BACKGROUND
sodium dodecyl sulfate  is a highly effective and widely used protein denaturant  <cit> .
our previous work has shown that amphipathic solvents, like 2-methyl- <dig> -pentanediol , a commonly used precipitant for crystallization studies  <cit> , can protect proteins from sds denaturation, and in several cases can drive the transition from the sds-denatured state to a functional folded state  <cit> .
this protecting effect of mpd is observed with a wide range of proteins including membrane proteins and soluble enzymes, and is not applicable if proteins are denatured in guanidine or urea, two other denaturant agents.
in the case of hen egg-white lysozyme, sds concentrations above  <dig>  mm abolished the activity of the enzyme in the absence of mpd.
however, in  <dig> m mpd, the activity was preserved in the presence of sds.
in addition, when the enzyme was first denatured with sds for  <dig> hours prior to adding mpd, full enzymatic activity was recovered in  <dig> m mpd following a further  <dig> hour incubation period.
although the persistence of the enzyme activity in the sds/mpd mixture was clearly established, preservation of its structure was only speculative until now.
in the present contribution, a detailed x-ray study addresses the pending question.
crystals of hen egg-white lysozyme could be grown for the first time in a sds/mpd medium, providing support that adding mpd to proteins can avoid sds denaturation.
the obtained complexes of sds-lysozyme and mpd-lysozyme give some insights in the interplay of protein-sds and protein-mpd interactions.
a previous report described the structure of cross-linked lysozyme crystals soaked in sds solutions  <cit> .
in the latter, unlike both structures described in this paper, sds was found at three different locations inside the protein inducing a major structural deformation in the interior of lysozyme.
RESULTS
crystallization
crystals of hen egg-white lysozyme solubilized in  <dig> mm sds, a usually denaturing condition, and  <dig> m mpd were obtained, without any cross-linking, contrary to the study of yonath et al.
<cit> , in two different conditions .
tetragonal crystals, called "form i" crystals, were obtained from a classical sodium acetate solution at ph  <dig> .
in this condition, few mpd, slightly volatile, is expected to remain in the drop after equilibration of the protein droplet with the mpd-free reservoir solution .
to enhance the concentration of sds and mpd into the drop, and taking into account results from the literature  <cit> , crystals were also obtained by equilibrating protein solutions with reservoir solutions of  <dig> mm tris ph <dig>  70% mpd and  <dig> mm sds resulting in "form ii" crystals.
both crystals belong to the same tetragonal space group, p <dig>  form i crystals diffracted x-rays to  <dig>  and form ii diffracted to  <dig> Å.
ramachandran plot
values listed in parentheses are for the highest resolution.
a rfactor = ∑||fo| - |fc||/∑|fo|.
rfree was calculated with 5% of the reflections set aside randomly throughout the refinement.
structure overview
unlike the one obtained by yonath et al  <cit> , the overall conformation of both structures is very similar to native lysozyme .
the rmsd values calculated using the cα atoms, after superimposition with the native structure, are  <dig>  and  <dig>  Å for form i and form ii crystals, respectively .
no large structural changes are observed and the interior of the enzyme is intact.
the most significant variations are observed at the surface of the proteins in loops where electron densities are less well-defined .
these differences are probably due to uncertainties from the model or intrinsic thermal behavior.
the presence of high concentration of mpd can therefore avoid disruption of the structure of the enzyme by sds.
indeed, as shown by yonath  <cit> , sds, by binding deeply into the hydrophobic core of the protein, induces separation of the two wings of lysozyme.
in addition, the form i crystal, where few mpd is expected to remain in the drop, shows that mpd was able to induce irreversibly the correct folding of lysozyme in the presence of a denaturing concentration of sds .
interaction of sds or mpd with lysozyme
depending on the used reservoir solution, different small molecules were found co-crystallized with lysozyme .
sds and mpd molecules could be identified unambiguously in the density maps due to their characteristic shape.
with condition ii buffer, a first mpd molecule, in the s configuration, occupies the c-subsite in the sugar-binding cleft of the protein formed by residues asn <dig>  trp <dig>  ile <dig>  ala <dig> and trp <dig> .
this was also found to be the binding site of alcohol molecules like ethanol, propanol, butanol and pentanol  <cit>  and of denaturants, used at non-denaturing concentrations, like dmso, guanidinium hydrochloride  <cit>  and urea  <cit> .
this is in agreement with the assumption that generally mpd molecules prefer to bind to hydrophobic site and mpd binding is penetrative, leading to displacement of water molecules in grooves and cavities  <cit> .
the hydrophobic part of the molecule interacts with trp <dig>  and both alcohol groups are h bonded to the backbone of asn <dig> and to trp <dig>  respectively.
a second mpd molecule, in the s configuration, is bound on the surface between two lysozyme molecules, close to arg <dig> .
no molecules of sds were observed in the electronic density map.
the s configuration of both mpd molecules is quite clear by lowering the contour level to around  <dig>  but at this stage, it is impossible to deduce that s-mpd is more stabilizing than the r one.
a comparison of the above structure with the one of lysozyme crystallized in the presence of mpd, without sds,   <cit>  yields rmsd of  <dig>  Ǻ for the superposition of cα atoms.
the location of the second mpd molecule in the present study matches the one previously described, except the configuration which is switched.
the water molecule between mpd and arg <dig> is also conserved.
moreover, two chloride ions were identified in the same binding site as in the 1dpw structure.
additionally, a sodium ion was observed ligated to the main chain carbonyl oxygen's of residues ser <dig>  cys <dig>  arg <dig>  oγ of ser <dig> side chain, and two water molecules.
this sodium ion binding site was present in the enzyme in the presence of low concentration of dmso and guanidinium chloride  <cit> .
with the condition i buffer, as expected, no electron density was observed for mpd whereas a sds molecule was found on the surface of lysozyme, exposed to the solvent.
as suggested by weiss et al, mpd may only be able to bind lysozyme at high ph values and/or at high concentration.
sds is loosely bound, as indicated by its temperature factors .
the sds sulphate is salt bridged to arg <dig>  and the hydrocarbon tail makes contacts with ser <dig>  cys64-pro <dig>  ser72-asn <dig>  the binding position is different from those described in the sds-lysozyme complex described earlier where the sds molecules bind deeply into the protein  <cit> .
indeed, the hydrophobic interactions of sds with the core of the protein are assumed to be lowered in the presence of mpd.
in addition, the binding of sds does not alter the structure of lysozyme.
in this structure, no sodium or chloride ions were observed.
it is worth noting that this study presents one of the very few structures of proteins co-crystallized with a denaturing concentration of sds in its native form.
CONCLUSIONS
this study, where crystals of lysozyme were grown for the first time in the presence of the amphipathic solvent mpd and denaturing concentrations of sds, clearly established the preservation of the enzyme structure in a sds/mpd mixture.
it is hypothesized that high concentrations of mpd, changing the properties of sds, would lower  or avoid  interactions between sds and the protein.
indeed, in the form ii crystal, even though it contains higher sds concentration, but also higher mpd concentration, than in the form i, no sds molecule was observed.
this assumption is in agreement with the size-exclusion chromatography study described earlier  <cit> , demonstrating that the protein no longer interacts strongly with sds in a mpd buffer system.
these structural data therefore support the hypothesis that mpd can protect proteins from sds denaturation.
in addition, it is worth noting that this contribution shows one of the very few structures of proteins, in its native form, co-crystallized with sds.

 sodium dodecyl sulfate is a highly effective and widely used protein denaturant. our previous work has shown that amphipathic solvents, like 2-methyl- -pentanediol, a commonly used precipitant for crystallization studies, can protect proteins from sds denaturation, and in several cases can drive the transition from the sds-denatured state to a functional folded state. this protecting effect of mpd is observed with a wide range of proteins including membrane proteins and soluble enzymes, and is not applicable if proteins are denatured in guanidine or urea, two other denaturant agents. in the case of hen egg-white lysozyme, sds concentrations above mm abolished the activity of the enzyme in the absence of mpd. however, in m mpd, the activity was preserved in the presence of sds. in addition, when the enzyme was first denatured with sds for hours prior to adding mpd, full enzymatic activity was recovered in m mpd following a further hour incubation period. although the persistence of the enzyme activity in the sds/mpd mixture was clearly established, preservation of its structure was only speculative until now. in the present contribution, a detailed x-ray study addresses the pending question. crystals of hen egg-white lysozyme could be grown for the first time in a sds/mpd medium, providing support that adding mpd to proteins can avoid sds denaturation. the obtained complexes of sds-lysozyme and mpd-lysozyme give some insights in the interplay of protein-sds and protein-mpd interactions. a previous report described the structure of cross-linked lysozyme crystals soaked in sds solutions. in the latter, unlike both structures described in this paper, sds was found at three different locations inside the protein inducing a major structural deformation in the interior of lysozyme.  crystallization crystals of hen egg-white lysozyme solubilized in mm sds, a usually denaturing condition, and m mpd were obtained, without any cross-linking, contrary to the study of yonath et al., in two different conditions. tetragonal crystals, called "form i" crystals, were obtained from a classical sodium acetate solution at ph. in this condition, few mpd, slightly volatile, is expected to remain in the drop after equilibration of the protein droplet with the mpd-free reservoir solution. to enhance the concentration of sds and mpd into the drop, and taking into account results from the literature, crystals were also obtained by equilibrating protein solutions with reservoir solutions of mm tris ph 70% mpd and mm sds resulting in "form ii" crystals. both crystals belong to the same tetragonal space group, p form i crystals diffracted x-rays to and form ii diffracted to Å. ramachandran plot values listed in parentheses are for the highest resolution. a rfactor = ∑||fo| - |fc||/∑|fo|. rfree was calculated with 5% of the reflections set aside randomly throughout the refinement. structure overview unlike the one obtained by yonath et al, the overall conformation of both structures is very similar to native lysozyme. the rmsd values calculated using the cα atoms, after superimposition with the native structure, are and Å for form i and form ii crystals, respectively. no large structural changes are observed and the interior of the enzyme is intact. the most significant variations are observed at the surface of the proteins in loops where electron densities are less well-defined. these differences are probably due to uncertainties from the model or intrinsic thermal behavior. the presence of high concentration of mpd can therefore avoid disruption of the structure of the enzyme by sds. indeed, as shown by yonath, sds, by binding deeply into the hydrophobic core of the protein, induces separation of the two wings of lysozyme. in addition, the form i crystal, where few mpd is expected to remain in the drop, shows that mpd was able to induce irreversibly the correct folding of lysozyme in the presence of a denaturing concentration of sds. interaction of sds or mpd with lysozyme depending on the used reservoir solution, different small molecules were found co-crystallized with lysozyme. sds and mpd molecules could be identified unambiguously in the density maps due to their characteristic shape. with condition ii buffer, a first mpd molecule, in the s configuration, occupies the c-subsite in the sugar-binding cleft of the protein formed by residues asn trp ile ala and trp. this was also found to be the binding site of alcohol molecules like ethanol, propanol, butanol and pentanol and of denaturants, used at non-denaturing concentrations, like dmso, guanidinium hydrochloride and urea. this is in agreement with the assumption that generally mpd molecules prefer to bind to hydrophobic site and mpd binding is penetrative, leading to displacement of water molecules in grooves and cavities. the hydrophobic part of the molecule interacts with trp and both alcohol groups are h bonded to the backbone of asn and to trp respectively. a second mpd molecule, in the s configuration, is bound on the surface between two lysozyme molecules, close to arg. no molecules of sds were observed in the electronic density map. the s configuration of both mpd molecules is quite clear by lowering the contour level to around but at this stage, it is impossible to deduce that s-mpd is more stabilizing than the r one. a comparison of the above structure with the one of lysozyme crystallized in the presence of mpd, without sds, yields rmsd of Ǻ for the superposition of cα atoms. the location of the second mpd molecule in the present study matches the one previously described, except the configuration which is switched. the water molecule between mpd and arg is also conserved. moreover, two chloride ions were identified in the same binding site as in the 1dpw structure. additionally, a sodium ion was observed ligated to the main chain carbonyl oxygen's of residues ser cys arg oγ of ser side chain, and two water molecules. this sodium ion binding site was present in the enzyme in the presence of low concentration of dmso and guanidinium chloride. with the condition i buffer, as expected, no electron density was observed for mpd whereas a sds molecule was found on the surface of lysozyme, exposed to the solvent. as suggested by weiss et al, mpd may only be able to bind lysozyme at high ph values and/or at high concentration. sds is loosely bound, as indicated by its temperature factors. the sds sulphate is salt bridged to arg and the hydrocarbon tail makes contacts with ser cys64-pro ser72-asn the binding position is different from those described in the sds-lysozyme complex described earlier where the sds molecules bind deeply into the protein. indeed, the hydrophobic interactions of sds with the core of the protein are assumed to be lowered in the presence of mpd. in addition, the binding of sds does not alter the structure of lysozyme. in this structure, no sodium or chloride ions were observed. it is worth noting that this study presents one of the very few structures of proteins co-crystallized with a denaturing concentration of sds in its native form.  this study, where crystals of lysozyme were grown for the first time in the presence of the amphipathic solvent mpd and denaturing concentrations of sds, clearly established the preservation of the enzyme structure in a sds/mpd mixture. it is hypothesized that high concentrations of mpd, changing the properties of sds, would lower or avoid interactions between sds and the protein. indeed, in the form ii crystal, even though it contains higher sds concentration, but also higher mpd concentration, than in the form i, no sds molecule was observed. this assumption is in agreement with the size-exclusion chromatography study described earlier, demonstrating that the protein no longer interacts strongly with sds in a mpd buffer system. these structural data therefore support the hypothesis that mpd can protect proteins from sds denaturation. in addition, it is worth noting that this contribution shows one of the very few structures of proteins, in its native form, co-crystallized with sds. 
it is hypothesized that high concentrations of mpd would change the properties of sds and lower or avoid interactions between the denaturant and the protein. in this paper, a detailed x-ray study addresses the pending question. crystals of hen egg-white lysozyme were grown for the first time in the presence of mpd and denaturing concentrations of sds. the conformation of both structures was very similar to the native lysozyme and the obtained complexes of sds-lysozyme and mpd-lysozyme give some insights in the interplay of protein-sds and protein-mpd interactions. recently, we reported a unique approach to preserve the activity of some proteins in the presence of the denaturing agent, sodium dodecyl sulfate. these structural data therefore support the hypothesis that mpd avoids disruption of the enzyme structure by sds and can protect proteins from sds denaturation. this study clearly established the preservation of the enzyme structure in a sds/mpd mixture. although the persistence of the protein activity in the sds/mpd mixture was clearly established, preservation of their structure was only speculative until now. depending on crystallization conditions, tetragonal crystals in complex with either sds or mpd were collected.
 recently, we reported a unique approach to preserve the activity of some proteins in the presence of the denaturing agent, sodium dodecyl sulfate. this was made possible by addition of the amphipathic solvent -methyl-2-pentanediol, used as protecting but also as refolding agent for these proteins. although the persistence of the protein activity in the sds/mpd mixture was clearly established, preservation of their structure was only speculative until now.  in this paper, a detailed x-ray study addresses the pending question. crystals of hen egg-white lysozyme were grown for the first time in the presence of mpd and denaturing concentrations of sds. depending on crystallization conditions, tetragonal crystals in complex with either sds or mpd were collected. the conformation of both structures was very similar to the native lysozyme and the obtained complexes of sds-lysozyme and mpd-lysozyme give some insights in the interplay of protein-sds and protein-mpd interactions.  this study clearly established the preservation of the enzyme structure in a sds/mpd mixture. it is hypothesized that high concentrations of mpd would change the properties of sds and lower or avoid interactions between the denaturant and the protein. these structural data therefore support the hypothesis that mpd avoids disruption of the enzyme structure by sds and can protect proteins from sds denaturation. 