advances-in-protein-chemistry

with the total energy of an isolated system are constant despite internal changes. Moreover, second law which is related to Entropy of the
system, states the mechanical work results when a body interacts with another body at lower temperature. Therefore, any autonomous
process shows an increase in Entropy. Another important law that deals with thermal equilibrium (zeroth law of thermodynamics) states
that if two objects are in thermal equilibrium with a third object then the first two objects would be in thermal equilibrium with respect
to each other. The Gibbs free energy (ΔG) equation is helpful in understanding of thermodynamic activities of a system. In general, free
energy is a thermodynamic quantity equivalent to the capacity of a physical system to do work. Thermodynamics of protein stability
reveals a general tendency for proteins that denature at higher temperatures to have greater free energies of maximal stability. There is a
constant equilibrium between proteins in denatured and native states. The conditions in the system determine the amount of one of the
species. There are usually one or few states of folded proteins and numerous states of unfolded proteins.
ΔG F
< ΔG D
All the systems desire for the lowest energy possible. There is a barrier between denatured and folded protein, a transition state, which
needs to be overcome to shift from one form to the other. Mathematical relation of thermodynamic parameters in the form of Gibbs free
energy equation is below:
ΔG = ΔH – TΔS
Where, ΔG-free energy is measure of protein stability. The more negative is the free energy, the more stable structure of protein is
observed, ΔH– enthalpy is bond formation and breaking in protein while ΔS-Entropy is degrees of freedom in protein. Free Energy
(ΔG) is a thermodynamic quantity equivalent to the capacity of a physical system to do work. Entropy (ΔS), quantity representing
the amount of energy in a system that is no longer available for doing mechanical work, favors unfolding of protein, and you have to
overcome its barrier by lowering the ΔG. On the other hand, Enthalpy (ΔH), a thermodynamic quantity equal to the internal energy of a
system plus the product of its volume and pressure, favors folding of protein, because the number of inter-atomic interactions increases
upon protein folding. Enthalpy (ΔH) and Entropy (ΔS) are about the same size usually, so they cancel each other showing proteins are
not particularly stable, which is very imperative for their biological function. The resulting ΔG is therefore rather small, although ΔH and
ΔS themselves are large.
Forces that Govern Protein Stability
Figure 1: Forces in Atoms of Amino Acids that governs Protein Stability.
Hydrophobic effect: This is the most significant force in protein stability. Hydrophobic residues tend to associate and turn to the
inside of the core of the protein molecule. Hydrophilic residues will face the outside (in contact with water). For example, water molecules
surround the oil and lose some degrees of freedom. When hydrophobic residues (oil) are buried inside the protein, the disorder of water
increases, and ΔG decreases- the reaction is more spontaneous.
Van der Waals (VDW) interactions: these are relatively weak attractive forces between neutral atoms and molecules arising from
polarization induced in each particle by the presence of other particles. Inside the protein, the residues are tightly packed due to Van der
Waals interactions. The types of Van der Waals interactions govern the stability of proteins e.g. London dispersion forces and dipoledipole
forces.
Van der Waals potential: If the atoms are too far, the interaction between them is not observable. If they are too close- they collide
with each other. It should be noted that there is some optimum distance in which attractive and repulsive forces are in equilibrium.
Hydrogen bonds: Hydrogen bonds are interaction between a hydrogen atom attached to an electronegative atom and a lone pair of
electrons (Also on an electronegative atom).
Practical/Experimental Approach
Fluorescence: The amino acid Tryptophan is hydrophobic in nature, if it is buried (inside the folded or native protein) the fluorescence
is small. In principle, when it is exposed to the surface (for instance in the unfolded state) fluorescence is increased, as a result.
Calorimetery: Heat the protein and measure denaturation over different temperature ranges. When protein unfolds, it produces
heat. Hence, calorimeter measures the released heat gives the indication for denaturation.
CD Spectroscopy: There are characteristic absorption spectra for different protein structures. Proteins of approximately 300 residues
other than small globular structures have been most studied and the results cannot be applicable to other larger proteins like fibrous
proteins and/or those present in plasma membrane. However, the basic rules of protein folding determined during the study of similar
globular proteins, could be applicable on the more complex protein structure and mechanism of folding could be revealed properly.
Other various techniques for analyzing protein dynamics:
1. Fluorescence recovery after photobleaching (FRAP)
2. Single particle tracking (SPT)
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