In a recent study posted to the bioRxiv* preprint server, researchers quantified hydrophobic interactions encoded by fusion peptide 1 (FP1) sequences of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Middle Eastern respiratory syndrome coronavirus (MERS-CoV) using single-molecule force measurements.
Study: Characterization of Hydrophobic Interactions of SARS-CoV-2 and MERS-CoV Spike Protein Fusion Peptides Using Single Molecule Force Measurements. Image Credit: Kateryna Kon/Shutterstock
This news article was a review of a preliminary scientific report that had not undergone peer-review at the time of publication. Since its initial publication, the scientific report has now been peer reviewed and accepted for publication in a Scientific Journal. Links to the preliminary and peer-reviewed reports are available in the Sources section at the bottom of this article. View Sources
Background
FP1 peptide sequences from SARS-CoV-2 and MERS-CoV are majorly conserved and encode hydrophobic interactions that regulate virus and host cell membrane fusion. Hydrophobic interactions between viral FPs and the host cell membranes regulate the viral membrane fusion process and determine their infectivity; however, it is not unknown how the amino acid sequences in FPs mediate hydrophobic interactions.
It is possible that the non-polar triad (LLF), common to both FP1 sequences, dominates the encoding of hydrophobic interactions. However, FP1 from SARS (SFIEDLLFNK) and FP1 from MERS (SAIEDLLFDK) vary at two amino acid residues – phenylalanine (Phe) 2 vs. alanine (Ala) 2 and asparagine (Asn) 9 vs. Aspartic acid (Asp) 9, respectively. Therefore, the authors investigated whether FP1 sequence variations translated to differences in their hydrophobic interactions.
About the study
In the current study, researchers examined 11-amino acid sequences (undecamer, S1 to K10) from the FP1 domain of SARS-CoV-2 and MERS-CoV, referred to as “SARS-2 FP1” and “MERS FP1”, respectively.
They synthesized FPs using solid-phase methods and then immobilized them onto mixed monolayers terminating in tetraethylene glycol (EG4) or aminotetraethylene glycol (EG4N) groups. In this way, they measured adhesive interactions between single FP molecules immobilized onto mixed monolayers and the non-polar adhesion force measurements (AFM) tips.
AFM tips used for experiments involving FPs were triangular-shaped cantilevers with nominal spring constants of 0.01 N/m, functionalized and transferred onto the AFM fluid cell. The spring constants of the cantilevers were calibrated using the thermal tuning method and determined to be 0.027 ±0.003 N/m.
The FPs used in the current study are oligomers of α-amino acids; accordingly, their secondary structure and spatial presentation depended on the solution environment and interaction with interfaces. Therefore, the researchers performed adhesion force measurements in aqueous phosphate-buffered saline (PBS) buffer or PBS with 60 vol % methanol (MeOH); PBS also contained calcium (Ca2+). The addition of 60 vol % MeOH to PBS eliminated a majority of hydrophobic interactions mediated by non-polar peptide domains without disrupting ionic and van der Waal interactions.
The researchers performed AFM at room temperature using a “J” type scanner to generate histograms, each of which represented over 3,000 pull-off force curves. The recorded force curves used a constant contact time of 500 ms and retraction and approach speeds of 1,000 nm/s.
The researchers used circular dichroism (CD) and attenuated total reflectance – Fourier transform infrared spectroscopy (ATR-FTIR) to characterize the conformations of FPs. ATR-FTIR spectra for each peptide showed a minimum of 300 scans at a resolution of 4 cm-1. To probe the statistical significance of differences in the measured mean adhesion forces, researchers performed t-tests.
Study findings
The authors observed that SARS-CoV-2 and MERS wild-type FP1 generated detectable hydrophobic interactions at the single-molecule level; however, SARS-CoV-2 FP1 encoded a much-stronger hydrophobic interaction with a pull-off force of 1.91 ± 0.03 nN compared to MERS-CoV FP1, whose pull-off force was barely 0.68 ± 0.03 nN.
Although the LLF motif was present in both FP1 sequences, AFMs showed that a single residue substitution (Phe 2 vs. Ala 2) caused almost a three-fold difference in the hydrophobic interaction strength generated by SARS-CoV-2 FP1 and FP1 of MERS-CoV.
Infrared spectroscopy and CD measurements further validated that the influence of Phe 2 versus Ala 2 on the hydrophobic interaction had arisen from variations in the secondary conformations adopted by FP1.
The average pull-off force of SARS-2 FP1 in PBS and PBS containing 60 vol % MeOH was 1.91 ± 0.03 nN and 0.47 ± 0.01 nN, respectively, indicating a decreased adhesion force in the presence of 60 vol % MeOH containing PBS.
The authors noted no overlap between histograms of pull-off forces measured in PBS or PBS containing 60 vol % MeOH. This suggested that pull-off forces measured in PBS were dominantly hydrophobic interactions, while these forces in 60 vol % MeOH corresponded to van der Waals and electrical double layer interactions.
The t-tests obtained p-values < 0.05 for all comparisons (except one) made in the present study, indicating statistically significant results with a significance level of 95%.
Conclusions
The study provided critical insights into the mechanisms by which the amino acid composition (even a single residue) influenced hydrophobic interactions encoded by FPs and their implications for viral fusion activity. To conclude, the strategies regulating hydrophobic interactions of FPs are applicable in a range of contexts and could help contain transmission of SARS-CoV-2.
This news article was a review of a preliminary scientific report that had not undergone peer-review at the time of publication. Since its initial publication, the scientific report has now been peer reviewed and accepted for publication in a Scientific Journal. Links to the preliminary and peer-reviewed reports are available in the Sources section at the bottom of this article. View Sources
Article Revisions
- May 12 2023 - The preprint preliminary research paper that this article was based upon was accepted for publication in a peer-reviewed Scientific Journal. This article was edited accordingly to include a link to the final peer-reviewed paper, now shown in the sources section.
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