Page 148 - Academic Press Encyclopedia of Physical Science and Technology 3rd BioChemistry

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Encyclopedia of Physical Science and Technology EN013H-614 July 27, 2001 10:29

188 Protein Folding

yield a heat-capacity maximum corresponding to the ther- the pathway for protein folding reactions and can provide
mal transition temperature, T G , and an enthalpy change, estimates of the unfolding equilibrium constant at individ-
H un , for the transition. The H un value can be deter- ual sites on the protein. 26,27
mined either by integration of the thermogram or by curve
fitting (i.e., fitting a van’t Hoff equation to the shape of the
6. Other Experimental Methods
thermogram). Referring to these two H un estimates as
the calorimetric and van’t Hoff values, the ratio of the Fourier transform infrared (FTIR) vibrational spec-
calorimetric and van’t Hoff H un values can be used to troscopy senses the hydrogen bonding pattern of the pep-
determine whether the transition is best described as a tide bonds of a protein and can detect unfolding transitions
two-state process. That is, a ratio of 1.0 (indicating that in terms of changes in the secondary structure patterns. 28
two H un estimates are essentially the same) means that As compared to CD, which also senses secondary struc-
the structural transition is two state. ture, FTIR is relatively more responsive to β-sheet struc-
tures. A disadvantage of FTIR is that it requires a higher
protein concentration and that it is more difficult to auto-
5. Nuclear Magnetic Resonance
mate for titration experiments.
Nuclear magnetic resonance (NMR) spectroscopy is a Light-scattering methods, such as small-angle X-ray
powerful method for studies with proteins, as there is such scattering, or quasi-elastic light scattering, can provide
a large number of resolved signals (due to the individual information about the size of a protein, in terms of its ra-
1
nuclei, such as the H and 13 C atoms in the backbone dius of gyration. Unfolding or aggregation reactions are
29
and/or side chains of the amino acids). 26,27 This gives the detected as increases in the hydrodynamic radius. These
potential to track conformational transitions by observ- scattering methods are also relatively difficult to adapt to
ing changes at a large number of individual sites on the temperature or titration experiments.
protein. This is further made possible by the fact that the The ability of a protein to bind a specific ligand or to
signals (peaks having various chemical shifts) are usu- have catalytic activity can be used to determine the popula-
ally widely dispersed in the native state of a protein, as a tion of native species. The possibilities are numerous, de-
consequence of the sensitivity of the resonance peak for pending on the way that activity and binding are measured.
individual nuclei to the local magnetic field, which in turn These activity/binding assays should be easy to automate
is related to the three-dimensional structure of the protein. for a series of denaturant concentrations or pH values.
Unfolded proteins, by comparison, usually have a much Size exclusion chromatography and gel or capil-
narrower range of resonance peaks for similar amino acid lary electrophoresis are methods that separate protein
components. molecules based on size (or size and charge). 30–32 In these
Tracking any of the individual resonance signals, such methods, the protein sample travels down the column, gel
as those assigned to histidine or tryptophan residues, as slab, or capillary and, for a pure protein, should exit as
a function of denaturing condition (e.g., temperature, pH, a single peak traveling past the detector. Denatured pro-
or added chemical denaturant) provides a way to study the teins should appear to have a larger hydrodynamic radius
unfolding process, as the signal is transformed from that of and should travel more slowly. If the kinetics of inter-
the native state to that of the unfolded state. An important conversion of the native and unfolded species is slower
difference between NMR and the above optical methods is than the time needed to travel through the column (gel
that NMR signals can be dynamically averaged signals or or capillary), then it is possible to detect individual peaks
individual signals can appear for the native and unfolded for the native and unfolded species. If the interconver-
states. The latter results if the rate of interconversion of sion is rapid, a kinetically averaged peak position will be
the conformational states is relatively slow in comparison observed.
to the difference in resonance frequencies of the signals An example of a potentiometric measurement is one in
for the two states. which the pH (or number of protons bound versus those
The use of NMR for proteins studies is usually limited bound at some reference condition) is measured as a func-
to proteins having molecular weight of about 25 kDa or tion of the denaturing condition. Such an approach would
less. The method requires a relatively high concentration require a difference in the pK a of one or more amino
of protein, compared to other methods. acid side chains in the native and unfolded state. Usually,
Besides the above application of NMR to track the pop- several such amino acid side chains are in a protein. How-
ulation of native and unfolded states, NMR also can pro- ever, the potentiometric approach requires technical skill,
vide very high-quality information about the tertiary and and it is difficult to use in combination with high concen-
secondary structure of proteins. In addition, pulsed iso- trations of chemical denaturants or temperatures far from
tope labeling experiments can provide information about ambient.