Circular dichroism (CD)
Circular dichroism (CD) spectroscopy measures differences in the absorption of left-handed polarized light versus right-handed polarized light which arise due to structural asymmetry. The absence of regular structure results in zero CD intensity, while an ordered structure results in a spectrum which can contain both positive and negative signals.
Circular dichroism spectroscopy is particularly good for:
comparing the structures of a protein obtained from different sources (e.g. species or expression systems) or comparing structures for different mutants of the same protein
demonstrating comparability of solution conformation and/or thermal stability after changes in manufacturing processes or formulation [comparability protocols, see presentations in Resources]
studying the conformational stability of a protein under stress -- thermal stability, pH stability, and stability to denaturants -- and how this stability is altered by buffer composition or addition of stabilizers and excipients
CD is excellent for finding solvent conditions that increase the melting temperature and/or the reversibility of thermal unfolding, conditions which generally enhance shelf life
determining whether protein-protein or protein-ligand interactions alter the conformation of protein.
If there are any conformational changes, this will result in a spectrum which will differ from the sum of the individual components. Small conformational changes have been seen, for example, upon formation of several different receptor/ligand complexes.
Determination of Protein Secondary Structure by Circular Dichroism:
Secondary structure can be determined by CD spectroscopy in the "far-UV" spectral region (190-250 nm). At these wavelengths the chromophore is the peptide bond, and the signal arises when it is located in a regular, folded environment.
Alpha-helix, beta-sheet, and random coil structures each give rise to a characteristic shape and magnitude of CD spectrum. This is illustrated by the graph to the right, which shows spectra for poly-lysine in these three different conformations. The approximate fraction of each secondary structure type that is present in any protein can thus be determined by analyzing its far-UV CD spectrum as a sum of fractional multiples of such reference spectra for each structural type.
Like all spectroscopic techniques, the CD signal reflects an average of the entire molecular population. Thus, while CD can determine that a protein contains about 50% alpha-helix, it cannot determine which specific residues are involved in the alpha-helical portion.
Far-UV CD spectra require 20 to 200 µl of solution containing 1 mg/ml to 50 µg/ml protein, in any buffer which does not have a high absorbance in this region of the spectrum. (High concentrations of DTT, histidine, or imidazole, for example, cannot be used in the far-UV region.) Note that for many formulated protein samples the absorbance due to the excipients prevents collecting spectra below 200 nm (and even 200 nm is often not possible). When that is true the accuracy reliability of secondary structure calculations (the actual percentages of different structures) is compromised, but the validity of spectral comparisons is not
Information About Protein Tertiary Structure from Circular Dichroism:
The CD spectrum of a protein in the "near-UV" spectral region (250-350 nm) can be sensitive to certain aspects of tertiary structure. At these wavelengths the chromophores are the aromatic amino acids and disulfide bonds, and the CD signals they produce are sensitive to the overall tertiary structure of the protein.
Signals in the region from 250-270 nm are attributable to phenylalanine residues, signals from 270-290 nm are attributable to tyrosine, and those from 280-300 nm are attributable to tryptophan. Disulfide bonds give rise to broad weak signals throughout the near-UV spectrum.
If a protein retains secondary structure but no defined three-dimensional structure (e.g. an incorrectly folded or "molten-globule" structure), the signals in the near-UV region will be nearly zero. On the other hand, the presence of significant near-UV signals is a good indication that the protein is folded into a well-defined structure.
The near-UV CD spectrum can be sensitive to small changes in tertiary structure due to protein-protein interactions and/or changes in solvent conditions.
The signal strength in the near-UV CD region is much weaker than that in the far-UV CD region. Near-UV CD spectra require about 1 ml of protein solution with an OD at 280 nm of 0.5 to 1 (which corresponds to 0.25 to 2 mg/ml for most proteins).
Demonstrating Comparability of Conformation
Often it is necessary to demonstrate that different lots of a protein have equivalent conformations, for example after a scale-up in the purification process or to qualify a new manufacturing site, and CD can be a good tool for this.
The data below show a case where the far-UV spectra show that the recombinant form of an enzyme clearly does not have the same secondary structure as the natural protein (i.e. the recombinant protein is not properly folded).
Such cases of significant differences in secondary structure are, however, unusual. More typically subtle differences in conformation do not produce a detectable difference in far-UV CD, but may produce a difference in near-UV CD. One such example, for different lots of a monoclonal antibody, is shown below. This small but reproducible difference at ~240 nm correlates with differences in the stability of different lots of this antibody.
Thermal Stability by Circular Dichroism
Thermal stability is assessed using CD by following changes in the spectrum with increasing temperature. In some cases the entire spectrum in the far- or near-UV CD region can be followed at a number of temperatures. Alternatively, a single wavelength can be chosen which monitors some specific feature of the protein structure, and the signal at that wavelength is then recorded continuously as the temperature is raised. CD is often used to assess the degree to which solution pH, buffers, and additives such as sugars, amino acids or salts alter the thermal stability.
This graph illustrates thermal scans done in our lab for the same recombinant protein in 3 different buffers. While unfolding is completely reversible under all these conditions, clearly there are quite significant differences in thermal stability.
Many proteins aggregate or precipitate quickly after they are unfolded ("melted"), making unfolding irreversible. The reversibility of the unfolding reaction can be assessed by cooling the sample and then heating again to see if the unfolding reaction is duplicated. Finding solvent conditions that make unfolding reversible may be actually be more important for long-term stability (shelf life) than raising the melting temperature.
If (and only if) the melting is fully reversible, the melting temperature is directly related to conformational stability, and the thermodynamics of protein folding can be extracted from the data. The fact that thermal unfolding can generally be measured by CD at much lower concentrations than by DSC increases the probability of reversible reactions and of thermodynamically interpretable data.
This graph illustrates a detailed analysis of one of the data sets shown above, using custom software developed in our lab. The data (+) were fitted to a simple thermodynamic unfolding model (solid line). The fit returns the melting temperature (midpoint of the transition) as 47.3 +/- 0.1 °C. The width of the transition region is related to the enthalpy of unfolding, DH, which the fit returns as 52 +/- 2 kcal/mol.
Fitting the data also allows a more reproducible measurement of the onset of unfolding, a temperature which is often more relevant for formulation and shelf-life considerations than the midpoint. The onset (defined as the temperature at which 5% of the protein is unfolded) occurs at 36.1 +/- 0.3 °C in this case.
If the protein precipitates or aggregates as it is unfolded, the melting reaction will be irreversible, and the melting temperature will reflect the kinetics of aggregation and the solubility of the unfolded form of the molecule as well as the intrinsic conformational stability.
The cooperativity of the unfolding reaction is measured qualitatively by the width and shape of the unfolding transition. A highly cooperative unfolding reaction indicates that the protein existed initially as a compact, well-folded structure, while a very gradual, non-cooperative melting reaction indicates that the protein existed initially as a very flexible, partially unfolded protein or as a heterogeneous population of folded structures.
Melting of Secondary Structure
Changes in secondary structure, monitored in the far-UV CD region, can be determined with as little as 50 µg of protein, at concentrations of 0.2 mg/ml. By following changes over the entire far-UV CD region we can determine whether at high temperatures the protein is losing all of its secondary structure, loses only a portion of its secondary structure, or simply undergoes conformational change involving a change in secondary structure. Occasionally the unfolded form of a protein will possess a defined but totally different secondary structure than the native form (e.g., TNF-alpha contains beta-sheet when folded, but alpha-helix when melted, and many proteins form amyloid-like aggregates following a transition from alpha-helix to beta-strand).
Melting of Tertiary Structure
Changes in tertiary structure can be followed by monitoring changes in the near-UV CD region. Due to the weaker signal in this region this requires 1-3 mg of protein. Such studies will reveal whether the melting of a protein occurs in a single step (with concurrent loss of both secondary and tertiary structure), or in a two-step reaction.
Melting of Protein Complexes
The effect of forming a protein-protein complex (e.g. ligand/receptor or antigen-antibody) on the thermal stability of the individual proteins in the complex can also be determined. This works best if the individual proteins have CD spectra which are quite different from each other, such that changes at specific wavelengths can be monitored to follow changes in the corresponding protein. In such cases it is possible to determine whether there is an increase in stability of one or both of the proteins following complex formation.