Knob-socket Investigation of Stability and Specificity in Alpha-helical Secondary and Quaternary Packing Structure

The novel knob-socket (KS) model provides a construct to interpret and analyze the direct contributions of amino acid residues to the stability in α-helical protein structures. Based on residue preferences derived from a set of protein structures, the KS construct characterizes intra- and inter-helical packing into regular patterns of simple motifs. The KS model was used in the de novo design of an α-helical homodimer, KSα1.1. Using site-directed mutagenesis, KSα1.1 point mutants were designed to selectively increase and decrease stability by relating KS propensities with changes to α-helical structure. This study suggests that the sockets from the KS Model can be used as a measure of α-helical structure and stability. The KS model was also used to investigate coiled-coil specificity in bZIP proteins. Identifying and characterizing the interactions that determine the dimerization specificity between bZIP proteins is a crucial factor in better understanding disease formation and proliferation, as well as developing drugs or therapeutics to combat these diseases. Knob-Socket mapping methods identified Asn residues at a positions within the helices, and were determined to be crucial factors in coiled-coil specificity. Site-directed mutagenesis was conducted to investigate the role of the Asn residues, as well as the role played by the neighboring residues at the g and b positions. The results indicate that the Asn at the a position defines coiled-coil specificity, and that the Knob-Socket model can be used to determine bZIP protein quaternary interactions

Study of Physical Protein-Protein Interactions Between the MaSp1 C-Terminal Domain and Small Cysteine-Rich Proteins Found in the Major Ampullate Gland of Latrodectus hesperus

Spiders spin a wide variety of different silk types with different biological functions that are known for their extraordinary mechanical properties. Dragline silk has predominantly captured the interest of researchers because it exhibits high tensile strength and toughness while maintaining its elasticity. This thesis has focused on the characterization of a family of small molecular weight proteins recently discovered in dragline silk. These proteins were discovered in the western black widow spider, Latrodectus hesperus, and have been termed Cysteine-Rich Proteins (CRPs) due to their high conserved cysteine content. CRP family members were used in protein-protein interaction studies to determine if there is any interaction with the major ampullate spidroins (MaSps). After affinity chromatography and co-expression studies in bacteria, there were no detectable interactions between the CRPs and MaSp1. Further studies which could be an important role in the natural silk assembly process. Further protein interaction studies in different salt and pH conditions can further determine the function of the CRPs in dragline silk formation

Study of Physical Protein-Protein Interactions Between the MaSp1 C-Terminal Domain and Small Cysteine-Rich Proteins Found in the Major Ampullate Gland of Latrodectus hesperus

Spiders spin a wide variety of different silk types with different biological functions that are known for their extraordinary mechanical properties. Dragline silk has predominantly captured the interest of researchers because it exhibits high tensile strength and toughness while maintaining its elasticity. This thesis has focused on the characterization of a family of small molecular weight proteins recently discovered in dragline silk. These proteins were discovered in the western black widow spider, Latrodectus hesperus, and have been termed Cysteine-Rich Proteins (CRPs) due to their high conserved cysteine content. CRP family members were used in protein-protein interaction studies to determine if there is any interaction with the major ampullate spidroins (MaSps). After affinity chromatography and co-expression studies in bacteria, there were no detectable interactions between the CRPs and MaSp1. Further studies which could be an important role in the natural silk assembly process. Further protein interaction studies in different salt and pH conditions can further determine the function of the CRPs in dragline silk formation

Knob-socket Investigation of Stability and Specificity in Alpha-helical Secondary and Quaternary Packing Structure

The novel knob-socket (KS) model provides a construct to interpret and analyze the direct contributions of amino acid residues to the stability in α-helical protein structures. Based on residue preferences derived from a set of protein structures, the KS construct characterizes intra- and inter-helical packing into regular patterns of simple motifs. The KS model was used in the de novo design of an α-helical homodimer, KSα1.1. Using site-directed mutagenesis, KSα1.1 point mutants were designed to selectively increase and decrease stability by relating KS propensities with changes to α-helical structure. This study suggests that the sockets from the KS Model can be used as a measure of α-helical structure and stability. The KS model was also used to investigate coiled-coil specificity in bZIP proteins. Identifying and characterizing the interactions that determine the dimerization specificity between bZIP proteins is a crucial factor in better understanding disease formation and proliferation, as well as developing drugs or therapeutics to combat these diseases. Knob-Socket mapping methods identified Asn residues at a positions within the helices, and were determined to be crucial factors in coiled-coil specificity. Site-directed mutagenesis was conducted to investigate the role of the Asn residues, as well as the role played by the neighboring residues at the g and b positions. The results indicate that the Asn at the a position defines coiled-coil specificity, and that the Knob-Socket model can be used to determine bZIP protein quaternary interactions

Rational Knob-Socket Predictions of Alpha-Helical Stability

A construct that simplifies the complexity of residue packing would significantly impact our understanding and analysis higher order protein structure in the same way that the covalent peptide bond allows linear comparisons of protein sequences and main-chain hydrogen bonding patterns identify secondary structure. The novel knob-socket (KS) model provides a construct to interpret and analyze the direct contributions of amino acid residues to the stability in α-helical protein structures. Based on residue preferences derived from a set of protein structures, the KS construct characterizes intra- and inter-helical packing into regular patterns of simple motifs. Intra-helical interactions consist of a regular pattern of three residue triangular motifs called sockets, which contribute to helical stability. For inter-helical interactions, a single amino acid knob from one α-helix packs into a three amino acid socket within another α-helix. Therefore, sockets are defined in three categories: (1) free, unpacked and favoring intra-helical interactions, (2) filled, packed and favoring inter-helical interactions, and (3) non, unpacked and disfavoring α-helical structure. The three amino acid composition of a socket serves as a code that can be used to predict protein packing and by extension, can also be used to understand individual amino acid contributions to helical stability. The KS model was used in the de novo design of an α-helical homodimer, KSα1.1. Using site-directed mutagenesis, KSα1.1 point mutants have been rationally chosen to increase and decrease stability by relating KS propensities with changes to α-helical structure. In the KS α-helical model, each point mutation affects six surrounding sockets by altering the free/filled propensity values. By analyzing the changes in the propensities of these six sockets, KS based structure predictions were made for each mutant that relate to their stability. These predicted values are compared to the experimentally determined structure and stability of each protein from chemical and thermal denaturation studies as measured by circular dichroism spectroscopy. This study serves as a starting point to reveal how residue packing contributes to protein stability

Rational Knob-Socket Predictions of Alpha-Helical Stability

A construct that simplifies the complexity of residue packing would significantly impact our understanding and analysis higher order protein structure in the same way that the covalent peptide bond allows linear comparisons of protein sequences and main-chain hydrogen bonding patterns identify secondary structure. The novel knob-socket (KS) model provides a construct to interpret and analyze the direct contributions of amino acid residues to the stability in α-helical protein structures. Based on residue preferences derived from a set of protein structures, the KS construct characterizes intra- and inter-helical packing into regular patterns of simple motifs. Intra-helical interactions consist of a regular pattern of three residue triangular motifs called sockets, which contribute to helical stability. For inter-helical interactions, a single amino acid knob from one α-helix packs into a three amino acid socket within another α-helix. Therefore, sockets are defined in three categories: (1) free, unpacked and favoring intra-helical interactions, (2) filled, packed and favoring inter-helical interactions, and (3) non, unpacked and disfavoring α-helical structure. The three amino acid composition of a socket serves as a code that can be used to predict protein packing and by extension, can also be used to understand individual amino acid contributions to helical stability. The KS model was used in the de novo design of an α-helical homodimer, KSα1.1. Using site-directed mutagenesis, KSα1.1 point mutants have been rationally chosen to increase and decrease stability by relating KS propensities with changes to α-helical structure. In the KS α-helical model, each point mutation affects six surrounding sockets by altering the free/filled propensity values. By analyzing the changes in the propensities of these six sockets, KS based structure predictions were made for each mutant that relate to their stability. These predicted values are compared to the experimentally determined structure and stability of each protein from chemical and thermal denaturation studies as measured by circular dichroism spectroscopy. This study serves as a starting point to reveal how residue packing contributes to protein stability

Empagliflozin in Patients with Chronic Kidney Disease

Background The effects of empagliflozin in patients with chronic kidney disease who are at risk for disease progression are not well understood. The EMPA-KIDNEY trial was designed to assess the effects of treatment with empagliflozin in a broad range of such patients. Methods We enrolled patients with chronic kidney disease who had an estimated glomerular filtration rate (eGFR) of at least 20 but less than 45 ml per minute per 1.73 m(2) of body-surface area, or who had an eGFR of at least 45 but less than 90 ml per minute per 1.73 m(2) with a urinary albumin-to-creatinine ratio (with albumin measured in milligrams and creatinine measured in grams) of at least 200. Patients were randomly assigned to receive empagliflozin (10 mg once daily) or matching placebo. The primary outcome was a composite of progression of kidney disease (defined as end-stage kidney disease, a sustained decrease in eGFR to < 10 ml per minute per 1.73 m(2), a sustained decrease in eGFR of & GE;40% from baseline, or death from renal causes) or death from cardiovascular causes. Results A total of 6609 patients underwent randomization. During a median of 2.0 years of follow-up, progression of kidney disease or death from cardiovascular causes occurred in 432 of 3304 patients (13.1%) in the empagliflozin group and in 558 of 3305 patients (16.9%) in the placebo group (hazard ratio, 0.72; 95% confidence interval [CI], 0.64 to 0.82; P < 0.001). Results were consistent among patients with or without diabetes and across subgroups defined according to eGFR ranges. The rate of hospitalization from any cause was lower in the empagliflozin group than in the placebo group (hazard ratio, 0.86; 95% CI, 0.78 to 0.95; P=0.003), but there were no significant between-group differences with respect to the composite outcome of hospitalization for heart failure or death from cardiovascular causes (which occurred in 4.0% in the empagliflozin group and 4.6% in the placebo group) or death from any cause (in 4.5% and 5.1%, respectively). The rates of serious adverse events were similar in the two groups. Conclusions Among a wide range of patients with chronic kidney disease who were at risk for disease progression, empagliflozin therapy led to a lower risk of progression of kidney disease or death from cardiovascular causes than placebo