Global rotation

We should note that the use of the Lipari-Szabo analysis implies that relaxation data are available at multiple magnetic fields. It provides a phenomenological description of the rotational motion that can be very useful for comparing systems with similar structure. Nevertheless, one should be aware of the limits of this approach and avoid direct comparison of local or global rotational correlation times for structurally very different compounds. 

Figure 11.2 shows the excitation polarization spectrum of protoporphyrin IX in propylene glycol at —55°C (full line) and bound to the heme pocket of apohemoglobin recorded at 20°C (dotted line). One can see that polarization at a low temperature is higher than that observed when porphyrin is embedded in the heme pocket of apohemoglobin. This is the result of fluorophore local motions within the pocket, independently of the global rotation of the protein. 

Whether fluorophores are intrinsic or extrinsic to the macromolecule (protein, peptide, or DNA), depolarization is the result of two motions, fluorophore local motions and macromolecule global rotation. 

The data yield a rotational correlation time equal to 38 ps instead of 5.9 ns calculated theoretically for the cytochrome b2 core, with an extrapolated value A(o) of 0.208, lower than that (0.265) usually found for Trp residues at Xex = 300 nm at —45°C. The fact that the extrapolated anisotropy is lower than the limiting anisotropy means that the system is depolarized as a result of global and local motions within the protein. In this case, the value of the apparent rotational correlation time ( a) calculated from the Perrin plot is lower than the global rotational time of the protein ( bp). However, the fact that 4>a is 1000 times lower than 4>p indicates that a third process different than the global and local rotations is... 

The fluorophore is bound tightly to the protein, so it will follow the motions of the protein. Therefore, the rotational correlation time calculated from the Perrin plot will be equal to the global rotational time of the protein. 

For the fi-CD complex, alcohol addition increased the distribution width and only slightly changed the center lifetime this indicates a disruption of the CD-4 complex through competitive binding to the cavity. The average rotational correlation time, recovered from steady-state anisotropy, indicated that global rotation dominated the rotational diffusion of the complexes. 

Biological systems (membranes, DNA, proteins, etc...) display specific motions, global rotation and local dynamics, that are dependent on the structure, the environment and the function of the system. These motions differ from a system to another and, within one system local motions are not the same. The most known example is that of membrane phospholipids where the hydrophilic phosphates are rigid and the hydrophobic lipid is highly mobile. Polarized light is a good tool to put into evidence and to study the different types of rotations a molecule can undergo. 

It is possible to obtain experimental the rotakuial ccHn lion time of the flucffuphore 1 increasing the medium viscosity in presence ofgb cend and/or sucrose. In this case, the global rotation of tbe protein will be complete hindered and only the local motions of the flucvufdiore will be observed I / P as a function of 1 /1] yields a 1 01 with two slopes. The rotational correlation times of the protdn ( p) and of the fluoropbore (4r) are calculated at h h and low T /. respectivaly (Fig. S. lO). 

Measiuements of the emission anisotropy A as a function of added collisional quencher are made with the steady fluorescence intensity, which integrates the different weighted fluorescence lifetimes. Quenching emission anisotropy plot of 1 /A vs I (Fig. 5.14) yields for A(o) a value of 0.246 and 0.243 for [L-Met2] DREK and DREK, respectively. These values, lower than that (0.278) measured at - 45 °C for tyrosine at 280 nm (Lakowicz and Maliwal, 1983), indicate that tyrosine residue in both peptides display residual motion independent of the global rotation of the peptide. It is possible to measure the relative importance of the mean residual motions of the tyrosine residues ... 

Where Aq, dp, dp and dx are the intrinsic anisotropy, the depolarization factor due to the global rotation of the protein, the depolarization factor due to the local motions of the fluorophore and the depolarization factor due to the energy transfer and Brownian motion, respectively. 

Time resolved anisotropy decay performed at different temperatures, reveals the presence of two rotational correlation times, one Op, corresponding to the global rotation of the protein and the second Oa, a shorter one, reveals the presence of local residual motions around and / or near the two tryptophan residues. Oa is an apparent rotational time that is a mathematical combination of the global rotation of the protein and the segmental motion of the fluorophore. 

Steady-state fluorescence anisotropy of Trp residues in both preparations of ai-acid glycoprotein was performed at different temperatures. The Perrin plots (Fig. 5.7 and 5.8) reveal that Trp residues of ai-acid glycoproteiii s display free motions while those of a 1-acid glycoprotein follow the global rotation of the protein. 

Thus, red- dge excitation spectra experiments indicate that TNS microenvironment has nwtions that are dependent of the global rotation of the protein, i.e., TNS will follow the motion of apacid glycoprotein. 

Figure 8.20 displays the normalized fluorescence emission spectra of 10 pM of calcofluor in the presence of 15 pM of ai -acid glycoprotein obtained at three excitation wavelengths. The maximum (440 nm) of the Calcofluor fluorescence does not change with the excitation wavelength (9iex 385, 395 and 405 nm). The results obtained clearly indicate that the microenvironment of calcofluor has residual motions independent of the global rotation of the protein, and this may induce the local motions of calcofluor. 

Steady-state fluorescence anisotropy of 10 pM of Calcofluor in the presence of 5 pM of ai -acid glycoprotein = 435 nm and Xqx 300 nm) was performed at different temperatures. A Perrin plot representation (Fig. 8.21a.) yields a rotational correlation time equal to 7.5 ns at 20 °C. This value is lower than that (16 ns) expected for a i-acid glycoprotein and thus indicates that calcofluor displays segmental motions independent of the global rotation of the protein. Thus, two motions contribute to the depolarization process, the local motion of the carbohydrate residues and the global rotation of the protein, i.e., a fraction of the total depolarization is lost due to the segmental motion, and the remaining polarization decays as a result of the rotational diffusion of the protein. 

The rotational correlation time ( = 7.5 ns) obtained from the Penin plot is the result of the global rotation of the protein and the local motion of Calcofluor White. 

The anisotropies of both Trp-residues of apacid glycoprotein measured at 20°C are very close (Table 8.7), (a result identical to that obtained with the Weber method, see Table 8.6) indicating that the two Trp residues are highly mobile. This result is confirmed by the values of the rotational correlation times (3 and 5 ns) lower than the global rotational correlation time of a i-acid glycoprotein. When the Trp residue is embedded in the protein core and does not show any residual motions such as in the Lens culinaris agglutinin, its rotational con elation time will be close to that of the protein and its anisotropy will be higher than that of the surface Trp residue. 

We notice however, that the anisotropies are more important in the Weber method. This can be explained by the fact that in the Weber method, anisotropies were calculated at low temperatures (-33 and -16°C for the buried and surface Trp residues, respectively), while in the QREA method, the anisotropies were measured at 20°C. At low temperatures, the global rotation of the protein and the local motions ai ound the Trp residues are decreased. 

The Oi term contains the global rotation of the protein, the residual motion of the Trp residues and the motion of the microenvironment around the Trp residues. In a system... 

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