145 research outputs found
Residue Specific Analysis of Frustration in Folding Landscape of Repeat Alpha/Beta Protein Apoflavodoxin
Macromolecular crowding modulates folding mechanism of alpha/beta protein apoflavodoxin
Protein dynamics in cells may be different from that in dilute solutions in
vitro since the environment in cells is highly concentrated with other
macromolecules. This volume exclusion due to macromolecular crowding is
predicted to affect both equilibrium and kinetic processes involving protein
conformational changes. To quantify macromolecular crowding effects on protein
folding mechanisms, here we have investigated the folding energy landscape of
an alpha/beta protein, apoflavodoxin, in the presence of inert macromolecular
crowding agents using in silico and in vitro approaches. By coarse-grained
molecular simulations and topology-based potential interactions, we probed the
effects of increased volume fraction of crowding agents (phi_c) as well as of
crowding agent geometry (sphere or spherocylinder) at high phi_c. Parallel
kinetic folding experiments with purified Desulfovibro desulfuricans
apoflavodoxin in vitro were performed in the presence of Ficoll (sphere) and
Dextran (spherocylinder) synthetic crowding agents. In conclusion, we have
identified in silico crowding conditions that best enhance protein stability
and discovered that upon manipulation of the crowding conditions, folding
routes experiencing topological frustrations can be either enhanced or
relieved. The test-tube experiments confirmed that apoflavodoxin's
time-resolved folding path is modulated by crowding agent geometry. We propose
that macromolecular crowding effects may be a tool for manipulation of protein
folding and function in living cells.Comment: to appear in Biophysical Journal (2009). to appear in Biophysical
Journal (2009
Folding, Stability and Shape of Proteins in Crowded Environments: Experimental and Computational Approaches
How the crowded environment inside cells affects folding, stability and structures of proteins is a vital question, since most proteins are made and function inside cells. Here we describe how crowded conditions can be created in vitro and in silico and how we have used this to probe effects on protein properties. We have found that folded forms of proteins become more compact in the presence of macromolecular crowding agents; if the protein is aspherical, the shape also changes (extent dictated by native-state stability and chemical conditions). It was also discovered that the shape of the macromolecular crowding agent modulates the folding mechanism of a protein; in addition, the extent of asphericity of the protein itself is an important factor in defining its folding speed
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