Peptide-MHC I complex stability measured by nanoscale differential scanning fluorimetry reveals molecular mechanism of thermal denaturation

Opening opportunities for Kd determination and screening of MHC peptide complexes

“…While the negative control YF9 clearly shows no stabilization ( ∆T m = 1 ± 1 K), the positive control NV9 shows a high ∆T m (23.4 ± 0.6 K) in agreement with published data; 12 this represents the maximum ∆T m possible since NV9 stabilizes the binding groove so strongly that upon heating, other domains unfold first (Fig. S 2 ) 40 , 41 . All other peptides show an excellent correlation between K d and ∆T m .…”

“…It was also found that K d values from nMS correlate well with the thermal stabilization ∆T m of dsA2 by peptide binding (protein-peptide ratio: 1:10, Fig. 4b ), i.e., the increase in the midpoint of thermal denaturation ( T m ) above that of the empty dsA2 (35.7 ± 0.6 °C) as measured by tryptophan nanoscale differential scanning fluorimetry (nDSF 40 , 41 ). While the negative control YF9 clearly shows no stabilization ( ∆T m = 1 ± 1 K), the positive control NV9 shows a high ∆T m (23.4 ± 0.6 K) in agreement with published data; 12 this represents the maximum ∆T m possible since NV9 stabilizes the binding groove so strongly that upon heating, other domains unfold first (Fig.…”

Rapid peptide exchange on MHC class I by small molecules elucidates dynamics of bound peptide

“…nDSF measures the spectral shift of the tryptophan and tyrosine side chain fluorescence emissions caused by their exposure to water upon protein unfolding ( Vivian and Callis, 2001 ). The change in the ratio of emissions at 350 and 330 nm (F350/F330) quantifies the spectral shift, and the apparent midpoint temperature of thermal denaturation (T m ) is determined from the peak of the first derivative (d(F350/F330)/dT) ( Saikia and Springer, 2021 ).…”

“…Proteins were then solubilized in urea buffer (8 M urea, 50 mM HEPES pH 6.5, and 100 μM β-mercaptoethanol). Further buffers and solutions used for the production and folding of MHC-I molecules are described in Saikia and Springer (2021) , Saini et al, 2013a , Saini et al, 2015 .…”

“…If an MHC-I presents non-self peptides to CTL, these will initiate cell destruction. To detect, isolate, and stimulate CTL, recombinant peptide/MHC-I complexes (rpMHC-I) are used in diagnostics and vaccine development, for example as MHC tetramers ( Klenerman et al, 2002 ; Saikia and Springer, 2021 ). To rapidly generate many different rpMHC-I, exchange of peptides is commonly used; this is currently achieved by UV cleavage ( Rodenko et al, 2006 ), heat ( Luimstra et al, 2019 ), or with dipeptides ( Saini et al, 2015 ).…”