Page 145 - Academic Press Encyclopedia of Physical Science and Technology 3rd BioChemistry

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Protein Folding 185

14
oftheunfoldedandnativestates.Pressure-inducedunfold- aromatic side chains to water. Tryptophan’s absorbance
ing studies require a specialized high pressure cell. 12,13 is also sensitive to the local electrostatic field; changes
5. Dissociation/unfoldingofoligomericproteins:Olig- in indole-charge interactions can cause either red or blue
omeric proteins are interesting as models for understand- shifts upon protein unfolding. 15
ing intermolecular protein-protein interactions. A general Table II gives the typical concentration range used for
question for oligomeric proteins, including the simplest unfolding studies with proteins using this and other meth-
dimeric (D) proteins, is whether the protein unfolds in a ods. The sensitivity of difference absorbance measure-
two-state manner, D ↔ 2U, or whether there is an inter- ments will depend on the molar extinction coefficient of
mediate state, which might be either an altered dimeric the chromophore and their number, but a concentration

state, D , or a folded (or partially folded) monomer range of 0.01 to 0.1 mM protein is usually needed for
species, M. Models for these two situations are as reasonable signal to noise with a 1-cm pathlength cell.
follows: Thermal scans, to induce the unfolding transition, are easy
to perform with accessories available for most absorbance
D ↔ D ↔ 2U (11a)

spectrophotometers. Chemical denaturant- or pH-induced
D ↔ 2M ↔ 2U (11b) transitions can be less convenient (unless one has auto-
mated titration equipment), since a series of solutions with
For a D ↔ 2U model, the relationships between the equal protein concentration and varying denaturant must
observed spectroscopic signal, S exp ; the mole fraction of be prepared. With any of these perturbing conditions, it
dimer, X D , and unfolded monomer, X U ; and the unfolding is important to realize that the variation in the conditions
2
equilibrium constant (K un = [U] /[D]) will be given by itself (i.e., varying temperature, pH, chemical composi-
Eq. (5) and tion) can lead to a “baseline” change in the absorbance
signal from the native and unfolded species. 16 So long as
1/2
2
K un + 8K un [P] 0 − K un
X U = ; X D = 1 − X U (12) these baseline trends are linear and not as large as the ab-
4[P] 0 sorbance change associated with the conformational tran-
sition, the baseline trends can be corrected for in the data
where [P] 0 is the total protein concentration (expressed as
analysis.
monomeric form), where S i is the relative signal of species
The advantages of absorbance measurements are the
i and where K un will depend on the perturbant as given by
ready availability, ease of use, and low cost of the in-
one of the above equations. That is, the transition should
strumentation. The biggest disadvantage is that it is less
depend on the total subunit concentration, [P] 0 , and on
sensitive than some other methods.
any other perturbation axis.
2. Circular Dichroism
C. Experimental Signals
Circular dichroism (CD) is a very commonly used method
1. Absorbance Spectroscopy
for studying protein conformational changes. The far UV
Absorbance spectroscopy (difference spectroscopy) mon- spectral region (180 to 250 nm) is dominated by ab-
itors conformational transitions in macromolecules by sorbance by peptide bonds, and there are signature spectra
measuring absorbance changes, usually in the aromatic for α-helix and other types of secondary structure in a pro-
region of the ultraviolet (UV) spectrum. The amino tein. Additionally, the aromatic CD spectral region of 250
acids tryptophan and tyrosine are the most impor- to 300 nm senses the chirality around the aromatic amino
tant chromophores in the UV region for proteins. As acid side chains and there is usually a structured aromatic
mentioned earlier, tryptophan residues are often engi- CD spectrum for the native state of a protein. 14,17,18
neered into proteins as reporters of local and/or global The effective sensitivity of CD is comparable to or
environment. slightly better than that of difference UV absorbance spec-
The indole ring of tryptophan and the phenol ring of troscopy. CD instruments can be purchased with ther-
tyrosine show sensitivity of their absorbance spectrum to moelectric cell holders for thermal scans and with au-
solvent polarity. There is a blue shift in the absorbance of tomated titrator syringe pumps for chemical denaturant
indole and phenol upon increasing solvent polarity. As a titrations. Since the far-UV spectral regions is important
result, there will often be a blue shift in the absorbance in protein unfolding studies, it is necessary to work with
of tryptophan (typically monitored as a decrease in ab- salts and buffers that have minimal absorbance in this
sorbance in the 291- to 294-nm region of the spectrum) region. When performing CD measurements, it is nec-
or tyrosine (at 285 to 288 nm) upon unfolding of a pro- essary to pay attention to the buffer and salts and other
tein and a consequent increase in the exposure of these solution components (e.g., chemical denaturants) being