A Review of Protein Research Conducted in Cell-Like Environments
Stephen T. LanierProtein function is intimately dependent on protein folding, shape, and diffusion. In the earliest exploration of protein function, researchers studied protein in “reductionist” and “high dilution,” or in vitro, environments, sometimes in quantities as small as only a few molecules. Christian Anfinsen revolutionized the field with his Nobel Prize winning work on ribonuclease and the effects a cell-like, or in vivo, environment had on its folding, and therefore function (Gershenson). His work made clear an integral consideration of protein studies today: density matters.
When the field changed its approach from in vitro to in vivo, research followed studying the best methodology for observing protein in high-density environments. Two popular approaches have been discovered to allow the researcher to observe a specific protein even when macromolecular crowding is present: fluorescence-based approaches, especially green fluorescent protein (GFP) spectroscopy, and nuclear magnetic resonance (NMR) spectroscopy. GFP spectroscopy is achieved by attaching green fluorescent protein to a protein of interest (POI) and then using a specific wavelength of light to observe the POI and distinguish it from the rest of the cell. While GFP spectroscopy has proven particularly advantageous when studying rotational protein diffusion, it has also shown to add as much as 27 kDa mass to the tagged POI: because many POI are between 5 kDA and 100 kDA, the added weight is substantial (Pielak “Protein”). Furthermore, GFP has been observed to affect translational protein diffusion, whether by increased mass and viscosity alone or by other (unknown) chemical interactions between the fluorescent protein and the POI. Because protein research now emphasizes the state of environmental density, the mass- and diffusional-effects of GFP are of critical concern. Alternatively, NMR spectroscopy is achieved by applying a magnetic field to a POI. The magnetic field interacts with proteins in ways specific to the nuclei of its atoms; to elaborate, the nucleus of an atom absorbs electromagnetic radiation at specific frequencies that the nuclei of other atoms do not. By using the specific frequency of a target atom in a POI, NMR can identify a protein from others by observing which proteins absorb the frequency and which do not, the POI and extraneous macromolecular environment respectively. NMR spectroscopy proves difficult for studying some types of diffusion (rotational) and very large proteins, but unlike fluorescence spectroscopy, does not affect protein or environment density, viscosity and diffusion, or folding (Ye, Pielak “Protein”). Due to the respective limitations of each, GFP spectroscopy and NMR spectroscopy have distinctly separate arrays of use: while GFP spectroscopy is advantageous for studying large proteins, as the added mass of the tagging protein has less effect than on small proteins, and rotational diffusion; NMR spectroscopy is best suited to observe small proteins and translational diffusion.
A combination of NMR and GFP spectroscopy has since been used to discover the specific impetus responsible for density-specific protein folding and diffusion in cells. In one study by Wang et al. using NMR to research two specific and differently shaped proteins, chymotrypsin inhibitor 2 (CI2) and α-synuclein, researchers discovered that while CI2 diffuses faster than α-synuclein in dilute environments, the opposite is true with crowding, in vivo: that protein shape contributes to diffusion was concluded. Rate of diffusion is important when studying protein function because a protein’s concentration is a key factor in determining its degree of activity. In Wang’s study, a cell-like environment was the difference between one protein diffusing (and therefore functioning) more quickly than another: thus, the study was able to connect the importance of a cell-like environment to shape-specific protein function.
Others are studying the in-cell environment’s impact on protein folding, another intrinsic attribute of protein function. A study of two homo logs of ribosomal protein S16, one thermophilic and one mesophilic, by Mikaelsson et al. sought to determine that relationship by firstly attaching markers to two different parts of the protein and secondly measuring the distance between those two markers at different environmental densities using fluorescence spectroscopy. The first protein was not affected: they hypothesized that, due to its thermophilic nature, its greater rigidity at room temperature and resistance to enthalpic interactions prevented it from being affected by the experiment’s room temperature crowding. They found that the second protein, however, a mesophile, became more tightly folded in higher densities, as the distance between the two markers was smaller under higher concentrations of crowding agent. At 0 mg/mL crowding agent the average distance was 20.6 Å, and at 100 mg/mL the average distance was 18.9 Å; two different locations on the protein were marked and the same results were produced, as at 0 mg/mL the average distance was 17.4 Å, and at 100 mg/mL the average distance was 16.1 Å. From this data, researchers were able to conclude that proteins occupy less area, or that they fold more compactly, when crowded. Follow-up research has not yet been produced to determine if thermophilic proteins behave similarly in relevantly high-temperature environments.
Mikaelsson et al.’s results, which were fluorescence-spectroscopy-based, mirror similar studies by Pielak et al. (“Macromolecular), which were NMR-based, but each proposes a different conclusion. While the fluorescence-based spectroscopy studies argue the importance of “dominant,” or “hard-core,” forces on protein folding, NMR-based spectroscopy studies argue the importance of “weak nonspecific,” or “chemical,” interactions. Differences in spectroscopies are perhaps the cause of the divergence. A separate study by Ye et al. conducted an experiment using NMR-spectroscopy and then compared its results to data previously obtained using GFP. They found that NMR was capable of measuring weak protein-protein interactions and environmental viscosity, while previous studies using GFP on the same two proteins had overestimated the viscosity of the cell because of the tagging-protein’s added weight, and therefore overestimated the force size and importance of hard-core repulsive interactions. Both studies by Mikaelsson et al. and Pielak et al., then, exhibit a focus that is both practical and biased towards the forces best suited to their respective methodologies.
As the scientific community continues its discussion on protein research, it must decide when fluorescence-based-spectroscopy’s advantages outweigh affecting protein mass and environmental density and viscosity. That fluorescence-based-spectroscopy perturbs authentic density and viscosity is concerning, considering that both factors are often pivotal when determining function, but as Mikaelsson’s research exemplified, fluorescence-based-spectroscopy may offer greater insight into a protein’s behavior than perhaps NMR spectroscopy, such as when observing protein folding. If, however, scientists are able to conclude the impetus of crowding’s affects as either “dominant” and favoring fluorescence-based observation or “weak” and “nonspecific” and favoring NMR observation, one methodology may take precedence over the other; until such time, researchers must continue to deliberately decide upon their methodology, as did Wang when studying diffusion by choosing NMR spectroscopy, as did Mikaelsson when observing folding, and as Mikaelsson, Pielak, and Ye yet debate.
Works Cited
Gershenson, Anne, and Lila M. Gierasch. “Protein Folding in the Cell: Challenges and Progress.” Current Opinion In Structural Biology, 21.1 (2011): 32-41. Web. 6 Sept. 2014.
Mikaelsson, Therese, et al. “Macromolecular Crowding Effects on Two Homo Logs of Ribosomal Protein S16: Protein-Dependent Structural Changes and Local Interactions.” Biophysical Journal, 107.2 (2014): 401-410. Web. 8 Sept. 2014.
Pielak, Gary J., et al. “Macromolecular Crowding and Protein Stability.” Journal of the American Chemical Society, 134.40 (2012): 16614-8. Web. 24 Sept. 2014.
Pielak, Gary J., et al. “Protein Nuclear Magnetic Resonance Under Physiological Conditions.” Biochemistry, 48.2 (2009): 226-234. Web. 6. Sept. 2014.
Wang, Yaqiang, et al. “Disordered Protein Diffusion Under Crowded Conditions.” Journal Of Physical Chemistry Letters, 3.18 (2012): 2703-2706. Web. 7 Sept. 2014.
Ye, Yansheng, et al. “F-19 NMR Spectroscopy as a Probe of Cytoplasmic Viscosity and Weak Protein Interactions in Living Cells.” Chemistry-A European Journal, 19.38 (2013): 12705-12710. Web. 7 Sept. 2014.