Physical Biochemistry

Objectives
Review of basic thermodynamics important for biochemistry and understanding how molecules function and what drives cellular processes. Concepts of thermodynamics are important for trying to comprehend the properties of complex molecules which may be poorly defined: e.g. the temperature dependence of the equilibrium constant for protein denaturation gives the enthalpy of that equilibrium without even knowing the composition or structure of the protein.

 Useful information is obtained by techniques that are based on thermodynamic principles: e.g. binding constants from equilibrium measurements.

First Law: conservation of energy.

For a change in state, the energy of the system change is

where the heat, q, absorbed by the system and work, w, done by the system are positive.

WHAT CRITERIA ESTABLISH A SYSTEM IS AT EQUILIBRIUM?

For a reversible process with only PdV work, the entropy change is dqrev/T , and the energy change becomes For an isolated system where the energy and V are constant, then a change in the system is an equilibrium process only if

 The previous equation means that the entropy of the system undergoing this process at constant E and V must be either a maximum or minimum, and it turns out that this condition refers to a maximum for the entropy (not a minimum).

Gibbs Free Energy

For biological systems, a more important condition is constant P and T, not constant V. Hence, to consider the direction of equilibrium for processes at constant P and T, a new state function, Gibbs free energy, G, is defined:

 And the change in G under any conditions becomes:

Therefore, imposing the constraint of constant P and T with the above equation, the criterion for equilibrium of the system is

Chemical Equilibrium and Binding

Objective: To determine the direction of equilibrium of chemical reactions and macromolecular associations and thereby determine relative stability.

 we learned that the Gibbs free energy function is the appropriate function to describe the behavior of physical systems at constant T and P. G= RTln(Keq) This relationship is a central concept to biological systems in that Keq is an experimentally accessible quantity and thus the above relationship allows determination of the key function, free energy.

 Consider the general chemical reaction, We start by simply stating that the free energy change can be expressed as

 where G0 is the standard-state free energy change , or the free energy change of the reaction at the selected standard conditions for a moles of A, etc. This equation is based on the chemical potential or partial molar Gibbs free energy, I, an intensive variable, dependent on T, P and composition of the system, but not on its size

 General case: define partial molar quantity:

where Xi is any extensive property, and ni is the number of moles of component i. Then, addition of i would change the value of property X by:

 Consider the Gibbs free energy function for a solution A solution is a multicomponent system, and thus more terms are needed in G than just P, V, S and T. The contribution to the free energy for component i is chemical potential or partial molar Gibbs free energy, i

 where ni is the number of moles of i. The subscripts indicate T, P and all other components are constant. Starting with the standard thermodynamic equation for free energy from a previous lecture and adding the chemical potential to account for variations in components, the total free energy change is

 Change in G when the variables T, P and n change. Consider the amount of substance n1 changes at constant T and P, then G changes by an amount given by the coefficient So,

Membrane dialysis
involves the transfer of components between different phases across a permeable membrane. the sum of all chemical potentials in the system is zero. chemical potential must be the same in both phases for all species that can pass through the membrane.

At equilibrium,

Membrane dialysis
Transfer between the inside and outside of species i gives the result

Mixing
no difference in interaction between solute and solvent, entropy change arises only from randomness

So, free energy of mixing is

 The Gibbs free energy fcns in terms of chemical potential is

and for ideal mixing is

Dilute ideal solutions

For a dilute ideal solution, the mole fraction can be converted to concentration, Ci For non-ideal solutions

chemical equilibrium
for a reaction carried out at equilibrium, or reversibly, at constant T and P

Temperature dependence of Keq and G0:

The temperature dependence of Keq and G0 can be used to determine H,

van t Hoff plot

lnKeq as a linear function of 1/T

Example Applications of Thermodynamics to Biophysical Problems

Conformational transitions
Thermodynamic principles are often applied to conformational transitions to describe the observed behavior and obtain better insight into the forces stabilizing the conformational states. Transitions:
dihedral angle rotation loop motion of a protein helix-coil transitions melting of tRNA protein unfolding

 Thermodynamics informs us about the direction a process will take, we learn about relative stability and relative populations for the two states of the transition. Thermodynamics informs us of nothing about what stabilizes one state relative to another, or how the transition process is achieved.

Thermodynamics applied to protein structure stability and disorder.

Thermodynamic stability of the folded state of proteins is key to function and tight control between ordered and disordered protein structure is maintained in the cell. Improper denaturation of proteins, associated with aggregation, underlies a large number of diseases.

 The equilibrium for protein conformation is measured in the direction of unfolding: The transition is described as a two-state system That is, the native and denatured forms of the protein are different macroscopic states amenable to the usual thermodynamic relationships.

 Keep in mind of course that the denatured state comprises many conformational forms of the molecule. The chain is flexible (higher entropy) but highly hydrated (lower entropy). The folded state compensates for the loss of chain entropy by more favorable interaction energies (lower enthalpy).

Contribution of configurational entropy to protein unfolding

The folded state of a protein is greatly reduced in order and thus has very small entropy. One measure of the difference in the degree of order between a random coil unfolded state and the highly restrained folded state was made by removing a disulfide bridge in the protein hen egg white lysozyme, HEWL

Hen Egg White Lysozyme (HEWL)

HEWL, 129-residue protein, has 4 disulfide bonds, one between 6 and 127 links residues close to the two chain ends and has high solvent exposure. Cooper et al (1991, JMB 225:939-943) prepared a 3disulfide form of HEWL, 3SS-HEWL, in which the 6127 SS bond was selectively reduced.

 They conducted thermal unfolding studies on wt HEWL and 3SS-HEWL. The results showed
3SS unfolding was two-state Hunf values were nearly equal for wt and 3SSHEWL at the same temperature

Sunf increased by ~25 cal mol-1 K-1 for 3SSHEWL, i.e. the entropy of unfolded 3SS-HEWL was greater than wt, as expected for uncrosslinking the chain.

Assessment:
recall the expression for entropy in terms of the number of ways of arranging particles,S=kln Similarly, the conformational entropy depends on the number of arrangements of a single molecule, i, in a given state i.

 Assume entropy of the folded state is the same for 3SS and wt. If so, then f terms cancel to give

 Substituting known quantities and evaluating on a per residue level,

Or, the change in number of arrangements per residue in the denatured state is

Conclusions
It is concluded that the entropy per residue for unfolding increases by approximately 10% upon removal of the S-S cross-link. How could one test if a per-residue value is legitimate and the entropy change is evenly distributed over the polypeptide?
Make similar derivatives of one of the other disulfide bonds.

The authors conclude disulfide bonds are significant in stabilizing the folded state of proteins.

Free energy to measure protein structural stability

The transition is usually studied in the direction written with denaturation induced by heating, adding denaturant, or changing pH. Mutation has also been key in trying to tease out the subtleness of structural stability. In practice, protein denaturation is often irreversible, but some proteins will renature and regain the native structure spontaneously when the conditions favoring the folded state are re-established.

 Folding experiments on proteins with reversible folding are amenable to thermodynamic analysis. Some physical property (e.g. calorimetry, CD, UV, NMR, fluorescence) is used to follow the transition. The transition is plotted as the fraction of folded protein as a function of some variable, similar to the manner in which the helix-coil transition was characterized.

 The transition between these two states, N and D, is determined by the usual differences in the thermodynamic parameters. Assuming constant T, P, pH and denaturant,

 The midpoint of the transition occurs at the temperature, TM, at which the concentrations of N and D are equal and set by the condition. gives the entropy of the transition from the enthalpy, a measurable quantity (by calorimetry):

Protein Folding
Proteins fold spontaneously under physiological conditions. In the equilibrium between the denatured state (unfolded or partially unfolded) and the native state (folded, biologically functional), under physiological conditions the vast majority of molecules are in the native state.

 PRIMARY STRUCTURE DETERMINES TERTIARY (AND QUATERNARY) STRUCTURES. demonstrated by the fact that many proteins can refold from a more or less "random coil" set of conformations without "instructions" from any other cellular components All the information for 3-dimensional structure is provided by the amino acid sequence.

 Proteins can be unfolded (denatured) in vitro by chemical agents like urea, or extremes of heat or pH, and then refolded (renatured) by diluting out the chemical denaturant, changing the pH, etc

 Proteins fold on a defined pathway (or a small number of alternative pathways); they don't randomly search all possible conformations until they arrive at the most stable (lowest free energy) structure.

 Proteins that don't (re)fold on their own, without assistance, don't need other "instructions" -- they just need "molecular chaperones" (which are also proteins) to keep them from slipping off the folding pathway or to help them to get back on it. Some chaperones require "expenditure" of energy currency (hydrolysis of ATP) to carry out their function.

 Many diseases are the result of defects in protein folding, e.g., the spongiform encephalopathies (human CJD, bovine mad cow disease), Alzheimer disease, Parkinson disease, Huntington disease Diseases involving deposits of misfolded proteins (amyloid deposits) result from aggregation of a specific protein, different for different diseases, that has misfolded and formed cross-beta structures that form higher order structures (protofibrils and fibrils/fibers) that are very stable. One hypothesis is that cellular degradation apparatus can t keep up with disposal of the abnormally folded protein.

Gfolding (change in free energy) between unfolded structure and folded structure is SMALL. Gfolding results from many contributions: enthalpy changes electrostatic effects (hydrogen bonds, salt bridges) solvation/desolvation of charged residues van der Waals interactions steric factors entropy change (2 sources): entropy (hydrophobic effect) conformational entropy (degrees of freedom, flexibility) Small differences in energy are important -- loss of 1 or 2 hydrogen bonds might shift equilibrium from folded state to unfolded form of protein.

Energy Landscape View of Protein Folding

Molten Globule
The goal is to find the lowest point in the landscape, the energy minimum of the system. This is generally assumed to be the native or functional state of the polypeptide. The first step is the movement of hydrophobic R-groups out of contact with water. This drives the collapse of the polypeptide into a compact and dynamic "molten globule".

 The path to the native state is not necessarily a smooth or predetermined one. The folding polypeptide can get "stuck" in a local energy minimum; there may not be enough energy (from thermal collisions) for it to get out again. If a polypeptide gets stuck, there are mechanisms to unfold it and let it try again to reach its native state.

Anfinsen's experiments: unfolding and refolding RNase

He unfolded RNase with denaturing agent (8 M urea). problem: 4 S S bonds in RNase (covalent crosslinks) "staple in" some of the 3-D structure even when backbone is unfolded. Solution: reducing agent ( mercaptoethanol) -- reduces disulfide bonds (S S --> 2 SH groups), so unfolded protein is entirely unfolded.

 Loss of native structure -- denaturation -inactivates RNase. slow removal of urea (by dialysis) --> refolded protein refolded in absence of reducing agent (so O2 in air could reoxidize SH groups to disulfides) Enzyme refolded and regained activity -proof the right combinations of S-S bonds had formed (i.e., structure was correct)

 SPECIFIC CONCLUSIONS: Correct tertiary structure of RNase backbone had returned. The right SH groups must have been adjacent to each other prior to reoxidation as a result of the backbone refolding correctly, because disulfide bonds formed spontaneously with the right combinations of Cys residues. MORE GENERAL CONCLUSIONS (Nobel Prize!): Native structure is the thermodynamically most stable (favored) state for most proteins. Native tertiary structure is determined by the primary structure (amino acid sequence) of a protein.

 Folding means arriving at the right combinations of and angles for every residue in the sequence. Proteins fold on a defined pathway (or a small number of alternative pathways); they don't randomly search all possible conformations until they arrive at the most stable (lowest free energy) structure. The code that dictates 3-dimensional structure from AA sequence seems to be redundant and more complex than we can currently understand or predict!

 Proteins that don't (re)fold on their own, without assistance, don't need other "instructions" -- they just need "molecular chaperones" (which are also proteins) to keep them from slipping off the folding pathway or to help them to get back on it.

Assisted Folding
Molecular Chaperones
interact with partially folded or improperly folded polypeptides; facilitate correct folding pathways they can also provide microenvironments in which folding can take place HSP70 (~ 70 000 Da)
class of chaperone bind to regions rich in non-polar residues and prevent inappropriate aggregation more abundant in cells stressed by high temperature (Heat Shock Proteins) can facilitate quaternary structure

 Chaperonins
complex structures polypeptides get trapped in pocket lid closes energy assisted folding takes place lid opens folded polypeptide released 10% of E. coli proteins depend on this process

GroEL

GroEL/GroES Complex

 Protein disulfide isomerases (PDI)

catalyze interchange or shuffling of disulfide bonds until native conformation is reached elimination of folding intermediates with inappropriate S-S bonds Peptide prolyl cis-trans isomerase (PPI) - catalyzes interconversion of the cis and trans isomers of proline peptide bonds

Protein Folding Kinetics Levinthal s Paradox

C. Levinthal (J. Chim. Phys. 1968, 65, 44) Consider a small protein (polypeptide) comprised of 100 amino acids. Protein Folding Kinetics - Levinthal s Paradox If each amino acid can assume 2 conformations, then the polypeptide can adopt 2100 1030 conformations. Assume that conformations interconvert with a time constant of 10-12 s. The time required to sample all conformations is roughly: (1030 conformations) r (10-12 s/conformation) = 1018 s 1010 years Age of the universe 1.2 r 1010 years proteins do not fold by a random search of conformation space

Nalepa et al. Nature Reviews Drug Discovery 5, 596613 (July 2006) | doi:10.1038/nrd2056

Ubiquitin Proteasome System

Protein degradation through the UPS is a highly regulated process, involving several steps
ubiquitin activation by E1 (ubiquitin-activating enzyme) followed by ubiquitin delivery to E2 complex formation by E2-CysUb, E3 (ubiquitin ligase) and the substrate transfer of ubiquitins to the substrate lysine(s) to earmark the substrate with a polyubiquitin chain polyubiquitylated substrate is released from the E3 Proteasomes recognize the polyubiquitin chain as a signal to deubiquitylate and destroy the substrate Proteasome unfolds the substrate in ATP-dependent manner, removes the ubiquitin chain through a proteasome-associated ubiquitin hydrolase activity, and threads the unfolded protein into the proteasome chamber, where the protease active sites are located

Many diseases are the result of defects in protein folding

Cystic fibrosis involves misfolding and resulting lack of a protein involved in Cl transport across membranes. Many neurodegenerative disorders involve abnormal protein aggregation. Prion diseases (e.g., CJD, Creutzfeldt-Jakob disease) = spongiform encephalopathies (also includes mad cow disease, chronic wasting disease in elk and deer, scrapie in sheep, etc.) Alzheimer disease Parkinson Disease Huntington Disease Partly folded or misfolded polypeptides or fragments may sometimes associate with similar chains to form aggregates. Partial unfolding of correctly folded proteins may also lead to aggregation. Aggregates vary in size from soluble dimers and trimers up to insoluble fibrillar structures (amyloid). Unlike most correctly folded proteins, both soluble and insoluble aggregates can be toxic to cells through unknown mechanisms.