Tracking Local Conformational Changes of Ribonuclease A Using Picosecond Time-Resolved Fluorescence of the Six Tyrosine Residues

Abstract

The six tyrosine residues of ribonuclease A (RNase A) are used as individual intrinsic probes for tracking local conformational changes during unfolding. The fluorescence decays of RNase A are well described by sums of three exponentials with decay times (tau(1) = 1.7 ns, tau(2) = 180 ps, and tau(3) = 30 ps) and preexponential coefficients (A(1) = 1, A(2) = 1, and A(3) = 4) at pH 7, 25 degrees C. The decay times are controlled by photo-induced electron transfer from individual tyrosine residues to the nearest disulphide (-SS-), bridge, which is distance (R) dependent. We assign tau(1) to Tyr-76 (R = 12.8 A), tau(2) to Tyr-115 (R = 6.9 A), and tau(3) to Tyr-25, Tyr-73, Tyr-92, and Tyr-97 (all four at R = 5.5 +/- 0.3 A) at 23 degrees C. On the basis of this assignment, the results show that, upon thermal or chemical unfolding only Tyr-25, Tyr-92, and Tyr-76 undergo significant displacement from their nearest -SS- bridge. Despite reporting on different regions of the protein, the concordance between the transition temperatures, T(m), obtained from Tyr-76 (T(m) = 59.2 degrees C) and Tyr-25 and Tyr-92 (T(m) = 58.2 degrees C) suggests a single unfolding event in this temperature range that affects all these regions similarly.

Thermally induced early events of ribonuclease A under reducing conditions: Evidenced by principal component analysis and two-dimensional correlation infrared spectroscopy

“…For the fully deuterated RNase A under non-reducing conditions, the fluorescence intensity decreased with temperature gradually from 31 to 61 8C, then increased a little bit from 63 to 65 8C, and finally decreased again from 67 to 75 8C. Similar dependences of the fluorescence quantum yield of RNase A on temperature were also observed by Noronha [25] and Gally [26] under non-reducing conditions, where the decreases of the fluorescence intensity was attributed to the quenching of the exposure of buried tyrosine residues to the aqueous solvent and the fluorescence increase arisen from a reduction of the quenching likely due to an increase in the distance from the tyrosine rings to the quenchers (disulfide bridges) during unfolding. It was reported that the thermal unfolding of RNase A induced a separation of the two highly quenched tyrosine residues from the nearby disulfide bridges via changes in their environment [25], and therefore the presented fluorescence increase must represent an unfolding process of RNase A.…”

“…As RNase A contains no tryptophan residues, the emission band centered at 303 nm is assigned to the tyrosine fluorescence [24,25]. The plots of the Tyr intensity as a function of temperature (T) in the different cases are shown in Fig.…”

Photo-synthesis of protein-based nanoparticles and the application in drug delivery

“…Recently, it was found that disulfide bonds buried in proteins can be reduced by UV illumination, which dramatically affects the protein conformations as well as their biological functions [15][16][17][18]30,31]. The absorption of UV light by Trp residues was responsible for the reductive splitting of the nearby disulfide bonds and the production of new free thiols in a lot of proteins such as Fusarium solani pisi cutinase [15], goat α-lactalbumin [16] and hen egg white lysozyme [32].…”

“…The photo-ionization electrons from Trp residues are transferred to the nearby disulfide bonds, resulting in the breakage of disulfide bonds and the formation of free thiols. The electron transfer rate and the reduction rate of disulfide bonds are quite dependent on the distance between Trp residues and the nearly disulfide bonds [16,30,31]. The newly formed free thiol groups can easily form a new intra/intermolecular disulfide bonds even under the weak oxidative condition of dissolved oxygen in protein solution.…”

Conformational Stability of Bovine Serum Albumin in Aqueous Amides: A Further Insight into the Mechanism of Urea Acting on the Protein

“…On the contrast, the fluorescence will red shift in hydrogen bond acceptor solvent, as the conjugation effect is increased. 21 There is considerable evidence that the maximum fluorescence emission of tyrosine occurred at 306 nm in the hydrophobic core of protein 22,23 and blue shifts to 302 nm in water. 11 Thus, it can be deduced that water molecule serves as hydrogen bond donor to tyrosine.…”