Browsing by Subject "conformation"
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Item Allostery governs Cdk2 activation and differential recognition of CDK inhibitors(2021-05) Majumdar, AbirCyclin-dependent kinases (CDKs) are the master regulators of the eukaryotic cell cycle. To become activated, CDKs require both regulatory phosphorylation and binding of a cognate cyclin subunit. Using a series of DEER and NMR experiments, we studied the activation process of the G1/S kinase Cdk2 in solution. We show that catalytically inactive Cdk2 readily adopts multiple active-like states for efficient dephosphorylation, and that regulatory phosphorylation on the activation loop enhances allosteric coupling with the cyclin subunit. We then used DEER and FRET experiments to measure the binding of multiple CDK inhibitors and developed a thermodynamic model that describes the allosteric coupling between regulatory phosphorylation, cyclin binding and inhibitor binding. We reveal that the allosteric coupling between these biochemical effectors is responsible for the differential recognition of Cdk2 and Cdk4 inhibitors. Finally, we used sequence analysis, DEER, FRET and activity assays to identify and measure the effects of mutating an allosteric hub that has diverged between Cdk2 and Cdk4. We demonstrate that this hub controls the strength of allosteric coupling, and that the altered architecture and allosteric wiring of Cdk4 leads to compromised activity toward generic peptide substrates and comparative specialization toward its primary substrate retinoblastoma (RB).Item Effect of the Thermodynamic and Physical State of the Freeze-Concentrate on Protein Stability(2017-12) Jena, SampreetiIn this dissertation research, specific interactions (excipient-excipient, excipient/protein-ice, protein-excipient) governing protein conformational stability and crystallization behavior of excipients in the freeze concentrate, were explored. Furthermore, the effects of formulation composition (type and mole fractions of excipients in the formulation) on afore-mentioned interactions, during freeze-thaw and freeze-drying of protein formulations, was investigated. Concentration dependent effects of excipients including the bulking agent, lyo/cryo-protectant and surfactant on the nucleation and growth of crystalline phases in the freeze concentrate were characterized and quantified. Changes in the secondary and tertiary conformations of model proteins (such as Bovine Serum Albumin and Immunoglobulin) due to crystallization of excipients, were determined as a function of formulation composition during freeze-thaw and freeze-drying. Infrared (IR) Spectroscopy was used to detect onset of crystallization the bulking agent and lyo/cryo-protectant. X-Ray Diffractometry (XRD) was used to characterize the polymorphic form of crystalline phases. Far UV circular Dichroism (CD) was used to characterize secondary conformation of protein in thawed and reconstituted (freeze-dried) formulations. IR Spectroscopy was used to characterize secondary conformation of protein in frozen and freeze-dried formulations. A bulking agent – lyo/cryo-protectant – protein system, a typical freeze-drying formulation, was chosen for characterization of frozen and freeze-dried formulations. It was observed that high concentrations of non-crystallizing components such as the protein and lyo/cryo-protectant (usually a disaccharide such as trehalose) inhibited crystallization of the (otherwise readily crystallizing) bulking agent (such as mannitol) and vice versa. At low concentrations, surfactants such as Polysorbate 20, prevented growth of crystalline phases due to amphiphilic interface coverage, but when their concentrations exceeded the critical micelle concentration (CMC), they enhanced degree of crystallinity in the formulation. Structural unfolding of the protein was detected upon crystallization of the lyo/cryo-protectant and micelle formation (when surfactant concentration exceeded CMC). Detection of protein aggregates in reconstituted solutions, confirmed that unfolding induced during freezing, thawing and drying processes, did not reverse upon reconstitution. Presence of ice surfaces and other crystalline interfaces (such as those introduced by the bulking agent) significantly contributed to protein degradation. In our model system, thawing induced stresses such as recrystallization were found to be more detrimental than the stresses induced by freezing and desiccation and hence, freeze-drying yielded better structural recovery of the protein than freeze-thaw in our model system. Secondary relaxations arising from the flexible polar groups on the protein surface (millisecond time scales) and dynamic ring flips of the monosaccharide units about the glycosidic linkage (microsecond time scales) of disaccharides (indicating flexibility of glycosidic linkage) were detected in our model freeze-dried system using Frequency Domain Dielectric Spectroscopy. In the presence of protein, flexibility of the glycosidic linkage was decreased and likewise, presence of disaccharides slowed down the dynamics of flexible protein groups, up to a critical protein to disaccharide mass ratio (= 0.5). Surfactant and higher protein to disaccharide mass ratios (≥ 0.5) produced the opposite effect. These secondary relaxations govern conformational stability of the protein and propensity of the disaccharide to crystallize during storage below the Tg. In the final part of the thesis, effects of slow freezing on lyo/cryo-protectant-protein formulations during cryo-vitrification was investigated. Chemical toxicity of cell penetrating lyo/cryo -protectants such as Dimethyl Sulfoxide (DMSO), frequently used for cryo-vitrification of organs and tissues, was shown to be dictated by their hydrogen bonding behavior (characterized by IR Spectroscopy). At temperatures where hydrogen bonding interactions between lyo/cryo-protectant and water were unfavorable, the lyo/cryo-protectant directly partitioned in the hydration shell of the protein and caused unfolding of the protein, potentially due to hydrophobic interactions. It was also ascertained that when the freeze concentrate is vitrified during freezing, rapid thawing is a necessity to minimize ice recrystallization during devitrification to minimize the damage to the proteins. This dissertation research has enhanced an overall understanding of interactions between the excipients, protein and crystalline interfaces (of ice and crystalline excipients such as bulking agent) as well as protein dynamics in the freeze-concentrate. This information is needed to identify stresses arising in the protein micro-environment that lead to conformational destabilization (and loss of activity) during preservation of protein formulations and is currently absent in literature.