A second look at mini-protein stability: analysis of FSD-1 using circular dichroism, differential scanning calorimetry, and simulations

Abstract

Mini-proteins that contain <50 amino acids often serve as model systems for studying protein folding because their small size makes long timescale simulations possible. However, not all mini-proteins are created equal. The stability and structure of FSD-1, a 28-residue mini-protein that adopted the betabetaalpha zinc-finger motif independent of zinc binding, was investigated using circular dichroism, differential scanning calorimetry, and replica-exchange molecular dynamics. The broad melting transition of FSD-1, similar to that of a helix-to-coil transition, was observed by using circular dichroism, differential scanning calorimetry, and replica-exchange molecular dynamics. The N-terminal beta-hairpin was found to be flexible. The FSD-1 apparent melting temperature of 41 degrees C may be a reflection of the melting of its alpha-helical segment instead of the entire protein. Thus, despite its attractiveness due to small size and purposefully designed helix, sheet, and turn structures, the status of FSD-1 as a model system for studying protein folding should be reconsidered.

Figure 1

Structure of FSD-1 (PDB: 1FSD). (A) For clarity, side chains of selected residues are shown. (B) The main-chain atoms in the β-hairpin of FSD-1 are shown colors and the hydrogen bonds between Y3 and F12 highlighted by black dashes. The α-helix is shown in light gray. Figures were generated using PyMOL (33). Sequence: QQYTAKIKGRTFRNEKELRDFIEKFKGR.

Figure 2

(A) Far-UV CD spectra of FSD-1 at 4°C and 80°C. Spectra were measured at 4°C premelting (solid) and postmelting (dotted). (B) Spectra of FSD-1 and an unfolded FSD-1 double mutant (I7PK^DP) at 4°C. ^DP denotes D-Proline.

Figure 3

Thermal unfolding of FSD-1 monitored by CD at 218 nm. The melting curve was fitted to a two-state model and the resulting T_m was 41°C and ΔH_vH was 18 kcal/mol.

Figure 4

DSC melting curve fitted to a two-state model. T_m was determined to be 41°C and ΔH_cal was determined to be 15 kcal/mol. Dark and gray circles represent two back-to-back DSC scans.

Figure 5

Potential-energy overlap between neighboring replicas during the last 10 ns of the simulation. Each distribution curve represents the potential-energy distribution at a single temperature. The left-most curve represents the potential energy of the lowest-temperature replica, and the right-most curve represents the potential energy of the highest-temperature replica.

Figure 6

(Top) Temperatures sampled by 3 of 64 representative replicas during the course of the simulation. Replicas 1, 31, and 62 started at 262.2 K, 337.4 K, and 437.8 K, respectively. (Bottom) RMSD of three replicas during the course of the REMD simulation. A folding event is observed in replica 62, and unfolding events are observed in replicas 1 and 31.

Figure 7

Thermal unfolding monitored by RMSD and RMSF. The data were fit to a two-state model. RMSF values were calculated for residues 3–25. The fitted melting temperatures T_m are shown in the panels.

Figure 8

Average number of hydrogen bonds formed between main-chain atoms in residues Y₃ and F₁₂ (x) or between residues in strand 1 (residues 2–6) and strand 2 (residues 9–13) of the hairpin (+) during the REMD simulation.