Unfolding of Acrylodan-Labeled Human Serum Albumin Probed by Steady-State and Time-Resolved Fluorescence Methods

Abstract

Steady-state and time-resolved fluorescence spectroscopy was used to follow the local and global changes in structure and dynamics during chemical and thermal denaturation of unlabeled human serum albumin (HSA) and HSA with an acrylodan moiety bound to Cys34. Acrylodan fluorescence was monitored to obtain information about unfolding processes in domain I, and the emission of the Trp residue at position 214 was used to examine domain II. In addition, Trp-to-acrylodan resonance energy transfer was examined to probe interdomain spatial relationships during unfolding. Increasing the temperature to less than 50 degrees C or adding less than 1.0 M GdHCl resulted in an initial, reversible separation of domains I and II. Denaturation by heating to 70 degrees C or by adding 2.0 M GdHCl resulted in irreversible unfolding of domain II. Further denaturation of HSA by either method resulted in irreversible unfolding of domain I. These results clearly demonstrate that HSA unfolds by a pathway involving at least three distinct steps. The low detection limits and high information content of dual probe fluorescence should allow this technique to be used to study the unfolding behavior of entrapped or immobilized HSA.

Anisotropy resolved multidimensional emission spectroscopy (ARMES): A new tool for protein analysis

“…Anisotropy plots for each component showed clearly that this was a single step transition between the native and the unfolded states, because the anisotropy change was the same for each emitter. This agreed with previously published results [53,54], for example, initial (0M)/final (4M) anisotropy ratios (~1.7) for both Trp components were close the mean lifetime ratio (1.9) from Flora et al [17] Comparing the MCR scores (Figure 3, and SI, Fig. S-12/13) shows very different behavior and it was interesting to note that the Comp3 (RTP) contribution increased in a sigmoidal fashion (centered at 2M GuHCl) as GuHCl concentration increased.…”

“…Here, the second option was more probable, as reports have shown that there was a decreased Trp lifetime for HSA denaturation due to increased solvent exposure after recovery from thermal denaturation. The increased anisotropy was ~11% which was the same as the mean lifetime decrease of 5.28 to 4.72 ns recorded by Flora et al [17]. This was supported by evaluation of MCR scores (Figure 3) where Comp4 scores were always much less after refolding which indicated higher quenching due to solvent exposure.…”

“…The anisotropy values recovered here at 20 °C were much higher than those reported by other groups such as Flora et al [17] (0.27 versus 0.18). This was because ARMES recovered only Trp fluorescence, which ensured minimal contribution from RTP or Tyr emission to the anisotropy measurement.…”

“…This agreed with the three-step model with the expanded form being generated up to ~40 °C, the second step between 50-60 °C with the unfolding of domain II, . Page 12 of 26 followed by complete, irreversible unfolding at temperatures above ~60 °C [17,18,[48][49][50]. Comp1/2 displayed similar yet non-identical changes in normalized MCR scores, signifying the different locations of the Tyr residues contributing to each signal.…”

Monitoring Local Unfolding of Bovine Serum Albumin During Denaturation Using Steady-State and Time-Resolved Fluorescence Spectroscopy

“…When GuHCl is used to induce protein unfolding in serum albumins it has led to the reporting of contradictory results in the literature with regard to the exact unfolding mechanism. There are reports that point induced unfolding HSA occurs in a single step [13,[21][22][23][24] while other studies indicate a multi-step process with the formation of stable intermediates [25][26][27][28][29].…”