BSc Module FS460
Investigating Protein Structure and Function
Proteins contain several chromophores that absorb light in the ultra-violet and infra red regions. Many also display fluorescence. The most important chromophores are the aromatic rings of Phe, Tyr and Trp.
UV absorbance and fluorescence are useful probes of structure and structural changes. This is due to the fact that chromophores display shifted spectra upon increasing or decreasing polarity of their environment, with changes in wavelength of maximum absorbance (lambda max) and molar extinction coefficient possible.
Spectral characteristics of chromophores in proteins
Quantitation of tryptophan and tyrosine
It is often very difficult to accurately determine the number of Trp and Tyr residues in a protein. Conventional amino acid analysis frequently underestimates the content of these two important amino acids which are frequently involved in enzymic catalysis.
A useful and simple approach is the use of 4th derivative spectroscopy of unfolded proteins. In this approach the fourth derivative of the uv-spectrum is calculated (usually automatically by the spectrophotometer). This results in a series of peaks and troughs rather than a broad absorbance peak.
One of the troughs on the trace is unique to tryptophan, the trough at 292 nm. The concentration of tryptophan in a sample of protein can then be determined with reference to a standard curve determined with the tryptophan model compound. If the concentration of protein is known then the number of tryptophans per molecule can be calculated.
The tyrosine content is a little more elusive as there is no absolutely unique peak or trough. The peak at 282 nm has a small contribution due to tryptophan – this can be taken into account by determining another standard curve for the tryptophan model compound at 282 nm. A correction factor can then be used to determine tyrosine concentration from the peak at 282. This is usually done by standard addition of tyrosine model compound to a solution of the protein.
The extrapolated x-axis intercept gives the number of tyrosines / molecule protein.
Determination of amino acids exposed to solvent
UV spectroscopy can be used to determine the number of aromatic amino acids that are exposed to the solvent. This is a very useful probe of conformational changes occurring in proteins and can be used to detect the presence of aromatic amino acids in active site clefts of enzymes.
The usual approach is to perform “difference spectroscopy” in a dual beam spectrophotometer. One cell of the spectrophotometer contains the protein dissolved in buffer and the other has the protein in buffer with a perturbant such as glycerol.
The difference in absorption is quantified and compared to the absorbance of completely denatured protein. This allows the determination of the relative amount of solvent- exposed aromatic amino acids.
Solvent perturbation method
- Calculate the content of significant chromophores (usually tryptophan and tyrosine) in the protein using a spectrum determined in water and by comparison with spectra of model tryptophan or tyrosines, or by amino acid analysis.
- Record the difference spectrum of denatured protein Vs native protein and identify extinction coefficient and lambda max.
- Record difference spectrum in the presence of a perturbant such as 20% ethylene glycol and determine extinction.
- Calculate ratio of extinction in 20% ethylene glycol to that of denatured protein (the fraction of the total that are exposed to solvent) and multiply by the number of chromophores known to be present to give the number of solvent exposed chromophores.
Surface exposed amino acids are frequently found buried in crevices on a proteins surface – particularly if they are involved in the active site of an enzyme. It is possible to gain information about the size of such a cleft by using a range of perturbants of differing molecular sizes and hence differing accessibilities.
A useful property of tryptophans is that they can be oxidised which destroys their absorbance. The amino acid sequence can be compared before and after oxidation to identify particular tryptophans – this can be useful to place particular sequences at the protein surface.
pH titration of surface exposed tyrosines
The lambda max of tyrosine shifts from 274 to 295 nm as pH increases:
Curve A: all of the tyrosines are on the surface
Curve B: some of the tyrosines are exposed and some become exposed at high pH when the protein becomes denatured
Curve C: internal tyrosines are in a polar environment and can be titrated
Circular dichroism (CD)
CD is a technique based upon the interactions of proteins with circularly-polarised light.
Circularly-polarised light consists of two components and components. These components interact differentially with amino acids resulting in an ellipse of polarisation. This is characterised as the molar ellipticity of a protein:
where [ ] is in degrees cm-2 Mole-1. The molar ellipticity varies with wavelength to describe the CD spectrum.
Distinct spectra are seen for polypeptides in random coil, alpha-helix and beta-sheet conformations. The CD signal at any given wavelength is given by the sum of the contributions of different secondary structures:
The spectra can be deconvoluted by computer to give accurate relative amounts of helix, coil and sheet. A simple estimation of alpha-helical content can be obtained at one wavelength. For instance at 208 nm the values of are -4,000 for beta sheet and random coil and -33,000 for alpha-helix:
Such estimates are fairly crude and most modern CD spectrophotometers have software to calculate secondary structure content.
This form of spectroscopy is based on the vibrational frequencies of atoms and bonds within a molecule. For proteins, seven major absorbance bands are seen, all derived from the peptide bond.
Amide bands I, II and III are most prominent and sensitive to secondary structure. Most studies use the amide band I derived from C=O stretching in the peptide bond. This depends on hydrogen bonding pattern, hence on secondary structure. For polylysine, a protein model compound:
alpha-helix frequency = 1650-1658
beta-sheet frequency = 1625-1640
random coil frequency = 1640-1648
In real proteins rather than polylysine, the absorbance maxima overlap
Second derivative spectroscopy is needed to deconvolute the spectrum
Infra-red spectroscopy is most sensitive to beta-sheet.