hypomurocin ( hm ) peptides are the members of peptaibol family [ 13 ] , which were originally isolated from the fungus hypocrea muroiana ( synonymous to trichoderma atroviride ) , and they showed antibiotic and hemolytic activities . in a recent study , the hm molecules were also detected in other hypocrea spp . , and based on the results , it was suggested that the production of peptaibols by trichoderma / hypocrea species may contribute to the colonization and defense of ecological niches for these fungi .
based on their sequence length , the hypomurocins can be divided into two distinct groups , namely , the short - sequence hm a peptides composed of 11 amino acids and the long - sequence hm b peptides consisting of 18 amino acid residues . the former group of hm molecules covers six different peptides ( see table 1 ) , which contain a nonproteinogenic amino acid ( i.e. , -aminoisobutyric acid , aib ) , and additionally , one of them ( i.e. , hm a-2 molecule ) contains a further nonproteinogenic residue ( i.e. , d - isovaline , d - iva ) .
as it can be seen , the n - terminal amino acid of hm a peptides is acetylated , and an amino alcohol ( i.e. , leucinol , leuol ) is linked by an amide bond at their c - terminal end . among the hm a molecules , for the hm a-1 peptide , the nmr measurement performed in dimethyl sulfoxide ( dmso ) revealed that this molecule adopted a mixed helical conformation , which contained - and 310-helices , as well as type i -turn structure .
nevertheless , this nmr measurement led to the observation that the hm a-1 peptide could be characterized predominantly by 310-helical structure in the presence of micelles . in a subsequent nmr study , the structural features of hm a-3 and hm a-5 molecules were investigated in dmso solution , and based on the results , it was suggested that these peptides possessed also a mixed helical conformation containing not only - and 310-helical parts , but also type i -turn structure . in our theoretical study , a detailed conformational analysis was performed on all six hm a peptides , in order to identify their characteristic structural properties , focusing on the backbone and side - chain conformations , as well as on the evolving intramolecular h - bonds .
the hm a molecules contain nonproteinogenic amino acids , such as aib and d - iva residues , and furthermore , they contain an amino alcohol , that is , leuol ; thus these nonstandard residues were parameterized by means of quantum chemical calculations . for the parameterization , the second - order mller - plesset perturbation method and the 6 - 311 g basis set were applied . in order to derive the partial atomic charges for the above - mentioned nonstandard residues , the restrained electrostatic potential ( resp )
method was used . to perform the conformational analysis of hm a peptides , the simulated annealing ( sa ) method
was applied , as it was previously used for the short - sequence trichobrachin molecules .
the sa calculations were carried out with the amber 9 program , in the course of which the amber 99sb force field   and the generalized born implicit solvent model [ 1113 ] were applied , and no - cutoff was used for the nonbonding interactions . in the case of each hm a molecule ,
first of all an initial energy - minimization was performed , and using this geometrically optimized structure , the sa procedure composed of three consecutive stages was applied , as follows : ( 1 ) heating to 1000  k for 1000  fs , ( 2 ) equilibration at 1000  k during 4000  fs , and ( 3 ) cooling from 1000  k to 50  k for 10000  fs . for the cooling stage ,
a near - exponential protocol was used , which consisted of the following linear phases , successively : ( 1 ) from 1000  k to 500  k during 1000  fs , ( 2 ) from 500  k to 200  k for 2000  fs , ( 3 ) from 200  k to 50  k during 7000  fs .
the sa procedure , composed of heating , equilibration , and cooling stages , was carried out 1000 times ; thus 1000 conformers were obtained in the case of each peptide , for which a final energy - minimization was performed .
this geometry optimization was carried out by the steepest descent method applied for the first 100 steps , which was followed by the conjugated gradient method , using the gradient convergence criterion of 0.001  kcal  mol   and the maximum number of iterations of 10000 .
in order to identify the characteristic structural features of hm a peptides , the backbone and side - chain conformations were studied , and the evolving intramolecular h - bonds were investigated , based on the conformers derived from the sa calculations . to characterize the backbone conformations of hm a molecules
, the presence of types i and iii -turns was examined along the entire sequence of peptides . for the determination of -turn structures , the typical ranges of  and  torsion angles with regard to the i + 1th and i + 2th residues of a tetrapeptide unit were used [ 14 , 15 ] .
they were as follows : ( 1 ) in the case of type i -turn : i+1 = 60  30 and i+1 = 30  30 , i+2 = 90  30 and i+2 = 0  30 ; ( 2 ) in the case of type iii -turn : i+1 = 60  30 and i+1 = 30  30 , i+2 = 60  30 and i+2 = 30  30. the populations of types i and iii -turns identified in certain tetrapeptide units of hm a molecules are represented in table 2 for the conformers obtained by the sa simulations .
based on these results , it could be concluded that types i and iii -turns could be detected for almost all tetrapeptide units of each molecule . in the case of five hm a peptides ( i.e. , hm a-1 , hm a-3 , hm a-4 , hm a-5 , and hm a-5a )
, a similar conformational pattern could be observed with regard to the populations of -turn structures .
accordingly , for the gln  xaa  xaa  aib and pro  leu  xaa  aib tetrapeptide segments , larger amount of types i and iii -turns could be found , in comparison with those detected in the case of other four tetrapeptide units ( i.e. , xaa 
xaa , xaa  aib  pro  leu , aib  pro  leu  xaa , and xaa  aib  pro  leuol ) . on the basis of data shown in table 2
, it could be seen that , for the hm a-2 molecule , the relationship between the populations of -turn structures was the opposite , considering the above - mentioned two groups of tetrapeptide segments .
consequently , types i and iii -turns appeared with lower frequency in the case of former two tetrapeptide units than for the latter four tetrapeptide segments . nevertheless , taking into account the different tetrapeptide units
, it could be seen that , in the case of hm a-2 molecule , the amount of -turn structures found in the gln  xaa  xaa  aib and pro  leu  xaa  aib segments were lower , while the populations of types i and iii -turns observed in the xaa 
leu , aib  pro  leu  xaa and xaa  aib  pro  leuol units were larger , as compared to those detected in the corresponding tetrapeptide segments of other five hm a molecules . on the whole , these results indicated that the hm a peptides could be characterized by types i and iii -turn structures . in order to further describe the backbone conformations of hm a molecules , the occurrence of 310- and
-helical conformations was also investigated . to identify these helical structures , the following characteristic ranges of  and  torsion angles
were applied : ( 1 ) for the 310-helix ,  = 60  30 and  = 30
 30 ; ( 2 ) for the -helix ,  = 60  30 and  = 50  30 . in the case of two types of the helical conformations mentioned above
, the typical ranges of  and  torsion angles overlap with each other ; thus the cumulative helical content was also calculated , using the combined ranges of  and  torsion angles characteristic to the 310- and -helices . in order to characterize the evolving helical structures , the percentages of helical contents ( i.e. , helicities )
were calculated for each conformer derived from the sa calculations , applying the following formula [ 17 , 18 ] : 
 ( 1)f = nhn100 , 
 where f is the helicity and nh is the number of amino acids satisfying the torsion angle criteria for the 310- and -helical conformations and the cumulative helical content , respectively . for simplicity , in this study
, n is not equal to the number of all amino acids ( i.e. , 11 residues ) found in the sequences of hm a peptides , as it was previously used [ 17 , 18 ] . in the present study ,
the n = 10 was applied , and the reasons for this were as follows : ( 1 ) the short sequence length of hm a molecules ; ( 2 ) the  torsion angle could not be defined in the case of leuol residue . based on the conformers obtained by the sa simulations , the distributions of helicity values were calculated , and the populations of conformers characterized by certain helical contents were determined . as a representative sample , the distributions of helicity values are illustrated in figure 1 for the hm a-1 peptide , and these distributions were found to be similar in the case of other five molecules . nevertheless , the populations of conformers possessing different helical contents are shown in table 3 for each hm a peptide .
since the values regarding the cumulative helical content proved to be equal to the values concerning the 310-helical content , the distribution of these helicity values was not demonstrated in figure 1 , and these helicity values were not included in table 3 .
as the distribution plot and the values with regard to the 310- and -helical contents indicated , differences could be observed between these two types of helicities .
accordingly , larger populations of conformers characterized by at least 30% helicity could be detected for the 310-helical content , as compared to those found in the case of -helical content . moreover , on the basis of data presented in table 3 , it could be concluded that a similar distribution of helicity values could be observed for all the hm a molecules , taking into account the 310- and -helical contents , respectively .
on the basis of these results , it could be suggested that the hm a peptides showed a preference for the 310-helical structure over the -helical conformation .
for the side - chains of amino acids , except for the aib and pro residues , the proportions of three rotamer states ( i.e. , g(+ ) , g( ) , and trans ) were determined ( see table 4 ) , and the preferred rotamers were identified , on the basis of conformers derived from the sa calculations . the side - chain of the d - iva amino acid of hm a-2 peptide showed a preference for the g(+ ) rotamer over the other two rotamer states ; nevertheless , the ratios of g( ) and trans rotamers proved to be similar .
in contrast , for the side - chains of gln , val , and val residues , mainly the g( ) rotamer state was favored , and similar proportions of g(+ ) and trans rotamers could be observed in the majority of cases .
for the side - chains of ile , ile , and ile amino acids , a decreasing tendency could be detected with respect to the ratios of three rotamer states , as follows : g(+ ) > trans > g( ) . in the case of the side - chains of leu , leu , and leu residues , similar proportions of g( ) and trans rotamers
could be observed in the majority of cases , and both of them were found to be preferred over the g(+ ) rotamer state .
for the side - chains of leuol amino acids , large , moderate , and small ratios were identified concerning the g( ) , trans , and g(+ ) rotamers , respectively .
since various intramolecular h - bonds could contribute to the stability of the conformational states of peptides , the appearance of h - bonds evolved between the backbone nh donor and co acceptor groups was investigated . among them , the i  i + 3 h - bonds formed between a nh group of i + 3th and a co group of ith amino acids play an important role in the structural stabilization of -turns , as well as of 310-helical conformation .
the other type of intramolecular h - bonds , such as the i  i + 4 h - bonds formed between a nh group of i + 4th and a co group of ith residues , contributes to the structural stability of -helical conformation .
the occurrence of i  i + 3 and i  i + 4 h - bonds was examined along the entire sequence of hm a peptides .
an intramolecular h - bond was assumed to exist if the n  o distance between the n atom of nh donor group and the o atom of co acceptor group was within 3.5   , and if the n  h  o angle subtended at the h atom by the bond to the n atom and the line joining the h and o atoms was larger than 120. the populations of the different types of i  i + 3 and i  i + 4 h - bonds observed for the conformers of each hm a molecule are represented in table 5 .
these results indicated that the i  i + 3 h - bonds appeared in all the tetrapeptide units , for which types i and iii -turns were identified .
the i  i + 4 h - bonds were also detected along the entire sequence of molecules ; however , their populations proved to be much smaller , as compared to those in the case of i  i + 3 h - bonds .
based on these results , it could be also suggested that the hm a peptides could be characterized by -turn or 310-helical structure rather than by -helical conformation .
in the present study , a conformational analysis was performed for all the hm a peptides , and the structural properties of these short - sequence peptaibols were characterized comprehensively . taking into account the backbone conformations ,
types i and iii -turns were identified in certain tetrapeptide units of each hm a molecule , and additionally , the 310- and -helical contents were determined .
these results indicated that the hm a peptides could be characterized by -turn structures or 310-helical conformation rather than by -helical structure .
nevertheless , the side - chain conformations of amino acids were investigated , and the ratios of three rotamer states , as well as the preferred rotamers , were identified . for the
hm a peptides , the appearance of i  i + 3 and i  i + 4 h - bonds was examined , and the results led to the conclusion that the i  i + 3 h - bonds could contribute to the stability of -turn and 310-helical structures . as it was mentioned previously , based on the data derived from the nmr measurements , it was suggested that the hm a-1 , hm a-3 , and hm a-5 peptides adopted a mixed helical conformation , for which - and 310-helices , as well as type i -turn structure , appeared [ 6 , 7 ] .
the results obtained by our structural investigation indicated that the hm a molecules could be characterized by types i and iii -turns or 310-helical conformation , and these peptides showed a preference for these two secondary structural elements over the -helical structure . in summary ,
our theoretical study provided a detailed description of the three - dimensional structure of short - sequence hm a peptides .
 in this theoretical study , a conformational analysis was performed on short - sequence hypomurocin a peptides , in order to identify their characteristic structural properties . for each hypomurocin a molecule , not only the backbone conformations , but also the side - chain conformations were examined . 
 the results indicated that certain tetrapeptide units could be characterized by types i and iii -turn structures , and considering the helical conformations , it could be concluded that the hypomurocin a peptides showed a preference for the 310-helical structure over the -helical structure . beside the backbone conformations , 
 the side - chain conformations were investigated , and the preferred rotamer states of the side - chains of amino acids were determined . 
 furthermore , the occurrence of i  i + 3 and i  i + 4 intramolecular h - bonds was studied , which could play a role in the structural stabilization of -turns and helical conformations . 
 on the whole , our theoretical study supplied a comprehensive characterization of the three - dimensional structure of short - sequence hypomurocin a peptides . 
1. Introduction
2. Methods
3. Results and Discussion
4. Conclusions