The secondary structure of the insect defensin A depends on its environment. A circular dichroism study

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Introduction
Insect defensins are inducible antibacterial proteins produced in response to bacterial injuries and secreted in the insect hernolymph (1].Defensin A has been iso lated and characteriwd from the larvae of the fleshfly Phormia terranovae [2].It is a small (40 residues) cationic prote i n, representative of a large family of insect defense proteins.Its conformation in water has already been determined by using 20 lH-NMR asso ciated with modeling techniques [3,4].Defensin A is organized i n three distinct regions: a C-terminal anti parallel (3-sheet linked to an amphipatic a-helix via two S-S bridges and a N-terminal loop linked to the � sheet by the third S-S bridge (fig 1).Defensin A be haves as an amphipathic protein able to spread as a monolayer at the air/water interface, and interacts with membrane lipids [5] by forming voltage depen dent channels [6].
Here we report circular dichroism (CD) data showing that the secondary structure of defensin A depends on the solvent nature and on the protein concentration, pH and presence of salts in aqueous solutions.

Materials and methods
Recombinant insect defensin A was prepared by Transgene (Strasbourg, France).Pure water was obtained from a Millipore (Milli Q) apparatus.Methanol and acetonitrile were purchased from BDH and hexaftuoroisopropanol (HFIP) from Merck.
Concentrations of defensin A were calculated from the absorbance at 275 nm using E = 1400 M-1 cm-t for the single Tyr29 chromophore.CD spectra were recorded at room tempe rature on a Jobin-Yvon IV autodichrograph using 0.1or0.01cm path length in older to measure absorbance values less than 1 on the whole range of wavelengths.Optical activities were reported as ellipticity per amino acid residue: 9 ,.. (unit: deg cm2 dmol-1).The a-helical content was roughly estimated from CD data according to the formula: % a-helix = 922i/ 3298x(-10) given by Zhong and Johnson on the basis of a 26-protein data set [7}.

Results
Nature of the solvent CD spectra of defensin A in various solvents are pres ented in figure 2. In acetonitrile the spectrum was cha� racterized by a weak positive band near 200 nm and a weak negative band centered at 228 nm.The spectrum obtained in water presented a negative band at 207 nm and a shoulder near 221 nm.Spectra obtained in HFIP and methanol had comparable shapes: a negative band centered near 208 nm (210 nm for methanol) and a negative shoulder near 218 nm (221 nm for methanol).They correspond to 15% for the turns, 25% for the strands, 27% for the a-helix, and 33% for the mobile loop.
Experimental conditions were: 5 to 9% defensin A in water, pH 4.9, temperature from 22 to 33°C.
The intensity of the negative dichroic bands increased in the following order: acetonitrile < water < methanol < HFIP.At a fixed concentration of 3.2 x 10-5 M and according to the ellipticity at 222 nm, the a-helix content was estimated to be about 15% in water and 20% in HFIP [7].ellipticity (-13% for 5 mM CaCl2) (fig Sa).From all these features, the ellipticity appears related to the presence of salts with a possible specific effect of Ca2+, but this independent from the ionic strength change.

Discussion
This study illustrates the great sensitivity of the secon dary structure of defensin A to environmental condi tions.CD spectra show clearly the solvent dependence of the helical content as monitored by l9222f.It is not surprising that the higher helical content was observed in HFIP since this solvent is known as a structure promoting solvent [8] in contrast to acetonitrile in which defensin A is weakly structured.Intermediate situations which clearly demonstrated the presence of an a-helix were observed for water and methanol.The propensity of defensin A to self-associate was previously demonstrated both by surface tension mea surements (M aget-Dana, Ptak, in preparation) and by fluorescence polarization experiments of the single Tyr residue as a function of viscosity (Talbot JC, per sonal communication).In this study, the dependence of the helicity percentage on defensin concentration clearly indicates that oligomerization plays a major role on the secondary structure formation.A helical content of about 20% at 10-3 M as extracted from the present study is in good agreement with the 25% value determined unambiguously from NMR data in the same range of concentrations [3].It is likely that this would be the maximum amount of helical content induced by the defensin oligomerization.In dilute solutions, we can assume that the helix fragment (16-20) between the two S-S bridges is invariant and corresponds to the • 10% of a .. helix content found at 10-5 M. It can be noticed that the well resolved signals detected in NMR experiments carried out at mM concentrations [3] ruled out the presence of large defensin A oligomers.Therefore, the evolution of the dichroic intensity as a function of defensin A concen tration would more cor!�spond to an increased amount of small oligomers than to oligomers growing in size .
Secondary structure is also very sensitive to other environmental parameters such as pH or the presence of mono-and divalent cations.As far as the pH is concerned, the maximum helical content was detected in the pH range 7-8 corresponding to the deprotona tion of the two His residues (pK • 7).From NMR data [3), His13 was located just before the beginning of the helix whereas His 19 belonged to the first tum of the helix.Thus, the ionisation state of the His residues may have a great influence on the secondary structure of defensin A. The other ionisable group involved in the helix (Arg23) is always protonated (pK 11111 12) in the pH range of this study.We can also consider that, in addition to a direct influence on defensin A folding, the pH of the solution might affect, as a first event, the assoc iated state of defensin and, as a consequence, the helix content.This is consistent with the observation that the pH for the maximum helicity corresponds also nearly to the isoelectric point of defensin A ( calcu lated p/ • 8.3).At this pH the electrostatic repulsions between defensin A molecules are weak and self-asso ciation should be favoured.Besides, the bell-shape of the curve (fig 4b) is in favour of a relation between the global charge of the protein and the helicity.Finally, the maximum helicity corresponds to a pH range where His residues are deprotonated and where the global charge of defensin A is weak.The KCI effect might be analyzed with respect to the self-association process as well.As a matter of fact, it has been shown that defensin A has less tendency to self-associate in the presence of monovalent salts [9].Here, the loss of ellipticity observed in the presence of KCI might be a consequence of the dissociation of defensin A oligo mers.
The optimum antibacterial activity has been observed at a pH ranging between 7.5 and 8 [6].This is the pH range for which we found a maximum helical content.Therefore, the biological activity of defensin A seems to be closely dependent on its secondary structure and may be on its autoassociation state since both appeared correlated.

Fig 1 .
Fig 1.Primary sequence of defensin A. Structure elements as determined by 2D IH-NMR and molecular modeling are indicated.They correspond to 15% for the turns, 25% for the strands, 27% for the a-helix, and 33% for the mobile loop.Experimental conditions were: 5 to 9% defensin A in water, pH 4.9, temperature from 22 to 33°C.

Fig 4 .
Fig 4. Effect of pH on the secondary structure of defensin A. a. CD spectra of defensin A as a function of pH in 5 mM Tris buffers.From top to bottom: pH 3.3, 10.6, 4.9, and 7.5.Defensin A concentration: 2 x 10-s M .b.192221 as a function of pH.
Fig S. Effect of salts on the secondary structure of defensin A. a. CD spectra of defensin A as a function of KCl concentration. 2 x 10-s M defensin A in 5 mM Tris (pH 7.5).KCI concentration from bottom to top: 0, 5, 10, and 100 mM.(---) additional effect of 5 mM CaCl 2 (100 mM KCI + 5 mM CaCii).b. 1922 2 1 as a function of KCl concen tration.