Mapping Site-Specific Changes that Affect Stability of the NTerminal Domain of Calmodulin

Biophysical tools have been invaluable in formulating therapeutic proteins. These tools characterize protein stability rapidly in a variety of solution conditions, but in general, the techniques lack the ability to discern site-specific information to probe how solution environment acts to stabilize or destabilize the protein. NMR spectroscopy can provide site-specific information about subtle structural changes of a protein under different conditions, enabling one to assess the mechanism of protein stabilization. In this study, NMR was employed to detect structural perturbations at individual residues as a result of altering pH and ionic strength. The N-terminal domain of calmodulin (N-CaM) was used as a model system, and the 1H-15N heteronuclear single quantum coherence (HSQC) experiment was used to investigate effects of pH and ionic strength on individual residues. NMR analysis revealed that different solution conditions affect individual residues differently, even when the amino acid sequence and structure are highly similar. This study shows that addition of NMR to the formulation toolbox has the ability to extend understanding of the relationship between site-specific changes and overall protein stability

Critical Review of Rasaratna Samuccaya: A Comprehensive Treatise of Indian Alchemy

Rasaratna Samuccaya (RRS) a 13th century C.E. alchemical treatise, authored by Vāgbhaṭa, is a useful compilation related to preparation and properties of drugs of mineral and metallic origin. This text throws light on the state of Indian expertise in the field of alchemy regarding the extraction, purification, conversion of metals/minerals into therapeutically suitable forms, various instruments developed for alchemical purposes and treatment of numerous diseases by using herbo-mineral preparations. The present work is an attempt to summarize the key features of RRS to highlight its utility and contribution in the development of Indian alchemy. To study and summarize the important, comprehensive and specific points mentioned in RRS and to elaborate the contribution of RRS in the field of Indian alchemy. A critical review of RRS from Suratnojjvalā Hindi commentary by Ambikadatta Shastri was done and the collected information was compared with other available literature of Rasaśāstra. Research of modern science was also utilized to explore some facts mentioned by Vāgbhaṭa. RRS is a precise treatise among available ancient literature. It comprises of all eight branches of Ayurveda, although it mainly deals with therapeutic aspects of Rasaśāstra and emphasizes the use of metals and minerals in treating nearly 68 types of ailments. It contains 30 chapters, 3871 verses and detailed description of 960 formulations. Classification of metals and minerals; description of some new instruments, formulations and averting use of metals and minerals in pregnancy are the key features of RRS

Effect of PEGylation on protein hydrodynamics

We studied the effect of PEGylation on protein hydrodynamic behavior using hen egg-white lysozyme (HEWL) as a model protein. HEWL was PEGylated with a linear, 20 kDa PEG using reductive amination to produce PEG1-, PEG2-, and PEG3-HEWL. Near- and far-UV–CD spectroscopy revealed no significant effect of PEGylation on HEWL higher order structure. SDS–PAGE, mass spectrometry, online static light scattering (SLS) and sedimentation velocity analytical ultracentrifugation (SV-AUC) were employed to characterize the heterogeneity and molecular weights of the purified PEG-HEWL molecules, the results of which underscored the importance of using first-principle based methods for such analyses along with the underlying complexities of characterizing PEG–protein conjugates. Hydrodynamic characterization of various linear and branched PEGs (5–40 kDa) and PEG-HEWL molecules was performed using dynamic light scattering (DLS) and SV-AUC. The PEG polymer exhibited a random-coil conformation in solution with the Mw ∝ Rhn scaling relationship yielding a scaling exponent (n) = 2.07. Singly branched PEGs were also observed to exhibit random-coil behavior with Stokes radii identical to those of their linear counterparts. SV-AUC studies of PEG-HEWL showed PEG has a “parachute” like effect on HEWL, and dramatically increases the frictional drag; PEG-HEWL also exhibited random-coil-like characteristics in solution (n = 1.8). The sedimentation coefficient (s) of PEG-HEWL remained invariant with increasing degree of PEGylation, indicating that the increase in molecular mass from PEG was compensated by an almost equivalent increase in frictional drag. Our studies draw caution to using SV-AUC for the characterization of size heterogeneity of PEG–protein mixtures