| dc.description.abstract | The goal of this dissertation was to develop a method to assess how solution conditions affect a protein's stability, and a well-characterized model protein, was used to accomplish this objective. Generally, stabilizing conditions for proteins are identified from screening a matrix of conditions using an array of biophysical methods that probe physical stability by determining melting temperatures (Tm) of secondary and tertiary structure. Although this approach has been invaluable for quickly obtaining stable formulations, these tools do not provide site-specific information, which is needed to gain mechanistic understanding of protein stability. Our methodology employs high-resolution solution NMR spectroscopy to acquire molecular information about how specific residues within the structure are impacted by solution conditions and how the conditions affect stability. The N-terminal domain of Calmodulin (N-CaM) was recombinantly expressed in 15N media along with supplemental calcium to enhance in vivo stability. Solution NMR spectroscopy was used to evaluate the role of site-specific changes in mobility and structure on the overall stability of this model system in different solution conditions. The 1H-15N HSQC experiment was used to investigate effects of pH and ionic strength on individual residues within or near the two homologous Ca2+-binding sites. Circular dichroism (CD) was also employed to assess the thermal stability of N-CaM and to help interpret the observed site-specific changes in 1H-15N HSQC spectra. To increase protein yield and produce a sufficient amount of protein for NMR analysis, varying amounts of calcium concentrations were screened in the expression media. Based on densitometric analysis of SDS-PAGE, 1.5 mM CaCl2 was determined to provide the optimal concentration for enhancing in vivo stability, leading to the highest yield of N-CaM expression. From 2D NMR experiments, quantitative information that reflects changes in dynamics was derived from changes in peak shape and chemical shift position to elucidate how the solution environment affects residues within N-CaM. 1H-15N HSQC experiments provided quantitative determination of specific residues near known degradation sites that undergo physical perturbations. Changes in the chemical shift position and peak shape observed during 1H-15N HSQC experiments confirmed that increasing pH and ionic strength affects a limited subset of residues within a localized region. Residues N60, G61, and D64 in the Ca2+-binding loop site I was affected, in increasing pH and high ionic strength, while residues in the corresponding position of Ca2+-binding loop site II remained unaffected. CD data confirmed that, at basic pH, increased ionic strength resulted in increased overall thermal stability. Our study used common methods of probing physical stability to evaluate the overall thermal stability of N-CaM in different solution conditions. Analysis by NMR spectroscopy provided more detailed, site-specific information to show that different solution conditions affect individual residues differently, even when the amino acid sequence and structure are highly similar. By utilizing NMR spectroscopy combined with CD analysis, we were able to show that a subset of individual residues play a role in overall stability. Employing 2D NMR experiments to provide molecular information can be utilized in evaluating solution conditions for formulation of proteins of therapeutic interest. | |
