This article describes a practical experiment for teaching basic spectroscopic techniques to introduce the topic of protein conformational change to students in the field of molecular biology, biochemistry, or structural biology. The spectroscopic methods employed in the experiment are absorbance, for protein concentration measurements, and fluorescence and circular dichroism, for probing protein conformational changes.
The use of spectroscopic methods for protein study is an extremely powerful and important technique. The interaction between light and matter has been widely adopted in science. An experiment that teaches the use of these methods for the purpose of studying proteins is essential for students in many fields of biochemistry. In the post-genome era, the study of protein folding, form, and function will become even more important than before. Because the function of a protein depends on its correct native structure , protein folding is one of the central processes in biology. To properly comprehend the folding phenomenon, the conformation of the native and the denatured states of proteins must be understood, as well as the partially folded intermediates that are sometimes present in the protein folding pathway .
A theoretical and practical experiment using spectroscopy for the study of protein conformation was created in an effort to introduce this topic to undergraduate students. The experiment was structured as follows: i) quick introduction to spectroscopy-based methods for protein studies; ii) practical use of the spectrophotometer for the accurate measurement of protein concentration; iii) practical use of fluorescence for local protein conformation studies; and iv) practical use of circular dichroism (CD)1 for protein secondary structure determination. This experiment takes up just 4 h of laboratory time.
MATERIALS AND METHODS
Lyophilized horse apomyoglobin (apoMb) and all chemicals utilized were of analytical grade and were purchased from Sigma (St. Louis, MO). ApoMb was ressuspended in water to a final concentration of about 2–4 mg/ml. Protein concentration stock buffer was comprised of 25 mM sodium phosphate, pH 6.5, 7.2 M guanidinium chloride (Gdm-Cl). Conformation buffers were prepared as follows: i) native, 20 mM Tris-Cl, pH 7.0; ii) intermediate, 10 mM acetate, pH 4.2; iii) unfolded, 20 mM sodium phosphate, pH 6.5, 4 M Gdm-Cl. All solutions were filtered after preparation and had their pH checked before and after filtration.
The dilutions for concentration measurement were prepared as described in Table I. The vials were mixed and left to equilibrate for 15 min at room temperature. The absorbance was then read with a Jasco V-530 spectrophotometer (Jasco, Easton, MD) at 280 nm in quartz cuvettes of 1-cm path length using the blank vial as a reference.
Three vials were prepared containing 2 ml of each of the conformational buffers at 4 °C. In each, apoMb was added to a final concentration of 2 μM (concentration measured in the previous step) and left to equilibrate for 20 min. CD measurements were read with a Jasco J-810 spectropolarimeter at 222 nm in quartz cuvettes of 1-cm path length.
Three vials were prepared as described for the CD method. A fourth vial with 4 μM of L-tryptophan (Sigma) in the native conformational buffer was also prepared. Fluorescence measurements were read with an ABL2 spectronic fluorometer (Applied Biosystems, Foster City, CA) in quartz cuvettes of 1-cm path length. Excitation was set at 280 nm, and the emission spectrum was collected from 300–500 nm.
RESULTS AND DISCUSSION
The laboratory started with a introduction to spectroscopic techniques for protein studies. It is beneficial to start by describing the wavelength regions of the electromagnetic spectrum, the absorption and emission of radiation, as well as introducing the Beer-Lambert law. A discussion then followed regarding the first Table in Part II of Cantor and Schimmel's notable book . This table ranks several techniques according to their structural information and experimental constraints for the study of biopolymers, stating clearly that techniques based on the use of light are the ranked best.
Protein Concentration Measurement—
As important as acquiring information about protein conformation is the knowledge of its correct concentration. Several concentration determination methods available make use of the absorbance properties of aromatic residues and are very accurate, and the use of this approach associated with the unfolding of the protein in the presence of a high concentration of Gdm-Cl is extremely efficient . The method described in Ref. 4 is simple and practical, making it easy to be employed in any experiment. It relies on the absorbance spectra of the protein's aromatic groups, tyrosine and tryptophan, and disulfides. Their molar absorbance coefficients (ϵ) at 280 nm are calculated in 20 mM phosphate buffer, pH 6.5, and 6 M Gdm-Cl , the number of such residues in a protein are calculated, and the protein ϵ calculated using the equation below:
It is understood from Eq. 1 that the protein molar absorbance coefficient ϵ is direct function of the number of tryptophans and tyrosines in the protein.
The students were introduced to the apoMb amino acid sequence (Fig. 1) and structure (available at the Protein Data Bank, www.rcsb.org/pdb/), which demonstrated that the majority of apoMb residues are involved in α-helical structures. ApoMb has eight α-helices named A to H and possesses two tryptophans (Trp) in the A helix [5, 6]. At this point, it is useful to introduce some of the characteristics of Trp, such as a blue-shifted fluorescence emission when in a hydrophobic environment such as the interior of a protein and red-shifted fluorescence emission when in a polar environment such as when exposed to solvent . Thus, Trp fluorescence is a good probe for local conformation in proteins.
The students were asked to calculate the apoMb ϵ, and the use of Gdm-Cl was also highlighted because it ensures that the protein is unfolded and that no changes in the spectrum due to interactions of residues in folded protein are present. After the ϵ calculation, the students prepared dilutions and were instructed to use the Beer-Lambert law to calculate the final protein concentration. They were also informed that the best results occur when the optical density at 280 nm absorbance of the dilution measured falls between the range of 0.1–0.8. Results outside of this range are inaccurate because both excess or absence of light increases the noise in the experiment. The measurements were corrected by dilution and were used for calculating the protein concentration using the Beer-Lambert law:
where A is the measured absorbance at 280 nm, ϵ is the extinction coefficient (M−1 cm−1), ℓ is the path length (cm), and C is concentration (M−1).
Conformation Analysis by Fluorescence—
Fluorescence spectroscopy is the phenomenon of light emission by an excited molecule when it undergoes a transition to a lower energy level . The main fluorophore in proteins is the residue Trp because of its high quantum yield and sensitivity to the structural and dynamic properties of proteins (see above). The quantum yield is the ratio of the number of photons emitted to the number of photons absorbed and can be lowered by quenching. The fluorescence studies of apoMb are shown in Figs. 2 and 3. At pH 7.0 and in the absence of denaturant, apoMb is folded and its Trps are buried in a hydrophobic environment (Fig. 2). These conditions result in a λemission maximum of around 330 nm (Table II). As the pH is lowered to 4.2, an intermediate species appears and the Trps are only partially buried, as demonstrated by an increase in the maximum λemission, and a second phenomenon is noticed, an increase in fluorescence intensity (Fig. 2). This increase in Trp fluorescence is likely to be the result of fluorescence quenching in the native state released in the intermediate . Fig. 3 shows normalized fluorescence spectra for comparison of relative fluorescence intensities at each wavelength; note that because ApoMb has 2 Trps and was used in the concentration of 2 μM, free Trp concentration used was 4 μM. When the apoMb is unfolded by the denaturant (Gdm-Cl), the compact structure is lost and the Trps are exposed to the solvent resulting in a maximum λ value of >350 nm (Table II). The spectrum for Trp alone is shown for comparison, and its λemission maximum is 354 nm, a value that is similar to the value of 350 nm for unfolded apoMb (Table II).
With this experiment, the students learn to use a probe that follows the protein conformational change as it unfolds. Once the students are instructed to understand that the Trp is experiencing a change in environment and that change is due to the unfolding of the protein as its chain looses structure and becomes more flexible, their comprehension of the phenomena is fast and effective.
Conformation Analysis by CD—
Additional methods for the evaluation of protein conformation can also be utilized. One of these methods is the measurement of CD at far-ultraviolet wavelengths (usually considered to be between 170 and 260 nm), where the protein spectral characteristics are determined by its secondary structure . Asymmetric molecules in solution are optically active, i.e. when they interact with circularly polarized light they absorb left and right polarized lights with different efficacy. This difference in absorption yields an elliptically polarized beam that can be measured by the CD spectropolarimeter, which converts it in ellipticity (Θ) as shown by the equation below:
where AL and AR are the absorbances for left and right circularly polarized light, respectively. One should note that sign of the CD signal is either negative or positive depending on the preferential absorption of left or right polarized light.
Peptide bonds are responsible for the CD signal between 170 and 260 nm, and this technique is therefore sensitive to the global secondary structure of a protein and is used for probing the amount of α-helices, β-sheets, and random coils present in a protein . α-Helices and β-sheets are very compact structures that are present in the folded protein, and the random coil is the shape of an unfolded protein. It soon becomes clear that this technique is useful for probing the amount of structure as the protein loses its native conformation due to unfolding. The data measured by CD is expressed as the ellipticity (Θ) in millidegree, which can be converted, for proteins, in mean residue molar ellipticity by the equation:
where C is the protein concentration in mol/liter and n is the number of residues in the protein. The mean residue molar ellipticity is given in deg·cm2·dmol−1 and usually is the choice for presenting data related to protein because it allows for quick information about the amount of structure when comparing different proteins or mutants.
The results of the CD measurement in the particular buffers are shown in Table II. The CD was measured at 222 nm because this is the wavelength at which α-helices present a maximum signal, and this secondary structure is the major component of apoMb. In native conditions, the protein shows a large signal that is partially lost as the pH is lowered to 4.2 (−18,000 to −13,000 deg·cm2·dmol−1; Table II). This occurs because the intermediate is a partially folded species that lies between the folded and unfolded states. As the protein is further unfolded by the strong denaturant, Gdm-Cl, the secondary structure is lost and only residual signaling is measured (−800 deg·cm2·dmol−1; Table II).
Instructing the students to create a table similar to Table II helped them to visualize the variation in conformational change followed by the two different probes and to reinforce the understanding of the loss of native conformation as the protein unfolds. Throughout this table, we were able to teach how important the knowledge of the conformation of a protein is and how to follow it using denaturants and spectroscopic probes as the protein unfolds. The experiment ended with some examples showing the use of these methods to study protein folding and stability [10, 11].
We herein describe a laboratory-based experiment for teaching protein structure principles and the use of simple spectroscopic techniques to measure protein conformation. This experiment may also be used to introduce the problem of protein folding and the biophysical methods to understand it. The methods used are simple and the experiments are quickly fulfilled. We suggest 4 h to perform it with the necessary introduction, but less time can be used if it is in the interest of the instructors. Unlike fluorometers, CD spectropolarimeters are sometimes not available in laboratories. However, the experiment can be performed without the CD, and this technique can easily be explained theoretically after the fluorometer experiment. The students easily understand the CD method. (In fact, the first experiment that I tutored was performed using only fluorescence as an example of conformational probing.) Our final conclusion is that the spectroscopic-based laboratory experiment described herein is valid for teaching important principles in protein science and can be very useful for biochemistry, biophysics, and structural biology courses.
I thank V. Soares for technical assistance and S. C. Farah, R. Meneghini, and W. Regis for helpful comments.