Biomedical subjects

Neeti Sinha

Publications and source records attributed to Neeti Sinha.

3 recordsLinked to original sources

PubMed2002

Differences in electrostatic properties at antibody-antigen binding sites: implications for specificity and cross-reactivity.

Antibodies HyHEL8, HyHEL10, and HyHEL26 (HH8, HH10, and HH26, respectively) recognize highly overlapping epitopes on hen egg-white lysozyme (HEL) with similar affinities, but with different specificities. HH8 binding to HEL is least sensitive toward mutations in the epitope and thus is most cross-reactive, HH26 is most sensitive, whereas the sensitivity of HH10 lies in between HH8 and HH26. Here we have investigated intra- and intermolecular interactions in three antibody-protein complexes: theoretical models of HH8-HEL and HH26-HEL complexes, and the x-ray crystal structure of HH10-HEL complex. Our results show that HH8-HEL has the lowest number and HH26-HEL has the highest number of intra- and intermolecular hydrogen bonds. The number of salt bridges is lowest in HH8-HEL and highest in HH26-HEL. The binding site salt bridges in HH8-HEL are not networked, and are weak, whereas, in HH26-HEL, an intramolecular salt-bridge triad at the binding site is networked to an intermolecular triad to form a pentad. The pentad and each salt bridge of this pentad are exceptionally stabilizing. The number of binding-site salt bridges and their strengths are intermediate in HH10-HEL, with an intramolecular triad. Our further calculations show that the electrostatic component contributes the most to binding energy of HH26-HEL, whereas the hydrophobic component contributes the most in the case of HH8-HEL. A "hot-spot" epitope residue Lys-97 forms an intermolecular salt bridge in HH8-HEL, and participates in the intermolecular pentad in the HH26-HEL complex. Mutant modeling and surface plasmon resonance (SPR) studies show that this hot-spot epitope residue contributes significantly more to the binding than an adjacent epitope residue, Lys-96, which does not form a salt bridge in any of the three HH-HEL complexes. Furthermore, the effect of mutating Lys-97 is most severe in HH26-HEL. Lys-96, being a charged residue, also contributes the most in HH26-HEL among the three complexes. The SPR results on these mutants also highlight that the apparent "electrostatic steering" on net on rates actually act at post-collision level stabilization of the complex. The significance of this work is the observed variations in electrostatic interactions among the three complexes. Our work demonstrates that higher electrostatics, both as a number of short-range electrostatic interactions and their contributions, leads to higher binding specificity. Strong salt bridges, their networking, and electrostatically driven binding, limit flexibilities through geometric constrains. In contrast, hydrophobic driven binding and low levels of electrostatic interactions are associated with conformational flexibility and cross-reactivity.

Animals↗

PubMed2002

Protein structure to function via dynamics.

Protein folding, binding, catalytic activity and molecular recognition all involve molecular movements, with varying extents. The molecular movements are brought upon via flexible regions. Stemming from sequence, a fine tuning of electrostatic and hydrophobic properties of the protein fold determine flexible and rigid regions. Studies show flexible regions usually lack electrostatic interactions, such as salt-bridges and hydrogen-bonds, while the rigid regions often have larger number of such electrostatic interactions. Protein flexible regions are not simply an outcome of looser packing or instability, rather they are evolutionally selected. In this review article we highlight the significance of protein flexibilities in folding, binding and function, and their structural and thermodynamic determinants. Our electrostatic calculations and molecular dynamic simulations on an antibody-antigen complex further illustrate the importance of protein flexibilities in binding and function.

Antibody Affinity↗

PubMed2002

Electrostatics in protein binding and function.

Protein electrostatic properties stem from the proportion and distribution of polar and charged residues. Polar and charged residues regulate the electrostatic properties by forming short-range interactions, like salt-bridges and hydrogen-bonds, and by defining the over-all electrostatic environment in the protein. Electrostatics play a major role in defining the mechanisms of protein-protein complex formation, molecular recognitions, thermal stabilities, conformational adaptabilities and protein movements. For example:- Functional hinges, or flexible regions of the protein, lack short-range electrostatic interactions; Thermophilic proteins have higher electrostatic interactions than their mesophilic counter parts; Increase in binding specificity and affinity involve optimization of electrostatics; High affinity antibodies have higher, and stronger, electrostatic interactions with their antigens; Rigid parts of proteins have higher and stronger electrostatic interactions. In this review we address the significance of electrostatics in protein folding, binding and function. We discuss that the electrostatic properties are evolutionally selected by a protein to perform an specific function. We also provide bona fide examples to illustrate this. Additionally, using continuum electrostatic and molecular dynamics approaches we show that the "hot-spot" inter-molecular interactions in a very specific antibody-antigen binding are mainly established through charged residues. These "hot-spot" molecular interactions stay intact even during high temperature molecular dynamics simulations, while the other inter-molecular interactions, of lesser functional significance, disappear. This further corroborates the significance of charge-charge interactions in defining binding mechanisms. High affinity binding frequently involves "electrostatic steering". The forces emerge from over-all electrostatic complementarities and by the formation of charged and polar interactions. We demonstrate that although the high affinity binding of barnase-barstar and anti-hen egg white lysozyme (HEL) antibody-HEL complexes involve different molecular mechanisms, it is electrostatically regulated in both the cases. These observations, and several other studies, suggest that a fine tuning of local and global electrostatic properties are essential for protein binding and function.

Hydrogen Bonding↗