Kroupskaya 1 and L.L. Sidorik 1

1Institute of Molecular Biology and Genetics NAS of Ukraine, 150 Zabolotnogo street,03680, Kyiv, Ukraine

2International Institute of Molecular and Cell Biology, Warsaw, Poland

3Nencki Institute of Experimental Biology, Warsaw, Poland

4Strazhesko Institute of Cardiology MAS of Ukraine, Kyiv, Ukraine The study of the mechanisms of anti-stress response is quite impor- tant for evaluation of heart failure origin and progression and for develop- ment of new effective therapeutic tools based on the apoptotic signaling blockage. Specialized family of anti-stress proteins including molecular chaperons, their co-chaperons, and target proteins plays a critical role in pro- and anti-apoptotic signaling.

The 90 kDa heat shock protein, Hsp90, is one of the most abundant eukaryotic proteins involved in various cellular processes such as protein fold- ing and protein degradation, apoptosis, molecular evolution, etc. and in the regulation of autoimmune diseases including cardiomyopaties. Molecular chaperone Hsp60, acting mainly in mitochondria, is of special interest, since it is capable to form complexes with proapoptotic proteins Bax. A decrease in Hsp60 level in cardiomyocytes is capable to launch the apoptosis. A recently discovered protein Sgt1 is a potential co-chaperon and/or target protein and possibly plays a role in Hsp90-related proteasome degradation of proteins.

Recently we observed changes of expression and cellular localization of Hsp70 in human hearts and identified specific anti-Hsp70 autoantibodies in patients sera bearing acute (myocarditis) and chronic (dilated cardio- myopathy) stages of heart failure. However, our knowledge about the possi-

ble role of Hsp60, Hsp90 and Sgt1 in the development of cardiovascular autoimmune diseases caused by chronic stress such as dilated (DCM) and ischemic cardiomyopathies (ICM) is quite limited.

We have examined the level of anti-Hsp60, anti-Hsp90 and anti-Sgt1 autoantibodies in sera of patients bearing DCM (39 patients), ICM (23 patients) and healthy donors (35) using ELISA. The possible changes of Hsp60, Hsp90 and Sgt1 protein expression have been revealed by Western-blot analysis.

The anti-Hsp90 and аnti-Sgt-1 autoantibodies level was significantly higher in the group of ICM patients in comparison with dilated and healthy ones. The anti-Hsp60 autoantibodies level was significantly higher in DCM patients sera. The increased Hsp60 expression was observed in DCM- affected heart in comparison with ischemic and normal ones since Hsp90 and Sgt1 expression were characterized by significant cellular re- localization rather than cellular content changes.

We suggest that changes in the level of specific anti-HSP autoanti- bodies could serve as possible diagnostic marker for cardiomyopathies of different genesis.

ALTERED CARDIAC TITIN EXPRESSION IN THE PATHOGENESIS OF HYPERTENSION

AND DURING HIBERNATION

E.V. Karaduleva¹, I.M. Vikhlyantsev¹, and Z.A. Podlubnaya^{1, 2}

1Institute of Theoretical and Experimental Biophysics, the Russian Academy of Sciences

2Pushchino State University, Pushchino, Moscow Region, Russia 142290 Gene expression can be controlled at different levels, including mRNA transcription, processing and stability, as well as translation, protein processing and stability, and post-translational modifications. Study of tran- scription and translation is of particular interest for characteristics of muscle functional state. Gene expression patterns for both hibernating phenotype and pathological state are left unstudied so far. In this work we have ana- lyzed changes of titin isoform composition in cardiac muscles of ground squirrels during hibernation and spontaneously hypertensive rats during the development of the disease.

Titin is a giant filamentous protein that forms a separate myofilament system in both skeletal and cardiac muscles. It is involved in assembling the sarcomere during myogenesis, stabilizing its structure, contributes to devel- opment of passive tension, regulation of actin-myosin interaction, partici- pates in regulation of gene expression, protein turnover, ion channel activity and signaling processes. Titin gene located in the chromosome 2 (region 2q31) contains 363 exons encoding 4200kDa protein (38138 amino acid residues). Alternative splicing of titin elastic zone in an I-disc of sarcomere is a basis of a variety of titin isoforms. Cardiac titin is expressed in two iso- forms: short N2B (~3000 kDa) and long N2BA (~3200-3400 kDa), with up

to seven variants of alternative splicing [1, 2].

In our experiments were used: ground squirrels Spermophillus undu- latus - summer active animals (heart temperature of 37°C) and hibernating animals (heart temperatures of 2-4°C), rats - normal rats (Wistar Kyoto) and spontaneously hypertensive rats (SHR). Hibernation was chosen as a unique model of adaptation to stress conditions and heart function depression. Win- ter sleep of Spermophillus undulatus lasts 5-6 months and consists of 1-to-3 weeks’ duration cycles (bouts) with short periods of arousal. During hiber- nation the frequency of respiration and the level of oxidative metabolism drop more than 10- or 100-fold. Upon awakening of animals the transition from almost complete suppression of all physiological processes to normal activity is very rapid (2-3 hours). Physiological systems ought to resist dras- tic hypothermia, hypoxia, ischemia, and oxidative stress. The most striking changes are observed in heart muscle functioning, as the heartbeat rate ele- vates from 4-20 to up to 400 beats per minute during arousal.

Hypertension was chosen as a model of pathology. The line of sponta- neously hypertensive rats (SHR) with elevated arterial pressure was divided into two groups: rats aged 15 weeks, at the early stage of the disease and rats aged 26 weeks, at a later stage of the disease. The Wistar Kyoto rats aged 17 weeks and with normal arterial pressure were used as a control. The research protocol was approved by the local Bioethical Committee. Arterial hyperten- sion, an elevation in the blood pressure in arteries, is an important symptom of the pathological states and diseases accompanied by either resistance to arte- rial blood flow or an increase in the heart output (or by both). A clinical symp- tom of this disease is heart hypertrophy, particularly pronounced in the left ventricle. At the ultrastructure level, individual dystrophic and necrotic lesions of muscle fibers develop in the some part of myocardium. Quantity of the connective tissue overgrows, which leads to focal and diffuse myocardial fi- brosis [3]. At the later stages, arterial hypertension can be complicated by car- diovascular insufficiency and ischemia.

To investigate protein isoforms titin, samples of fresh muscle tissue were incubated for 30 min at a room temperature in the solubilizing solution and SDS-PAGE of these samples was carried out by using agarose- strengthened 2-2.3% polyacrylamide gels according to the method of Tatsumi and Hattori (1995) with our modifications [4]. Immunoblotting of titin with monoclonal antibodies (AB5, 9D10 and T11) was carried out in the way de- scribed in [5]. Densitometry of protein bands and estimation of the molecular mass of titin bands in gel were performed using TotalLab software (Phoretix).

Bands of myosin heavy chains (MHC) (205 kDa), nebulin (770 kDa), and titin-2 (~2300 kDa) were used as standards for estimating the molecular weights. Heart RNA was isolated using the Total RNA Fatty and Fibrous Tis- sue Kit (BIO-RAD, #732-6830) according to the producer’s protocol. Reverse transcription was carried out by standard procedure using the MINT-Universal

cDNA synthesis kit (Evrogen, #SK002). RT-PCR primers were designed on the basis of rat genomic sequences (Fw 5'-ccaacgagtatggcagtgtca-3' and Rv 5'- tgggttcaggcagtaatttgc-3' for exons 50-219 (N2B titin isoform); Fw 5'- cggcagagctcagaatcga-3' and Rv 5'-gtcaaaggacacttcacactcaaaa-3' for exons 107108 (all N2BA titin isoforms)) [6]. Quantitative real-time PCR was con- ducted in a thermocycler DT-322 (DNA-Technology, Russia) with SYBR Green using the Tersus PCR kit (Evrogen, #PK021). PCR products were elec- trophoretically resolved in 5% polyacrylamide gel and visualized using ethy- dium bromide staining.

The results of the qRT-PCR showed a four-fold decline in mRNA con- tent for N2BA and a two-fold decline for N2B titin isoform in the hearts of hibernating ground squirrels as compared to that in the hearts of summer ac- tive animals. The overall decrease in mRNA level may be explained by re- pressed transcription or mRNA degradation in the cell during hibernation. It is known that enlarged methylation of promoters is a cause of differences in gene expression as a result of reduced transcription, rather than increased deg- radation [7]. Moreover, mRNA transcripts are protected from degradation by the RNA binding proteins, showing overexpression during hibernation, and by the long-sized Poly(A)tails which stabilize them [8]. Many genes are down- regulated during winter sleep and titin gene is no exception.

The study of titin at protein level also revealed the small decrease in the relative content of titin in cardiac muscles of hibernating ground squir- rels. At the same time, we have shown a two-fold increase in N2BA/N2B ratio in the hearts of ground squirrels during torpor in comparison with the ratio found in the hearts of nonhibernating summer animals. In view of the inhibited translation during winter sleep which may arise from reduced mRNA availability and inactivation of translational factors of initiation (eIF- 2) and elongation (eEF-2) by reversible phosphorylation [9], the discrepancy in protein and mRNA levels may be explained by an increased synthesis of N2BA-titin during the process of preparation for hibernation and going into the torpid state. This assumption was fully confirmed by our investigations, which showed that the increase in the content of the long N2BA-titin iso- form in the myocardium of ground squirrels occurred before hibernation period. It is known that the predominance of the long N2BA-titin isoform determines a higher degree of elasticity and, consequently, extensibility of the myocardium [10], which increases the force of heart contractions ac- cording to the Frank–Starling law [11]. We believe that the increase of the content of the long N2BA-titin isoform fulfils the adaptation function and facilitates the release of more viscous blood from heart chambers during hibernation. The enhanced extensibility of the myocardium in hibernating ground squirrels can also adapt the myocardium to greater mechanical loads during arousal when the heart rate reaches 400 beats/min and more.

In contrast to adaptive seasonal changes of isoform titin expression in

the hearts of hibernating mammals, development of disease in the hearts of SHR was attended by significantly drop (three times less) of titin portion as compared to myosin-heavy chains in rats aged 15 weeks (an early stage of the disease) [12]. Surprisingly, the data for qRT-PCR showed a four- and seven-fold increase in mRNA level, respectively for N2B and N2BA iso- forms of titin, in the hypertrophic heart in comparison with their levels in a normal one. This inverse relationship between the levels of gene expression and protein synthesis may be an effect of “anticipatory up-regulation of genes”, when the level of gene transcripts is elevated but there is no in- crease in the corresponding protein product [13]. We see the growth of mRNA-titin level as a sign of a compensation stage of disease.

What happens subsequently during the pathogenesis of hypertensive heart disease?

We have revealed that in the left ventricle of SHR aged 26 weeks at a later stage of the disease in accordance with data about titin destruction the mRNA level become three times lower for N2BA-isoform and four times lower for N2B-isoform.

Thus, our results indicate that the development of a pathological process in the hypertrophic heart muscle was accompanied with strong degradation of titin and depression of its expression at a later stage of the disease. Undoubtedly, these changes, combined with cardiovascular insuf- ficiency and ischemia, contribute to further aggravation of disease state. In contrast to pathological changes of SHR, adaptive changes occurring in torpid ground squirrels are aimed at increasing the portion of the long iso- form of cardiac N2BA-titin used for regulating the heart function. More- over, the decline in RNA and in protein synthesis during hibernation may be regarded as the accommodation for minimization of energetic expendi- tures.

We are sincerely grateful to Prof. Olga N. Ozoline and Dr. Maria N.

Tutukina (Institute of Cell Biophysiсs RAS) for using of the thermocycler (DT-322, DNA-Technology, Russia) and for expert technical assistance and valuable discussion.

This research was supported by grant of the Russian Foundation for Basic Research № 07-04-00479 and by grant of the President of the Russian Federation “Leading Scientific Schools” № 217.2008.4.

References

1. Freiburg А., Trombitas K., W. Hell W. et al., Circ. Res., V. 86, P. 1114-1121 (2000).

2. Bang M. L., Centner T., Fornoff F. et al., Circ. Res., V. 89, P. 1065-1072 (2001).

3. Brilla, C.G., Janicki, J.S., and Weber. K.T., Circ. Res., V. 69, P. 107–115 (1991).

4. Vikhlyantsev I.M., Podlubnaya Z.A., Biofizika, V. 52, No 6, P. 1030-1040 (2007).

5. Vikhlyantsev I.M., Karaduleva E.V., Podlubnaya Z.A. et al., Biophysics, V. 53,

№6: 592-597 (2008).

6. Opitz C.A., Kulke M., Leake M.C., Proc. Natl. Acad. Sci. USA, 100: 12688-12693 (2003).

7. Clifford C.P. and Nunez D.J., Cardiovascular Res., 38: 736-743 (1998).

8. Knight J.E., Narus E.N., Martin S.L. et al., Mol. Cell. Biol., V. 20, №17: 6374- 6379 (2000).

9. Frerichs K.U., Smith C.B., Brenner M. et al., Proc. Natl. Acad. Sci. USA, 95:

14511-14516 (1998).

10. Cazorla O., Freiburg A., Helmes M. et al., Circ. Res., 86: 59-67 (2000).

11. Granzier H. and Labeit S. // Exerc. Sport Sci. Rev., V. 34, No 2, P. 50-53 (2006).

12. Vikhlyantsev I.M., Podlubnaya Z.A., Karaduleva E.V. et al., Dokl. Akad. nauk RF, V. 417, №3: 403-406 (2007).

13. Storey K.B., Adv. Exp. Med. Biol., 543: 21-38 (2003).

EFFECTS OF HYDROGEN SULFIDE ON FROG MYOCARDIUM AFTER INHIBITION

OF ATP-DEPENDED POTASSIUM CHANNELS N.N. Khaertdinov, A.V. Yakovlev., G.F. Sitdikova Kazan State University, 18, Kremlevskii str., Kazan, 420008, Russia

Hydrogen sulfide (H2S) has been best known for decades as the toxic gas dubbed “gas of rotten eggs”. Recently it was shown that H2S endogenously generated from cysteine in a reaction catalysed by cys- tathionine β-synthase (CBS) and/or cystathionine γ-lyase (CSE) (Li L, Moore PK, 2008). By analogy to other endogenous gaseous molecules, such as nitric oxide (NO) and carbon monoxide (CO) H2S at physiological concentrations regulates cardiovascular functions in different animals (Li, Moore, 2008, Dombkowski et al., 2005). The vasodilator effect of H2S has been ascribed to its ability to open ATP-sensitive potassium channels (KATP channels) in vascular smooth muscle cells (SMCs) (Zhao et al., 2001). The purpose of the present study was to assess the role of ATP- depended potassium channels in the effects of H2S on frog myocardium contractility.

Methods

Experiments were held on frog heart ventricle using Powerlab 14s set-up. Muscular fiber prepared from ventricle had length 4-6 mm and diameter 1 mm. Isolated stripes were plunged vertically in 20 ml reservoir with Ringer solution for cold-blooded animals, containing in mM: 118,0 NaCl, 2,5 KCI, 1,8 CaCl, 10 Trizma (pH - 7,3-7,4 T=200C). Basal tip fas- tened to rubber block, upper tip fastened to non-rusting core joined with transducer perceptibility 0-50 g (AD Instrument). Preparation was stimu- lated through two silver-plated electrodes by electric impulses with dura- tion of 5 ms, amplitude 10 V with frequency 0,1 Hz. Muscle contractions

were recorded by Chart program. Sodium hydrogen sulfide (NaHS) used as donor of H2S, because in water medium it dissociated to Na2+ and HS-, then HS- bound with H+ and form undissotiated H2S. In neutral solution, one-third of NaHS exists as H2S and the remaining two-thirds are present as HS− (Beauchamp R.O. et al., 1984). This provides a solution of H2S at a concentration that is about 66% less compared to the original concentra- tion of NaHS. Glibenclamid was used as inhibitor of ATP-dependent po- tassium channels and was dissolved in DMSO to make stock solution. The final concentration of DMSO in bath solution did not exceed 0.1%. All used chemicaks were obtained from Sigma (USA).

Results

Bath application of NaHS in concentration 100 mkM decreased the am- plitude of contraction of isolated ventricle stripes. By 3 min of experiment the amplitude decreased by 95,22±1,91 of control and achieved 76,88±1,64 by 20 min of application (n=8, p<0.05) (fig.1 A, B). The effect of NaHS was reversi- ble and dose-dependent, EC50 = 102 mkM (fig.1 B). Thus, NaHS exerts negative inotropic effect to the frog heart contractility. In recent studies, a vasodilator action of H2S has also been reported in lower vertebrates, such as trout, pacific hagfish, sea lamprey, sandbar shark, marine toad, American alligator, and Pekin duck. In contrast to mammalian other classes of vertebrae

Fig. 1. The negative inotropic effect of NaHS in frog myocardium.

A – The original representative traces showing the decrease of amplitude of contraction of myocardium tissue, B – The decrease of force of contraction of the ventricular stripe by NaHS in concentration 100 mkM, B – The dose- dependent of negative inotropic effect of NaHS.

showed the different H2S effects on the smooth muscle. It was shown, that H2S led to both the contraction and relaxation of the isolated vascular muscu- lar cells at the fish and amphibian (Dombkowski et al., 2005).

One of the possible mechanisms of H2S effect at the vascular sys- tem may be the activation of КАТP channels H2S opens KATP channels in vascular SMCs, cardiomyocytes, pancreatic β-cells, gastro-intestinal SMCs, thereby regulating vascular tone, myocardial contractility, insulin secretion, gastrointestinal contractility (Ali et al., 2006, Cheng et al., 2004, Moore et al. 2003). The activation of the КАТP channels leads to the hyperpolariza- tion of the plasmatic membrane, which in turn inhibits the potential- dependent Са⁺-channels and decreases the vascular tension (Standen et al., 1989, Zhao et al., 2001). In order to inhibit KATP channels we used glibenclamid in concentration 50 mkM. Glibenclamid did not change the contraction force of ventricle stripes in control conditions during 20 min of application (fig.2 A) (n=15, p>0.05). NaHS in this condition increased the contraction force by 3 min of experiment to 110,18±2,6, then the am- plitude decreased by 76,62±0,87% (n=9, p<0.05). The reduction of con- traction force was the same as in control condition.

Thus, there were no any changes in contractility of myocardium after inhibition of KATP channels, probably these kind of channels did not play significant role in regulation of myocardium contractility in normal conditions compare to vessels. However, the inhibition of KATP channels induced the changes in NaHS action. We observed the increase of force tension in first 3-5 min of application, following by the decrease of contractility by 20 min of experiment, which was the same as in control. It was suggested that NaHS had several targets of action in frog myocardium and the late negative inotropic effect did not depend on the activity of АТP-dependent К⁺-channels.

Fig. 2. Effect of NaHS after inhibition of KATP channels.

A – Effect of glibenclamid in concentration 50 mkM on contractility of the ventricular stripe of frog myocardium, B – Effect of NaHS (100 mkM) on force of contraction after preliminary application of glibenclamid (50 mkM).

Acknowledgement: This study was funded by RFBR №09-04-00748 and Ministry of Education № 2.1.1/786.

References

1. Dombkowski, R.A., Russell, M.J., Schulman, A.A., Doellman, M.M., and Olson, K.R. Vertebrate phylogeny of hydrogen sulfide vasoactivity. Am. J. Physiol. Regul.

Integr. Comp. Physiol. 2005, 288, R243 – R252.

2. Zhao W., Zhang J., Lu Y., Wang R. The vasorelaxant effect of H2S as a novel en- dogenous gaseous KATP channel opener. EMBO J. 2001; 20(21): 6008-16.

3. Standen N.B., Quayle J.M., Davies N.W., Brayden J.E., Huang Y., Nelson M.T.

Hyperpolarizing vasodilators activate ATP-sensitive K+ channels in arterial smooth muscle. Science 1989; 245: 177–180.

4. Ali M.Y., Ping C.Y., Mok Y.Y. et al. Regulation of vascular nitric oxide in vitro and in vivo; a new role for endogenous hydrogen sulphide? Br. J. Pharmacol. 2006;

149(6): 625-34.

5. Cheng Y., Ndisang J.F., Tang G., Cao K., Wang R.. Hydrogen sulfide-induced re- laxation of resistance mesenteric artery beds of rats. Am. J. Physiol. Heart Circ.

Physiol. 2004; 287(5): H2316-23.

6. Beauchamp R.O. Jr., Bus J.S., Popp J.A., Boreiko C.J., Andjelkovich D.A. A criti- cal review of the literature on hydrogen sulfide toxicity. Crit. Rev. Toxicol. 1984.

V.13. P. 25-97.

7. Zhao W., Zhang J., Lu Y., Wang R. The vasorelaxant effect of H(2)S as a novel endogenous gaseous K(ATP) channel opener. EMBO J., 2001, 20:6008–6016.

8. Li L., Moore P.K. Putative biological roles of hydrogen sulfide in health and dis- ease: a breath of not so fresh air? Trends Pharmacol. Sci. 2008,29:84–90.

CONFORMATION OF ACTIN MONOMER IN CRYSTALS AND IN SOLUTION

S Yu. Khaitlina Institute of Cytology RAS,

Tykhoretsky pr., 4, Sankt-Peterburg 194064, Russia

Actin belongs to a superfamily of ATP-binding proteins that also in- cludes actin-related proteins, prokaryotic actin homologues, Hsp70-related proteins, hexokinases and other kinases. These proteins share a common fold with two large domains connected by a hinge and a nucleotide binding site located in the cleft at the domain interface (Bork et al., 1992; Kabsch, Holmes, 1995; Hurley, 1996; Egelman, 2003; Simanshu et al., 2005). Enzymes of this family catalyze ATP transfer or hydrolysis coupled to a large conformational change in which the two domains undergo a transition between the open and closed nucleotide binding cleft (Kabsch, Holmes, 1995; Hurley, 1996). Simi- lar conformational changes have been suggested to occur in G-actin (Tirion, ben-Avraham, 1993; Page et al., 1998) and F-actin (Lorenz et al., 1993; Miki, Koyama, 1994; Tirion et al., 1995). Consistently with this suggestion, the open/closed transition of the nucleotide binding cleft upon replacement in G-

actin of tightly bound cation or nucleotide can be evident in significantly faster release of ADP compared to ATP in both Ca- and Mg-actin (Kinosian et al., 1993), in a diminished susceptibility of segment 61-69 to trypsin in Mg- ATP-actin versus Ca-ATP-actin and a higher susceptibility of these residues in Mg-ADP-actin versus Mg-ATP-actin (Strzelecka-Golaszewska et al., 1993) as well as in a strong protection of the cleft-located residues in Mg-actin com- pared to Ca-actin against oxidative modifications by hydrohyl radicals gener- ated by synchrotron X-ray radiolysis (Guan et al., 1993). The results of these studies imply a high level of structural dynamics of actin molecule in solution, with the cleft closure/opening being favorable for actin polymerization and monomer dissociation, respectively (Strzelecka-Golaszewska, 2001).

On the other hand, most actin crystal structures available to date show an invariant conformation with the nucleotide binding cleft closed independent of the type of the tightly bound cation and nucleotide. The only open actin conformation was found in profilin-bound actin crystals (Chik et al., 1996). This conformation may, however, be maintained by profilin rather than be inherent to actin itself because removal of profilin at molecu- lar dynamics simulations transformed this open conformation to the closed one (Minehardt et al., 2006; Splettstoesser et al., 2009). Only closed mono- mer conformation has also been detected in the molecular dynamics analysis of the ATP- and ADP-actin crystal structures (Zheng et al., 2007; Dalhaimer et. al., 2008). Most strikingly, the crystal structure of G-actin cleaved by ECP 32/grimelysin between Gly 42 and Val 43 (Khaitlina et al., 1991), which increases accessibility of the nucleotide containing cleft to proteolysis and accelerates the nucleotide exchange indicating that ECP actin has a more open conformation than the non-modified protein (Strzelecka- Golaszewska et al., 1993; Khaitlina and Strzelecka-Golaszewska, 2002), was found to be in a typical closed conformation similar to all other actin crystal structures available (Klenchin et al., 2006). Consistently with the closed ECP actin conformation in the crystals, the nucleotide cleft stayed closed after the protein backbone was broken between Gly 42 and Val 43 at molecular dynamic simulation (Dalhaimer et al., 2008). Thus, the high plas- ticity exhibited by G-actin in solution is not detected upon crystallization.

This may be due to a specificity of actin that can be crystallized in only one conformation or to the crystallization conditions including the presence of precipitants and salt indispensable for crystallization (Klenchin et al., 2006).

Usually various ligands and salt are also included in solvent at molecular dynamics simulations to mimic physiological conditions (Dalhaimer et al., 2008; Splettstoesser et al., 2009; Pfaendtner et al., 2009 )

Effects of precipitants such as polyethylene glycol on actin structure are still not clearly understood. It has been shown however that polyethylene glycol enhances the extent and rate of actin polymerization (Tellam et al., 1983; Strömqvist et al., 1984) and tends to protect the native structure of G-