Understanding the Effects of Molecular Crowding on the Structure and Stability of Proteins Using NMR Spectroscopy

Abstract

Much of the research on biological proteins is performed in vitro (under artificial conditions) involving the isolation of the protein from the cell. The living cell, however, constitutes a very complex system, and a protein's structure and stability may be influenced by its native environment. An understanding of the effects of molecular crowding will provide important information regarding a protein's structure, dynamics, and stability in vivo.This study involves the titration of 15N FGF-1 with various intracellular components (to simulate an in-cell environment) followed by NMR spectroscopy to determine any chemical shift perturbation corresponding to shifting amino acid residues. Fibroblast Growth Factor 1 (FGF-1) is a protein involved in cellular proliferation, wound healing, and cancer development and metastasis. Little information is known regarding FGF-1's interactions inside the cell, as it follows a non-classical secretion pathway. To better understand the role of intracellular proteins on the structure of FGF-1, several experiments were carried out using multi-dimensional NMR spectroscopy; to the FGF-1 sample were added (1) intracellular proteins (from the purification of unlabeled FGF), (2) intracellular proteins and lysozyme, and (3) lysozyme alone (added as a control). HSQC data was obtained at regular intervals and processed using XWIN-NMR and Sparky software. A chemical shift perturbation plot was constructed from the data to show the (individual and combined) effects of the addition of the intracellular proteins and/or lysozyme on FGF-1. The preliminary results of this study indicate that a moderate number of amino acid residues were perturbed with the addition of intracellular proteins. This implies that molecular crowding plays a role in the structural conformation of FGF-1 and possibly other proteins in vivo.