactions, (2) filled, packed with a knob, and favoring inter-helical interactions, and (3) non, unpacked and disfavoring α-helical structure. Data within the Protein Data Bank was used to develop propensity libraries for each type of secondary structures. An α-helical propensity library was used to determine the relative frequency in which specific amino acid composition of sockets were free or filled. From this library and use of the KS model, a novel anti-parallel α-helical homodimer, KSα1.1, was designed. A single point mutation in the KSα1.1 sequence was incorporated in order to change the propensities of the surrounding six sockets and named a ‘hexagon.’ Values calculated from the difference between the total socket propensities for KSα1.1 and its corresponding mutated versions were used to predict changes in alpha helical content. Negative values corresponded to a predicted decrease in alpha helical content whereas positive values corresponded to a predicted increase in alpha helical content. Point mutations were made in the KSα1.1 sequence through the use of site-directed mutagenesis. To obtain high amounts of the desired mutated protein, plasmid vectors containing the specific point mutations in KSα1.1 sequence were transformed and expressed in E.coli. The transformed cells were induced for protein expression with Isopropyl β-D-1-thiogalactopyranoside (IPTG) and purified via column chromatography. The mutated versions of KSα1.1 were analyzed via circular dichroism (CD) spectroscopy to confirm predictions made using the KS model and propensity libraries. Deconvolutions were used to analyze the CD graphs and determine the percent content of alpha helix, beta sheet, and random coil structures. Mutant KSα1.1 proteins were compared to wild-type KSα1.1 protein in order to analyze changes in higher ordered protein packing and alpha helical structure

Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET

Tumor-derived exosomes are emerging mediators of tumorigenesis with tissue-specific addresses and messages. We explored the function of melanoma-derived exosomes in the formation of primary tumor and metastases in mouse and human subjects. Exosomes from highly metastatic melanoma increased the metastatic behavior of primary tumors by permanently “educating” bone marrow (BM) progenitors via the MET receptor. Melanoma-derived exosomes also induced vascular leakiness at pre-metastatic sites, and reprogrammed BM progenitors towards a c-Kit(+)Tie2(+)Met(+) pro-vasculogenic phenotype. Reducing Met expression in exosomes diminished the pro-metastatic behavior of BM cells. Importantly, MET expression was elevated in circulating CD45(−)C-KIT(low/+)TIE2(+) BM progenitors from metastatic melanoma subjects. RAB1a, RAB5b, RAB7, and RAB27a were highly expressed in melanoma cells and Rab27a RNA interference decreased exosome production, preventing BM education, tumor growth and metastasis. Finally, we identified an exosome-specific “melanoma signature” with prognostic and therapeutic potential, comprised of TYRP2, VLA-4, HSP70, an HSP90 isoform and the MET oncoprotein