Nivedita Mitra

Determinants of quaternary association in legume lectins

It is well known that the sequence of amino acids in proteins code for its tertiary structure. It is also known that there exists a relationship between sequence and the quaternary structure of proteins. The question addressed here is whether the nature of quaternary association can be predicted from the sequence, similar to the three‐dimensional structure prediction from the sequence. The class of proteins called legume lectins is an interesting model system to investigate this problem, because they have very high sequence and tertiary structure homology, with diverse forms of quaternary association. Hence, we have used legume lectins as a probe in this paper to (1) gain novel insights about the relationship between sequence and quaternary structure; (2) identify the sequence motifs that are characteristic of a given type of quaternary association; and (3) predict the quaternary association from the sequence motif.

Structural studies on peanut lectin complexed with disaccharides involving different linkages: further insights into the structure and interactions of the lectin

Crystal structures of peanut lectin complexed with Galbeta1-3Gal, methyl-T-antigen, Galbeta1-6GalNAc, Galalpha1-3Gal and Galalpha1-6Glc and that of a crystal grown in the presence of Galalpha1-3Galbeta1-4Gal have been determined using data collected at 100 K. The use of water bridges as a strategy for generating carbohydrate specificity was previously deduced from the complexes of the lectin with lactose (Galbeta1-4Glc) and T-antigen (Galbeta1-3GalNAc). This has been confirmed by the analysis of the complexes with Galbeta1-3Gal and methyl-T-antigen (Galbeta1-3GalNAc-alpha-OMe). A detailed analysis of lectin-sugar interactions in the complexes shows that they are more extensive when the beta-anomer is involved in the linkage. As expected, the second sugar residue is ill-defined when the linkage is 1-->6. There are more than two dozen water molecules which occur in the hydration shells of all structures determined at resolutions better than 2.5 A. Most of them are involved in stabilizing the structure, particularly loops. Water molecules involved in lectin-sugar interactions are also substantially conserved. The lectin molecule is fairly rigid and does not appear to be affected by changes in temperature.

Probing into the role of conserved N-glycosylation sites in the Tyrosinase glycoprotein family

N-linked glycosylation has a profound effect on the proper folding, oligomerization and stability of glycoproteins. These glycans impart many properties to proteins that may be important for their proper functioning, besides having a tendency to exert a chaperone-like effect on them. Certain glycosylation sites in a protein however, are more important than other sites for their function and stability. It has been observed that some N-glycosylation sites are conserved over families of glycoproteins over evolution, one such being the tyrosinase related protein family. The role of these conserved N-glycosylation sites in their trafficking, sorting, stability and activity has been examined here. By scrutinizing the different glycosylation sites on this family of glycoproteins it was inferred that different sites in the same family of polypeptides can perform distinct functions and conserved sites across the paralogues may perform diverse functions.

Protein stabilization through phage display

RNase S consists of two proteolytic fragments of RNase A, residues 1–20 (S20) and residues 21–124 (S pro). A 15‐mer peptide (S15p) with high affinity for S pro was selected from a phage display library. Peptide residues that are buried in the structure of the wild type complex are conserved in S15p though there are several changes at other positions. Isothermal titration calorimetry studies show that the affinity of S15p is comparable to that of the wild type peptide at 25°C. However, the magnitudes of ΔH° and ΔC p are lower for S15p, suggesting that the thermal stability of the complex is enhanced. In agreement with this prediction, at pH 6, the T m of the S15p complex was found to be 10°C higher than that of the wild type complex. This suggests that for proteins where fragment complementation systems exist, phage display can be used to find mutations that increase protein thermal stability.

Purification of G-Protein Coupled Receptors Using Nanodiscs

G-Protein Coupled Receptors (GPCRs) are seven-transmembrane (7-TM) proteins and belong to the largest gene family in the human genome. They mediate intracellular signaling in response to extracellular stimuli such as light, small molecules, peptides, etc.GPCRs are expressed in a wide variety of cell types and modulate cellular and physiological responses to the stimuli, which make them ideal drug targets. Despite their importance, GPCRs are not well understood at the molecular level in terms of their activation mechanism because they are notoriously difficulty to purify for quantitative biophysical studies. Due to their 7-TM hydrophobic domain, they need to be purified in detergents, which are often incompatible with their stability. Currently, GPCRs are purified using detergent conditions determined individually for each GPCR using an empirical approach. Thus, only a handful of GPCRs have been purified, which is an impediment to obtaining a molecular understanding of GPCRs. Here, we have developed a method for purifying GPCRs using nanodiscs, which are nanometer sized, disc-shaped, and self-contained lipid bilayer particles. In our method, we incorporate GPCRs straight from the cell membrane of a mammalian expression system into nanodiscs to minimize the amount of time the protein is in contact with detergent. Using this approach, we have successfully purified a family B GPCR, parathyroid hormone 1 receptor (PTH1R). We have investigated the binding of purified PTH1R to its native ligand PTH1 (1-34) using fluorescence anisotropy and obtained a dissociation constant of ∼29 nM, in agreement with previous reports. We propose that our method could be a general approach to purify GPCRs that will enable quantitative biophysical studies to yield a better molecular understanding of their activation mechanisms and interactions with downstream signaling proteins.