Food Protein – Derived Peptides as Calmodulin Inhibitors
3. Flaxseed Protein – Derived Peptides Flaxseed is recognized as an excellent source of
omega - 3 fatty acids as well as bioactive phenolic compounds. In addition to these properties, however, fl axseed proteins have desirable levels of hydropho- bic and positively charged amino acids that could enhance the production of CaM - binding peptides through enzymatic hydrolysis. Flaxseed proteins were hydrolyzed with alcalase, and the resultant peptide - rich digest was recovered as a supernatant after centrifugation of the reaction mixture (Omoni and Aluko 2006a ). The supernatant was passed through a 1 - kDa ultrafi ltration membrane to isolate low molecular weight peptides in the permeate, which was then freeze - dried. The freeze - dried per- meate was dissolved in ammonium acetate buffer,
I in causing protein unfolding in nNOS (Table 5.2 ).
In fact at a relatively high level of fraction II, there was a reduction in fl uorescence intensity, indicating some structural adjustment (probably folding) that moved the tryptophan residues into the enzyme protein core. Such an effect could be the result of excessive binding of water molecules by the highly charged peptides such that the enzyme molecule is deprived of appropriate solvation and hence forma- tion of a more closed structure. It is evident, there- fore, that the peptides with high density of positively charged groups as shown for fraction II of the fl ax- seed peptides could have substantial effects on enzyme protein structure accompanied by reduction in catalytic effi ciency.
The effect of the fl axseed protein – derived pep- tides on structural and catalytic properties of eNOS was also investigated. Unlike nNOS, the inhibitory effect of fraction I peptides against eNOS was com- pletely overcome at 50 nM CaM concentration (Omoni and Aluko 2006b ). In contrast, fraction II retained its inhibitory property in the presence of 50 nM CaM level and with greater potency; IC 50 values range from 3.2 to 14.51 mg/ml for fraction I when compared to 0.32 – 1.06 for fraction II. eNOS was inhibited mostly through a mixed type of enzyme inhibition, which suggests that the peptides inhibited catalysis by binding to CaM alone or the CaM/eNOS complex. Inhibition constants (Ki) were 2.13 and 0.01 mg/ml for fraction I and II, respec- tively (Omoni and Aluko 2006b ). When compared to the Ki values obtained for nNOS, it is evident that the fl axseed peptides have greater affi nity for eNOS (low Ki) than nNOS (high Ki). An implication of the results is that when used as a therapeutic product, the fl axseed peptides will affect eNOS to a greater extent than the nNOS.
The effects of fl axseed peptides on CaM - dependent eNOS protein molecule seem to be dif- ferent from those observed for nNOS, though structural changes were greater in the presence of fraction II than fraction I (Table 5.2 ). Since CaM contains no tryptophan residue, the changes obtained at 295 nm represent the effects of peptides on eNOS protein molecules. The fl axseed peptides did not produce any substantial change in the wavelength of this is probably the ideal structure required for catal-
ysis. Addition of fl axseed peptide fractions led to further increases in tryptophan fl uorescence accom- panied by a red shift in wavelength of maximum emission (Table 5.2 ), which is consistent with protein unfolding and interactions of the tryptophan residues with a hydrophilic environment. In this regard, fraction II was more effective than fraction
Figure 5.2. Kinetics of the inhibition of neuronal nitric oxide synthase by fl axseed protein – derived cationic peptide frac- tions. V o is the initial velocity, which is the net decrease in absorbance at 340 nm after a 30 - minute reaction time.
(A) Fraction I (42% content of positively charged amino acid residues) peptide concentrations (mg/ml): 0.0, 䊐 ; 1.5, 䊏 ; 2.5, 䊊 ; 3.5, 䊉 . (B) Fraction II (50% content of positively charged amino acid residues) peptide concentrations (mg/ml):
0.0, 䊐 ; 0.25, 䊏 ; 0.75, 䊊 ; 1.0, 䊉 . From Omoni and Aluko 2006a . Reproduced with copyright permission of Springer Publishers.
were more active against eNOS because of the more globular structural conformation that results from the interactions. It is possible that the globular struc- ture shields the active site of eNOS from the sub- strates, which leads to reduced catalysis. In contrast, nNOS structural changes in the presence of fl axseed peptides seem to be mostly of the protein unfolding type, as shown by the > 1.0 Fmax/Fo values (Omoni and Aluko 2006a ). Pea protein – derived peptides also produced increases in Fmax/Fo values for CaMKII interactions (Li and Aluko 2005 ), which are similar to the values obtained for nNOS in the presence of fl axseed peptides. Thus it is reasonable to suggest that protein folding (increased globular conformation) may be a more effective way for inhibitory agents to modulate the catalytic activities of NOS isoforms and CaMKII.
Apart from the effects on enzyme activities and protein structure, the fl axseed peptides were also investigated for their ability to modulate the struc- ture of CaM alone (Table 5.3 ). Similar to the results obtained for the pea protein – derived peptides (Li and Aluko 2005 ), the fl axseed peptides induced protein unfolding (Fmax/Fo > 1.0) when added to CaM, which is also consistent with previous reports.
Therefore, it is possible that one of the multiple maximum emission, which shows that the microen-
vironment of the tryptophan residues in eNOS was not affected (Table 5.2 ). However, fl uorescence intensity was greatly affected by peptides depending on the NOS isoform. At 5 μ g/ml peptide concentra- tion, the fl uorescence intensity of CaM/nNOS complex was about 50% of the value obtained for CaM/eNOS complex, indicating a more open struc- ture for the latter (Omoni and Aluko 2006a, 2006b ).
The Fmax/Fo value (ratio of fl uorescence inten- sity in the presence and absence of peptides) pro- vides a numerical indication of the structural changes that occur when proteins bind to ligands. A value of 1.0 indicates no effect of ligand on protein structure, while a higher value indicates protein unfolding that leads to a loose structural conformation with greater numbers of exposed aromatic groups. In contrast, if the Fmax/Fo ratio is less than 1.0, then there is protein folding into a more globular structure that shields the aromatic group from the environment.
Overall, CaM/eNOS complexes with fl axseed pep- tides produced Fmax/Fo values that were less than 1, whereas similar nNOS complexes had values that were greater than 1.0, except at 5 μ g/ml peptide concentration (Omoni and Aluko 2006a, 2006b ).
Therefore, it is evident that the fl axseed peptides
Table 5.2. Effect of fl axseed protein - derived cationic peptide fractions on the intrinsic fl uorescence of C a 2+ / C a M - dependent nitric oxide synthases. *
Treatment
λ max (nm) Fmax/Fo a
nNOS eNOS nNOS eNOS
None 331 331 — —
CaM 330 334 1.07 0.99
CaM + 12.5 μ g Fraction I b 334 333 1.62 0.97 CaM + 25 μ g Fraction I 337 331 1.96 1.22 CaM + 1.25 μ g Fraction II c 333 333 1.22 0.92 CaM + 2.5 μ g Fraction II 334 330 2.08 0.94 CaM + 5 μ g Fraction II 329 330 0.73 0.85 * Adapted from Omoni and Aluko 2006a, 2006b .
a Excitation wavelength of 295 nm; Fmax and Fo (arbitrary units) represent the maximum fl uorescence intensity of nNOS or eNOS in the presence and absence of peptides, respectively.
b First eluted fraction from SP - Sepharose column and contained 42% (w/w) of positively charged amino acid residues.
c Second eluted fraction from SP - Sepharose column and contained 50% (w/w) of posi- tively charged amino acid residues.
Table 5.3. Effect of fl axseed protein – derived peptides on the intrinsic fl uorescence of calmodulin. *
Treatment λ max (nm) Fmax/Fo a
none 302 —
Ca 2+ 315 1.60
12.5 μ g Fraction I b 342 1.75
25 μ g Fraction I 305 2.75
1.25 μ g Fraction II c 349 9.73 2.5 μ g Fraction II 343 4.61
5 μ g Fraction II 338 1.86
* Adapted from Omoni and Aluko 2006b .
a Excitation wavelength of 275 nm; Fmax and Fo (arbitrary units) represent the maximum fl uorescence intensity of CaM in the presence and absence of Ca 2+ or peptides, respectively.
b First eluted fraction from SP - Sepharose column and contained 42% (w/w) of positively charged amino acid residues.
cSecond eluted fraction from SP - Sepharose column and con- tained 50% (w/w) of positively charged amino acid residues.
Table 5.4. Effect of fl axseed protein – derived peptides on secondary structure fractions of calmodulin. *
Treatment α - Helix (%) β - Sheet (%) β - Turn (%) Random (%)
None 49 13 15 24
Ca 2+ 62 11 13 16
0.5 mg/ml Fraction I a 53 28 4 19
0.5 mg/ml Fraction II b 54 21 6 19
* Adapted from Omoni and Aluko 2006b .
a First eluted fraction from SP - Sepharose column and contained 42% (w/w) of positively charged amino acid residues.
b Second eluted fraction from SP - Sepharose column and contained 50% (w/w) of positively charged amino acid residues.
ways through which the peptides inhibited CaM - dependent enzymatic reactions is excessive unfold- ing such that the CaM protein molecule loses its ability to effectively bind to and activate target enzymes. This is further evident in the results that showed changes in the wavelength of maximum emission depending on the peptide concentration.
When compared to the active form of CaM where the tyrosine residues had maximum emission at 315 nm, all the peptide concentrations (with the exception of 25 μ g/ml of fraction I) produced emis- sions at wavelengths of between 338 and 349 nm, indicating a red shift that is typically observed during protein unfolding (Table 5.3 ).
Secondary structure of CaM as measured by cir- cular dichroism (CD) showed that helical conforma-
tion (62%) increased in the presence of calcium when compared to 49% helical conformation for CaM alone (Table 5.4 ). The CD result confi rms pre- vious reports from far - UV data (LaPorte et al. 1980 ; Malencik and Anderson 1982 ) that have showed the need for higher α - helix content if CaM is to effec- tively modulate activity of target enzymes. The α - helix content of CaM was reduced to ∼ 54% when peptides were added to the calcium/CaM complex (Table 5.4 ), confi rming that structural change in CaM protein molecule is a possible mechanism through which peptide - mediated inhibition of CaM - dependent enzymatic reactions can occur. Also important is the fact that addition of calcium reduced the proportion of random structures in CaM but peptides had the opposite effect (Table 5.4 ), which indicates that a certain degree of orderly protein conformation is necessary for CaM to activate target enzymes.
The near - UV data of CaM refl ects the tertiary conformation, which was changed by calcium and peptides as shown by the intensities obtained for phenylalanine and tyrosine. In general, fraction II of the fl axseed peptides produced greater effect on phe- nylalanine intensity when compared to fraction I (Omoni and Aluko 2006b ). Similar results were also obtained upon addition of pea protein – derived pep- tides to CaM (Li and Aluko 2005 ), which again confi rms the fl uorescence data that point to struc- tural changes in CaM structure as a possible mecha- nism for the peptide - induced modulation of CaM activities. The results also confi rm that increased levels of positively charged amino acid residues contribute to greater interactions between the food protein – derived peptides and CaM, ultimately
(Table 5.5 ). This demonstrates that the observed inhibitory effects were due mainly to the ability of pea protein – derived peptides to bind with CaM and inhibit activation of CaMKII. With an increase in CaM concentration, inhibition of CaMKII by frac- tion I peptides was severely reduced, suggesting that the peptides probably compete with the enzyme for binding onto CaM. By contrast, the inhibition of CaMKII activity by fraction II was not substantially infl uenced by the level of CaM present (Table 5.2 ).
Therefore, it is possible that the affi nity between fraction II peptides and CaM was very strong because of the high levels of lysine and arginine, and increasing the levels of CaM could not easily displace the peptides from the enzyme - binding site on CaM molecule. Proof of strong interactions between fraction II and CaM was obtained from the inhibition constant (Ki), which was 0.019 mg/ml when compared to the value of 0.47 mg/ml for frac- tion I (Li and Aluko 2005 ). A lower value of Ki indicates higher affi nity of the peptides for CaM and consequently less activation of CaMKII. Both frac- tions inhibited CaM in a competitive way, suggest- ing that enzyme binding sites on CaM were occupied by the peptides. Km values were signifi cantly higher (p < 0.05) for fraction II, which reinforces the fact that CaMKII activity was inhibited to a much greater extent by high density of positively charged groups than by a lower density.
In order to further determine the molecular basis for the inhibition of CaMKII by pea protein – derived translating to decreased ability of CaM to modulate
target enzymes.