Native States

One of the well known advantages of resonance Raman spectroscopy is that samples dissolved in water can be studied since water is transparent in the visible region. Furthennore, many molecules of biophysical interest assume their native state in water. For this reason, resonance Raman spectroscopy has been particularly strongly embraced in the biophysical connnunity. 

MES)==10 These results suggest tliat C(MES) grows (in all likelihood) only as In N with N. Thus tlie restriction of compactness and low energy of tlie native states may impose an upper bound on tlie number of distinct protein folds. 

The key question we want to answer is what are the intrinsic sequence dependent factors tliat not only detennine tire folding rates but also tire stability of tire native state It turns out tliat many of tire global aspects of tire folding kinetics of proteins can be understood in tenns of tire equilibrium transition temperatures. In particular, we will show tliat tire key factor tliat governs tire foldability of sequences is tire single parameter... 

Figure C2.5.6. Thennodynamic functions computed for the sequence whose native state is shown in figure C2.5.7. (a) Specific heat (dotted curve) and derivative of the radius of gyration with respect to temperature dR /dT (broken curve) as a function of temperature. The collapse temperature Tg is detennined from the peak of and found to be 0.83. Tf, is very close to the temperature at which d (R )/d T becomes maximum (0.86). This illustrates...

Figure C2.5.9. Examples of folding trajectories iT=T derived from the condition = 0.21. (a) Fast folding trajectory as monitored by y/t). It can be seen that sequence reaches the native state very rapidly in a two-state manner without being trapped in intennediates. The first passage time for this trajectory is 277 912 MCS. (b) Slow folding trajectory for the same sequence. The sequence becomes trapped in several intennediate states with large y en route to the native state. The first passage time is 11 442 793 MCS. Notice that the time scales in both panels are dramatically different.

For these sequences the value of Gj, is less than a certain small value g. For such sequences the folding occurs directly from the ensemble of unfolded states to the NBA. The free energy surface is dominated by the NBA (or a funnel) and the volume associated with NBA is very large. The partition factor <6 is near unify so that these sequences reach the native state by two-state kinetics. The amplitudes in (C2.5.7) are nearly zero. There are no intennediates in the pathways from the denatured state to the native state. Fast folders reach the native state by a nucleation-collapse mechanism which means that once a certain number of contacts (folding nuclei) are fonned then the native state is reached very rapidly [25, 26]. The time scale for reaching the native state for fast folders (which are nonnally associated with those sequences for which topological fmstration is minimal) is found to be... 

For sequences with folding is extremely slow and these sequences may not reach the native state in a... 

In tliis section we have not discussed experimental advances tliat are offering extraordinary insights into tlie way tlie denatured molecules reach tlie native state. Two remarkable experimental approaches hold tlie promise tliat in short order we will be able to watch tlie folding process from submicrosecond time scale until tlie native state is reached. A brief summary of tliese follow. 

Apart from tliese mainstream metliods enabling one to gain a comprehensive and detailed stmctural picture of proteins, which may or may not be in tlieir native state, tliere is a wide variety of otlier metliods capable of yielding detailed infonnation on one particular stmctural aspect, or comprehensive but lower resolution infonnation while keeping tlie protein in its native environment. One of tlie earliest of such metliods, which has recently undergone a notable renaissance, is analytical ultracentrifugation [24], which can yield infonnation on molecular mass and hence subunit composition and their association/dissociation equilibria (via sedimentation equilibrium experiments), and on molecular shape (via sedimentation velocity experiments), albeit only at solution concentrations of at least a few tentlis of a gram per litre. 

A completely new method of determining siufaces arises from the enormous developments in electron microscopy. In contrast to the above-mentioned methods where the surfaces were calculated, molecular surfaces can be determined experimentally through new technologies such as electron cryomicroscopy [188]. Here, the molecular surface is limited by the resolution of the experimental instruments. Current methods can reach resolutions down to about 10 A, which allows the visualization of protein structures and secondary structure elements [189]. The advantage of this method is that it can be apphed to derive molecular structures of maaomolecules in the native state. 

The biologiccJ function of a protein or peptide is often intimately dependent upon the conformation(s) that the molecule can adopt. In contrast to most synthetic polymers where the individual molecules can adopt very different conformations, a protein usually exists in a single native state. These native states are found rmder conditions typically found in Uving cells (aqueous solvents near neutred pH at 20-40°C). Proteins can be unfolded (or denatured) using high-temperature, acidic or basic pH or certain non-aqueous solvents. However, this unfolding is often reversible cind so proteins can be folded back to their native structure in the laboratory. 

Sali A, E Shakhnovich and M Karplus 1994b. PCinetics of Protein Folding. A Lattice Model Study of Requirements for Folding to the Native State, journal of Molecular Biology 235 1614-1636. 

Protein tertiary structure is also influenced by the environment In water a globu lar protein usually adopts a shape that places its hydrophobic groups toward the interior with Its polar groups on the surface where they are solvated by water molecules About 65% of the mass of most cells is water and the proteins present m cells are said to be m their native state—the tertiary structure m which they express their biological activ ity When the tertiary structure of a protein is disrupted by adding substances that cause the protein chain to unfold the protein becomes denatured and loses most if not all of Its activity Evidence that supports the view that the tertiary structure is dictated by the primary structure includes experiments m which proteins are denatured and allowed to stand whereupon they are observed to spontaneously readopt their native state confer matron with full recovery of biological activity... 

Section 27 22 Many proteins consist of two or more chains and the way in which the various units are assembled m the native state of the protein is called its quaternary structure... 

We have so far described the structure of DNA as an extended double helix The crys tallographic evidence that gave rise to this picture was obtained on a sample of DNA removed from the cell that contained it Within a cell—its native state—DNA almost always adopts some shape other than an extended chain We can understand why by doing a little arithmetic Each helix of B DNA makes a complete turn every 3 4 X 10 m and there are about 10 base parrs per turn A typical human DNA contains 10 base parrs Therefore... 

Through combined effects of noncovalent forces, proteins fold into secondary stmctures, and hence a tertiary stmcture that defines the native state or conformation of a protein. The native state is then that three-dimensional arrangement of the polypeptide chain and amino acid side chains that best facihtates the biological activity of a protein, at the same time providing stmctural stabiUty. Through protein engineering subde adjustments in the stmcture of the protein can be made that can dramatically alter its function or stabiUty. 

Gofactors. Frequendy proteins exist in their native state in association with other nonprotein molecules or cofactors, which are cmcial to their function. These may be simple metal ions, such as Fe " in hemerythrin or Ca " in calmodulin a heme group, as for the globins nucleotides, as for dehydrogenases, etc. 

Cellulose I. The majority of celluloses in the native state were previously thought to have the same crystal stmcture (Cellulose I), varying only in perfection of the crystaUites. Now, at least two different crystal stmctures are known for these materials, named la and ip. These two phases coexist in... 

It should be noted that in almost all cases only one fold exists for any given sequence. The uniqueness of the native state arises from the fact that the interactions that stabilize the native strucmre significantly destabilize alternate folds of the same amino acid sequence. That is, evolution has selected sequences with a deep energy minimum for the native state, thus eliminating misfolded or partly unfolded structures at physiological temperatures. 

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