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The stability and activity of human neuroserpin are modulated by a salt bridge that stabilises the reactive centre loop.

Noto R, Randazzo L, Raccosta S, Caccia S, Moriconi C, Miranda E, Martorana V, Manno M - Sci Rep (2015)

Bottom Line: Further, MD predictions were tested in vitro by purifying recombinant Glu289Ala NS from E. coli.The thermal and chemical stability along with the polymerisation propensity of both Wild Type and Glu289Ala NS were characterised by circular dichroism, emission spectroscopy and non-denaturant gel electrophoresis, respectively.Our results showed that deletion of the salt bridge leads to a moderate but clear reduction of the overall protein stability and activity.

View Article: PubMed Central - PubMed

Affiliation: National Research Council of Italy, Institute of Biophysics, Palermo, Italy.

ABSTRACT
Neuroserpin (NS) is an inhibitory protein belonging to the serpin family and involved in several pathologies, including the dementia Familial Encephalopathy with Neuroserpin Inclusion Bodies (FENIB), a genetic neurodegenerative disease caused by accumulation of NS polymers. Our Molecular Dynamics simulations revealed the formation of a persistent salt bridge between Glu289 on strand s2C and Arg362 on the Reactive Centre Loop (RCL), a region important for the inhibitory activity of NS. Here, we validated this structural feature by simulating the Glu289Ala mutant, where the salt bridge is not present. Further, MD predictions were tested in vitro by purifying recombinant Glu289Ala NS from E. coli. The thermal and chemical stability along with the polymerisation propensity of both Wild Type and Glu289Ala NS were characterised by circular dichroism, emission spectroscopy and non-denaturant gel electrophoresis, respectively. The activity of both variants against the main target protease, tissue-type plasminogen activator (tPA), was assessed by SDS-PAGE and chromogenic kinetic assay. Our results showed that deletion of the salt bridge leads to a moderate but clear reduction of the overall protein stability and activity.

No MeSH data available.


Related in: MedlinePlus

Thermal and chemical stability.(a) Far-UV CD spectra of WT NS (black lines) and E289A NS (red lines) at 25 °C before (solid lines) and after the temperature ramp from 25 to 90 °C at 1 °C/min. (b) First moment of emission spectra upon excitation at 275 nm as a function of Gnd HCl concentration: WT NS (black circles) and E289A NS (red circles). The points are averages over triplicate experiments. Solid lines are sigmoidal fit.
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f6: Thermal and chemical stability.(a) Far-UV CD spectra of WT NS (black lines) and E289A NS (red lines) at 25 °C before (solid lines) and after the temperature ramp from 25 to 90 °C at 1 °C/min. (b) First moment of emission spectra upon excitation at 275 nm as a function of Gnd HCl concentration: WT NS (black circles) and E289A NS (red circles). The points are averages over triplicate experiments. Solid lines are sigmoidal fit.

Mentions: The correct folding of E289A NS was verified by far-UV circular dichroism (CD), a technique able to quantify the secondary structure content of protein folds. Figure 6a shows the CD spectra of both WT and E289A NS, which exhibited no differences, in agreement with the conformational stability in MD simulations. Also, the thermal stability of both NS variants, monitored by CD at 216 nm during a thermal ramp, displayed no relevant differences. Indeed, the main transition typically observed in a thermal ramp for NS is due to the formation of latent or polymer NS, rather than to thermal unfolding1821. Figure 6a also shows the spectra for NS after a temperature ramp up to 90 °C, and therefore mainly related to polymeric NS, coherent with previously reported spectra of NS polymers23. Again, no significant changes were observed in the folding of both variants. The chemical stability of the native conformation was assessed by incubating NS with different amounts of a denaturant agent, guanidine hydrochloride (Gnd-HCl), and monitoring the intrinsic fluorescence of aromatic residues. In particular, the photoluminescence emission of tryptophans depends upon their exposure to a polar environment31; hence, the red-shift of the emission band (or more concisely of its first moment) can be used as a measure of protein unfolding32. Figure 6b shows that both E289A and WT NS presented a two-step unfolding transition at 0.8 M and 2.6 M Gnd-HCl concentration, as previously observed for WT NS1518. In comparison to WT NS, E289A NS exhibited a decrease of cooperative behaviour to reach the final unfolding state.


The stability and activity of human neuroserpin are modulated by a salt bridge that stabilises the reactive centre loop.

Noto R, Randazzo L, Raccosta S, Caccia S, Moriconi C, Miranda E, Martorana V, Manno M - Sci Rep (2015)

Thermal and chemical stability.(a) Far-UV CD spectra of WT NS (black lines) and E289A NS (red lines) at 25 °C before (solid lines) and after the temperature ramp from 25 to 90 °C at 1 °C/min. (b) First moment of emission spectra upon excitation at 275 nm as a function of Gnd HCl concentration: WT NS (black circles) and E289A NS (red circles). The points are averages over triplicate experiments. Solid lines are sigmoidal fit.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4556959&req=5

f6: Thermal and chemical stability.(a) Far-UV CD spectra of WT NS (black lines) and E289A NS (red lines) at 25 °C before (solid lines) and after the temperature ramp from 25 to 90 °C at 1 °C/min. (b) First moment of emission spectra upon excitation at 275 nm as a function of Gnd HCl concentration: WT NS (black circles) and E289A NS (red circles). The points are averages over triplicate experiments. Solid lines are sigmoidal fit.
Mentions: The correct folding of E289A NS was verified by far-UV circular dichroism (CD), a technique able to quantify the secondary structure content of protein folds. Figure 6a shows the CD spectra of both WT and E289A NS, which exhibited no differences, in agreement with the conformational stability in MD simulations. Also, the thermal stability of both NS variants, monitored by CD at 216 nm during a thermal ramp, displayed no relevant differences. Indeed, the main transition typically observed in a thermal ramp for NS is due to the formation of latent or polymer NS, rather than to thermal unfolding1821. Figure 6a also shows the spectra for NS after a temperature ramp up to 90 °C, and therefore mainly related to polymeric NS, coherent with previously reported spectra of NS polymers23. Again, no significant changes were observed in the folding of both variants. The chemical stability of the native conformation was assessed by incubating NS with different amounts of a denaturant agent, guanidine hydrochloride (Gnd-HCl), and monitoring the intrinsic fluorescence of aromatic residues. In particular, the photoluminescence emission of tryptophans depends upon their exposure to a polar environment31; hence, the red-shift of the emission band (or more concisely of its first moment) can be used as a measure of protein unfolding32. Figure 6b shows that both E289A and WT NS presented a two-step unfolding transition at 0.8 M and 2.6 M Gnd-HCl concentration, as previously observed for WT NS1518. In comparison to WT NS, E289A NS exhibited a decrease of cooperative behaviour to reach the final unfolding state.

Bottom Line: Further, MD predictions were tested in vitro by purifying recombinant Glu289Ala NS from E. coli.The thermal and chemical stability along with the polymerisation propensity of both Wild Type and Glu289Ala NS were characterised by circular dichroism, emission spectroscopy and non-denaturant gel electrophoresis, respectively.Our results showed that deletion of the salt bridge leads to a moderate but clear reduction of the overall protein stability and activity.

View Article: PubMed Central - PubMed

Affiliation: National Research Council of Italy, Institute of Biophysics, Palermo, Italy.

ABSTRACT
Neuroserpin (NS) is an inhibitory protein belonging to the serpin family and involved in several pathologies, including the dementia Familial Encephalopathy with Neuroserpin Inclusion Bodies (FENIB), a genetic neurodegenerative disease caused by accumulation of NS polymers. Our Molecular Dynamics simulations revealed the formation of a persistent salt bridge between Glu289 on strand s2C and Arg362 on the Reactive Centre Loop (RCL), a region important for the inhibitory activity of NS. Here, we validated this structural feature by simulating the Glu289Ala mutant, where the salt bridge is not present. Further, MD predictions were tested in vitro by purifying recombinant Glu289Ala NS from E. coli. The thermal and chemical stability along with the polymerisation propensity of both Wild Type and Glu289Ala NS were characterised by circular dichroism, emission spectroscopy and non-denaturant gel electrophoresis, respectively. The activity of both variants against the main target protease, tissue-type plasminogen activator (tPA), was assessed by SDS-PAGE and chromogenic kinetic assay. Our results showed that deletion of the salt bridge leads to a moderate but clear reduction of the overall protein stability and activity.

No MeSH data available.


Related in: MedlinePlus