Nanomolar Protein Thermal Profiling with Modified Cyanine Dyes

Abstract

Protein properties and interactions have been widely investigated by using external labels. However, the micromolar sensitivity of the current dyes limits their applicability due to the high material consumption and assay cost. In response to this challenge, we synthesized a series of cyanine5 (Cy5) dye-based quencher molecules to develop an external dye technique to probe proteins at the nanomolar protein level in a high-throughput one-step assay format. Several families of Cy5 dye-based quenchers with ring and/or side-chain modifications were designed and synthesized by introducing organic small molecules or peptides. Our results showed that steric hindrance and electrostatic interactions are more important than hydrophobicity in the interaction between the luminescent negatively charged europium-chelate-labeled peptide (Eu-probe) and the quencher molecules. The presence of substituents on the quencher indolenine rings reduces their quenching property, whereas the increased positive charge on the indolenine side chain improved the interaction between the quenchers and the luminescent compound. The designed quencher structures entirely altered the dynamics of the Eu-probe (protein-probe) for studying protein stability and interactions, as we were able to reduce the quencher concentration 100-fold. Moreover, the new quencher molecules allowed us to conduct the experiments using neutral buffer conditions, known as the peptide-probe assay. These improvements enabled us to apply the method in a one-step format for nanomolar protein-ligand interaction and protein profiling studies instead of the previously developed two-step protocol. These improvements provide a faster and simpler method with lower material consumption.

Figure 1

(A) Protein-probe principle: intact protein has minimal interaction with the Eu-probe, and the presence of Cy5 quenches the TRL-signal of the probe. When the protein is thermally denatured, the Eu-probe binds to the exposed hydrophobic regions. As a result, the interaction between Cy5 and the Eu-probe is reduced, leading to a high TRL-signal measurement. (B) The peptide-probe principle: the modified and synthesized quencher, known as a peptide quencher, exhibits a high affinity with the Eu-probe, resulting in efficient quenching of the TRL-signal. Thermally denatured protein disrupts the interaction between negatively and positively charged peptides, leading to a high TRL-signal. (C) Chemical structures of the Eu-probe and Cy5 (quencher).

Figure 2

Structures of Cy5 dye quenchers 1–14 (Q1–Q14).

Figure 3

(A) Melting curves of KRAS in both the absence and presence of MgCl₂ (1 mM) using the one-step peptide-probe assays with the Eu-probe (1 nM) and Q14 (10 nM) in HEPES buffer (10 mM, pH 7.5, 10 mM NaCl, 0.01% Brij 30). The T_m values were 47.6 ± 0.6 and 56.7 ± 0.5 °C for KRAS and KRAS + MgCl₂ (1 mM), respectively. (B) Detection of nucleotide exchange using 100 nM GDP-loaded GαS (R201C, C237S) in the absence and presence of additional GDP, GppNHp, and GTPγS (5 μM) with the one-step peptide-probe assays with the Eu-probe (7.5 nM) and Q14 (7.5 nM) at HEPES buffer (10 mM, pH 7.4, 10 mM NaCl, 1 mM MgCl₂, 0.01% Triton X-100). The T_m values for GαS (R201C and C237S) alone with GDP, GppNHp, and GTPγS were 44.4 ± 0.2, 45.5 ± 0.1, 48.2 ± 0.3, and 54.3 ± 1.0 °C, respectively. The initial first phase T_m value for GTPγS was 45.5 ± 0.3. All data are expressed as mean ± SD.

Figure 4

(A) Melting curves of Lrrk2 (wt) were measured in both the absence and presence of 200 μM GDP using the one-step peptide-probe and DSF methods. The one-step peptide-probe assay yielded T_m values of 40.2 ± 0.1 and 47.7 ± 0.1 for Lrrk2 (500 nM) without and with GDP, respectively. In comparison, DSF resulted in T_m values of 30.4 ± 0.1 and 46.5 ± 0.0 for Lrrk2 (4000 nM) without and with GDP, respectively. (B) Melting curves of various proteins, including TpeL, 7202 mAb, elF4A1, MDH, and G(i)α, were measured using the one-step peptide-probe assay with the Eu-probe (1 nM) and Q14 (10 nM) in HEPES buffer (10 mM, pH 7.5, 10 mM NaCl, 0.01% Brij 30). The T_m values for TpeL, 7202 mAb, elF4A1, MDH, and G(i)α were 46.9 ± 0.7, 77.3 ± 0.4, 42.4 ± 0.2, 50.0 ± 0.2, and 42.9 ± 0.3 °C, respectively. The data are presented as the mean ± SD.