they govern the specific folding of polypeptide chains into distinct 3d structures , build complementary binding interfaces between members of macromolecular complexes or create enzyme catalytic sites by spatially arranging together different chemical groups ( 1 ) . despite the large diversity of existing proteins ,
it is clear that nature has not exhaustively sampled the set of macromolecules with specific functions that can be chemically synthesized .
this is especially true because proteins are made out of a limited number ( twenty in most cases ) of genetically encoded amino acids .
although this limited number may confer advantages in terms of genetic encoding and synthesis of proteins in vivo , it clearly restricts protein s functional diversity .
introducing new chemistry into existing proteins is therefore an attractive strategy for biochemical studies , protein engineering and human therapeutics .
a promising approach is to expand the repertoire of amino acids by introducing non - natural amino acids into existing proteins or peptides ( 2 ) .
thereby , new regions of the chemical space can be explored that have not been already sampled along evolution .
non - natural amino acids , and especially l - alpha amino acids where the sidechain has only one single bond with the backbone ( i.e. excluding amino acids with branching at c or bonds with other backbone atoms ) , are particularly interesting because they do not affect the chemical properties of the protein backbone . as such they are more likely to keep the global fold and secondary structure elements of a polypeptide chain unchanged .
for instance , in a recent study , it was shown that non - natural sidechains dramatically increase the affinity of amyloid fiber inhibitors ( 3 ) .
similarly , several cyclic and other kinds of modified peptides with non - natural amino acid sidechains have been developed for therapeutics use , such as cilengitide ( 4 ) or crafilzomib ( 5 ) .
for instance , l-3,4-dihydroxyphenylalanine is a non - natural amino acid used in parkinson disease treatment ( 6 ) , and 5-hydroxytryptophan ( oxitriptan ) has been used as an antidepressant ( 7 ) .
another commonly used type of non - natural sidechains consists of d - amino acids .
d - amino acids are enantiomers of l - amino acids where the sidechain and the hydrogen atom attached to the c atom have been switched .
d - amino acids are known to increase resistance to protease degradation and are thus frequently used to enhance the biological stability of peptide - based drugs or biological tools ( 8,9 ) .
in addition to therapeutics use , non - natural sidechains have found many other applications in biochemistry and protein studies ( 10 ) .
these include photo - crosslinking amino acids to probe in vivo protein interactions ( 11,12 ) , fluorescent amino acids used as markers of specific proteins ( 13 ) or phosphorylated amino acid mimetics to probe the effect of post - translational modifications ( 14 ) . the importance of expanding the building blocks of proteins beyond the 20 natural ones is further emphasized by noting that a remarkably high degree of optimization is often observed in the choice of amino acid sequences for many proteins .
optimized sequences stand as an important hurdle to protein or peptide engineering based only on genetically encoded amino acids
. experimental research on non - natural sidechains is based mainly on solid - phase synthesis technology ( 15 ) and genetically encoding non - natural sidechains into micro - organisms or cell cultures ( 2 ) .
the former technique uses in vitro chemical peptide synthesis to assemble polypeptide chains with both natural and non - natural residues .
this technology is routinely used to synthesize non - natural peptides and can nowadays be applied to small proteins of up to hundred amino acids ( 16 ) .
trna pairs , together with the corresponding aminoacyl trna synthetases in a given organism ( 2 ) .
this approach enables production of non - natural sidechain containing polypeptide chains in micro - organisms that can be used as in vivo biological tools ( 13 ) or as modified peptide libraries for ligand screening ( 17 ) .
recent progresses currently enable encoding simultaneously hundreds of different non - natural sidechains in the same organism ( 18 ) .
bio- and chemo - informatics , structural modeling and visualization tools are powerful to guide and interpret experimental studies with both natural and non - natural amino acids .
recently , we and others developed different computational tools to better characterize non - natural sidechains and predict the effect of introducing them into proteins or peptides ( 1922 ) .
this includes sidechain 3d coordinates , biomolecular parameters describing properties such as bond length flexibility , as well characterization of conformational diversity in terms of rotamers ( 23 ) . for the latter
, different strategies have been elaborated to predict rotamer probabilities , using either solely free energy 
based calculations ( 20,22 ) , or combining physics - based calculations together with statistical analysis of conserved properties in natural sidechain rotamer libraries ( 19 ) . here
, we introduce the swisssidechain database , a unified and integrated resource providing access to curated biochemical and structural information for 210 non - natural sidechains .
many of the data are unique to swisssidechain ( e.g. rotamers , biomolecular parameters ) , whereas the rest has been collected from existing resources to provide a rapid access to this information . the swisssidechain database further includes visualization and molecular modeling tools . different options to browse the non - natural sidechains based on their chemical properties or their families
the swisssidechain database contains molecular and structural data for 210 non - natural alpha amino acid sidechains , both in l- and d - configurations , in addition to the 20 natural ones .
these amino acids have been selected based on two main criteria : first , the presence of non - natural sidechains in publicly available protein structures in the pdb ( protein data bank ) ( 24 ) and second , the commercial availability . each amino acid in swisssidechain
for all sidechains present in the pdb , we kept the existing three - letter code . for other sidechains ,
these include many d - amino acids whose l - configuration is present in the pdb . for them , a 
d was simply added in front of the three - letter code ( e.g. nle stands for l - norleucine and dnle for d - norleucine ) . for the rest , four - letter codes were chosen so as to recall as much as possible the full chemical name ( e.g. azda for l - azido - alanine ) , and always starting with a 
d for non - natural sidechains in d - configuration ( e.g. dzda for d - azido - alanine ) .
swisssidechain data are available in different files describing the chemical structure ( smiles and 2d chemical drawing ) and 3d coordinates ( pdb , mol2 ) of these sidechains , files describing their physico - chemical properties ( molecular weight , volume , logp , pka , partial charges , bond / angle / torsion constants ) , as well as files describing the probability of their possible conformations ( rotamers ) . among the different experimentally measurable chemical properties of non - natural sidechains in swisssidechain , volumes have been calculated with charmm ( chemistry at harvard molecular mechanics ) ( 25 ) and molecular weights with openbabel ( 26 ) .
experimental logp for both the full amino acid and the sidechain only have been manually collected from literature when available . for the rest of the sidechains , logp values
pka values for each protonatable group of the full amino acids have been retrieved from literature when available and computed with marvinsketch ( chemaxon , http://www.chemaxon.com ) otherwise .
model parameters , such as bond , angle and torsion constants , which provide information about sidechain flexibility and dynamics , are provided as parameter files compatible with the charmm force field ( 28 ) and can be readily used in the charmm ( 25 ) and gromacs ( groningen machine for chemical simulations ) ( 29 ) molecular mechanics software .
similarly , partial atomic charges , which are useful to predict possible interactions ( e.g. h - bond , electrostatic ) of a sidechain with its surrounding atoms , are provided as topology files in the charmm ( 25 ) and gromacs ( 29 ) format .
, we developed the current version of swisssidechain based on an up - to - date rotamer library for natural sidechains , instead of using rotamer libraries published in 2002 ( 23 ) . in practice , we performed a global analysis of natural sidechain conformations in all crystallographic structures available in the pdb ( as on may 2012 ) with resolution lower or equal to 1.75 .
this updated rotamer library of natural sidechains , and especially the conserved probabilities of the first dihedral angles of long sidechains , was used in all our analysis to predict rotamers for non - natural sidechains , as described in gfeller et al .
we further expand rotamer libraries to d - amino acids , both natural and non - natural .
for non - chiral sidechains , rotamer probabilities can be readily generated using the properties of mirror images .
in particular , a d - configuration characterized by backbone dihedral angles   and   and sidechain dihedral angles i is equivalent to the l - configuration with dihedral angles  ,  and i ( i = 1 , 
n , where n stands for the number of freely rotating dihedral angles along the sidechain ) . in other words , , where pd stands for the conformational probability of a d - amino acid and pl stands for the conformational probability of the same amino acid in l - configuration ( 30 ) .
note that this does not consider interactions with other residues , neither whether the previous and the next residues are in l- or d - configuration .
however , such information is typically not explicitly considered in rotamer libraries . for chiral sidechains ,
the previous relation does not hold because of the additional asymmetric centers . therefore , we used the same strategy based on molecular dynamics simulations and renormalization that was used for l - sidechains in gfeller et al .
( 19 ) to predict the rotamer libraries for chiral d - sidechains . to help exploring the structural environment of non - natural sidechains incorporated in polypeptide chains , we developed visualization plugins for pymol and ucsf chimera ( 31 ) .
these plugins enable users to mutate residues in protein structures to any sidechain ( natural and non - natural , in both l- and d - configurations ) present in the swisssidechain database .
this is particularly useful to evaluate the structural environment of a new residue and detect possible clashes that may arise on mutation .
moreover , the resulting structures can be readily used for future investigations with molecular mechanics software .
detailed instructions for installing and running these plugins are provided on the swisssidechain web site , together with short movies describing standard use for mutating a residue to a non - natural sidechain in a protein structure . for each sidechain ,
all data are collected in dedicated web pages containing 2d and 3d structures , the different names , biochemical properties , links to existing databases and download options .
links to existing databases include the pdb ( 24 ) , the pdb ligand expo ( 32 ) and pubchem ( 33 ) . files to be downloaded consist of structure files containing 3d coordinates ( pdb and mol2 formats ) , 2d structures , rotamers ( backbone dependent and independent ) and topology files for molecular mechanics software such as charmm ( 25 ) and gromacs ( 29 ) parameter files are available at the molecular dynamics ( md ) simulations pages .
the amino acid 3d structures for the l - configuration can be visualized online thanks to the open - source java viewer for chemical structures jmol ( http://www.jmol.org ) .
in addition we provide bulk download options for all data at the download page of swisssidechain . 

names , smiles and several biochemical parameters are listed on the right part of the page , as well as links to other structural or chemical databases .
names , smiles and several biochemical parameters are listed on the right part of the page , as well as links to other structural or chemical databases .
swisssidechain data can be browsed in large alphabetical tables containing the three- or four - letter codes and the sidechain full names , 2d structures , internal links to the sidechain pages , external links to the pdb , as well as download links for structural files .
both a full table and tables restricted to sub - families of natural sidechain derivatives ( e.g. methionine derivatives ) are provided .
two particularly important parameters are the volume and the hydrophobicity ( logp ) of the sidechains .
this enables users to zoom , visualize and select specific subsets of non - natural sidechains ( see figure 2 ) .
sidechains can be selected by clicking on the corresponding circles or selecting a region of the graph and using the 
select all button . selected sidechains appear in the box below the graph and links to sidechain pages can be followed from there .
this approach is especially useful for protein engineering and ligand design , to select a subset of sidechains with specific properties . 

each sidechain is represented by a yellow circle positioned according to the sidechain volume and logp .
select all button to select all sidechains in the plot . selected sidechains appear in the box below the plot , where users can follow the links to the sidechain web page ( see figure 1 ) or download all structural and molecular files .
users can zoom on the graph by selecting subregions ( see red rectangle in the overview panel ) .
each sidechain is represented by a yellow circle positioned according to the sidechain volume and logp .
selected sidechains appear in the box below the plot , where users can follow the links to the sidechain web page ( see figure 1 ) or download all structural and molecular files .
users can zoom on the graph by selecting subregions ( see red rectangle in the overview panel ) .
the swisssidechain database contains molecular and structural data for 210 non - natural alpha amino acid sidechains , both in l- and d - configurations , in addition to the 20 natural ones .
these amino acids have been selected based on two main criteria : first , the presence of non - natural sidechains in publicly available protein structures in the pdb ( protein data bank ) ( 24 ) and second , the commercial availability . each amino acid in swisssidechain
for all sidechains present in the pdb , we kept the existing three - letter code . for other sidechains ,
these include many d - amino acids whose l - configuration is present in the pdb . for them , a 
d was simply added in front of the three - letter code ( e.g. nle stands for l - norleucine and dnle for d - norleucine ) . for the rest
, four - letter codes were chosen so as to recall as much as possible the full chemical name ( e.g. azda for l - azido - alanine ) , and always starting with a 
d for non - natural sidechains in d - configuration ( e.g. dzda for d - azido - alanine ) .
swisssidechain data are available in different files describing the chemical structure ( smiles and 2d chemical drawing ) and 3d coordinates ( pdb , mol2 ) of these sidechains , files describing their physico - chemical properties ( molecular weight , volume , logp , pka , partial charges , bond / angle / torsion constants ) , as well as files describing the probability of their possible conformations ( rotamers ) . among the different experimentally measurable chemical properties of non - natural sidechains in swisssidechain ,
volumes have been calculated with charmm ( chemistry at harvard molecular mechanics ) ( 25 ) and molecular weights with openbabel ( 26 ) .
experimental logp for both the full amino acid and the sidechain only have been manually collected from literature when available . for the rest of the sidechains , logp values
pka values for each protonatable group of the full amino acids have been retrieved from literature when available and computed with marvinsketch ( chemaxon , http://www.chemaxon.com ) otherwise .
model parameters , such as bond , angle and torsion constants , which provide information about sidechain flexibility and dynamics , are provided as parameter files compatible with the charmm force field ( 28 ) and can be readily used in the charmm ( 25 ) and gromacs ( groningen machine for chemical simulations ) ( 29 ) molecular mechanics software .
similarly , partial atomic charges , which are useful to predict possible interactions ( e.g. h - bond , electrostatic ) of a sidechain with its surrounding atoms , are provided as topology files in the charmm ( 25 ) and gromacs ( 29 ) format .
, we developed the current version of swisssidechain based on an up - to - date rotamer library for natural sidechains , instead of using rotamer libraries published in 2002 ( 23 ) . in practice , we performed a global analysis of natural sidechain conformations in all crystallographic structures available in the pdb ( as on may 2012 ) with resolution lower or equal to 1.75 .
this updated rotamer library of natural sidechains , and especially the conserved probabilities of the first dihedral angles of long sidechains , was used in all our analysis to predict rotamers for non - natural sidechains , as described in gfeller et al .
we further expand rotamer libraries to d - amino acids , both natural and non - natural .
for non - chiral sidechains , rotamer probabilities can be readily generated using the properties of mirror images .
in particular , a d - configuration characterized by backbone dihedral angles   and   and sidechain dihedral angles i is equivalent to the l - configuration with dihedral angles  ,  and i ( i = 1 , 
n , where n stands for the number of freely rotating dihedral angles along the sidechain ) . in other words , , where pd stands for the conformational probability of a d - amino acid and pl stands for the conformational probability of the same amino acid in l - configuration ( 30 ) .
note that this does not consider interactions with other residues , neither whether the previous and the next residues are in l- or d - configuration .
however , such information is typically not explicitly considered in rotamer libraries . for chiral sidechains , the previous relation does not hold because of the additional asymmetric centers . therefore , we used the same strategy based on molecular dynamics simulations and renormalization that was used for l - sidechains in gfeller et al .
( 19 ) to predict the rotamer libraries for chiral d - sidechains . to help exploring the structural environment of non - natural sidechains incorporated in polypeptide chains , we developed visualization plugins for pymol and ucsf chimera ( 31 ) .
these plugins enable users to mutate residues in protein structures to any sidechain ( natural and non - natural , in both l- and d - configurations ) present in the swisssidechain database .
this is particularly useful to evaluate the structural environment of a new residue and detect possible clashes that may arise on mutation .
moreover , the resulting structures can be readily used for future investigations with molecular mechanics software .
detailed instructions for installing and running these plugins are provided on the swisssidechain web site , together with short movies describing standard use for mutating a residue to a non - natural sidechain in a protein structure .
for each sidechain , all data are collected in dedicated web pages containing 2d and 3d structures , the different names , biochemical properties , links to existing databases and download options .
links to existing databases include the pdb ( 24 ) , the pdb ligand expo ( 32 ) and pubchem ( 33 ) . files to be downloaded consist of structure files containing 3d coordinates ( pdb and mol2 formats ) , 2d structures , rotamers ( backbone dependent and independent ) and topology files for molecular mechanics software such as charmm ( 25 ) and gromacs ( 29 ) parameter files are available at the molecular dynamics ( md ) simulations pages .
the amino acid 3d structures for the l - configuration can be visualized online thanks to the open - source java viewer for chemical structures jmol ( http://www.jmol.org ) .
in addition we provide bulk download options for all data at the download page of swisssidechain . 

names , smiles and several biochemical parameters are listed on the right part of the page , as well as links to other structural or chemical databases .
names , smiles and several biochemical parameters are listed on the right part of the page , as well as links to other structural or chemical databases .
swisssidechain data can be browsed in large alphabetical tables containing the three- or four - letter codes and the sidechain full names , 2d structures , internal links to the sidechain pages , external links to the pdb , as well as download links for structural files .
both a full table and tables restricted to sub - families of natural sidechain derivatives ( e.g. methionine derivatives ) are provided .
two particularly important parameters are the volume and the hydrophobicity ( logp ) of the sidechains . in swisssidechain
this enables users to zoom , visualize and select specific subsets of non - natural sidechains ( see figure 2 ) .
sidechains can be selected by clicking on the corresponding circles or selecting a region of the graph and using the 
selected sidechains appear in the box below the graph and links to sidechain pages can be followed from there .
this approach is especially useful for protein engineering and ligand design , to select a subset of sidechains with specific properties . 

each sidechain is represented by a yellow circle positioned according to the sidechain volume and logp .
selected sidechains appear in the box below the plot , where users can follow the links to the sidechain web page ( see figure 1 ) or download all structural and molecular files .
users can zoom on the graph by selecting subregions ( see red rectangle in the overview panel ) .
each sidechain is represented by a yellow circle positioned according to the sidechain volume and logp .
selected sidechains appear in the box below the plot , where users can follow the links to the sidechain web page ( see figure 1 ) or download all structural and molecular files .
users can zoom on the graph by selecting subregions ( see red rectangle in the overview panel ) .
exploiting naturally evolved , and often well optimized , proteins or peptides while not being restricted to the limited set of 20 natural amino acids is a promising approach for protein engineering with applications in biochemistry , protein structure and function analysis , and for drug design ( 3,10 ) . toward this goal , non
the swisssidechain database provides a unified web resource for hundreds of non - natural sidechains .
the biochemical parameters allow researchers to rapidly retrieve sidechains with specific properties , such as volume or hydrophobicity .
the different visualization tools enable seamless introduction of non - natural sidechains in protein and peptide structures .
the model parameters , such as bond and angle flexibility or partial atomic charges are useful to investigate non - natural sidechains with molecular mechanics software .
most of the non - natural sidechains are commercially available , so that researchers interested in using the predictions obtained with the swisssidechain data can easily test them experimentally .
the naming is consistent with the pdb so that published experimental structures can be readily analyzed with the tools provided in swisssidechain . in the current version of swisssidechain ,
we focus on alpha amino acid sidechains that do not modify the backbone atoms ( i.e. without branching at c , modification of the nitrogen atom or longer backbones such as beta amino acids ) . these amino acids in their l - configuration are less likely to lead to large structural remodeling when incorporated into proteins or peptides . therefore they are often used in protein engineering experiments , and predictions of their effect on protein structure and function are in general more accurate .
the same amino acids in d - configuration have found many applications in biochemistry and drug discovery .
better characterizing d - amino acid properties is also useful to study d - retro - inverso peptides that can provide mimetics of l - peptides ( 34 ) , or d - peptides obtained by screening l - peptides against d - targets and using the mirror - image property of d - enantiomers ( 35 ) .
structural modeling of these types of non - natural sidechains is becoming feasible , although it is still more challenging .
this is for instance the case with peptides binding to peptide recognition domains , such as post synaptic density protein , drosophila disc large tumor suppressor , and zonula occludens-1 protein or src homology 3 domains ( 36,37 ) .
as the binding interface is often relatively short , one may be able to probe in silico the effect of alpha - amino acids in d - configuration ( 38 ) .
for these different reasons , we have included data for the d - configuration of all amino acid sidechains present in the current version of swisssidechain . as experimental techniques
keep progressing , more and more non - natural sidechains will become commercially available or be encountered in protein structures .
other developments may include focusing on different families of non - natural sidechains , such as fluorescent or photo - cleavable amino acids .
european molecular biology organization long - term fellowship [ altf 241 - 2010 to d.g . ] ; swiss institute of bioinformatics . funding for open access charge : swiss institute of bioinformatics .
 amino acids form the building blocks of all proteins . naturally occurring amino acids 
 are restricted to a few tens of sidechains , even when considering post - translational modifications and rare amino acids such as selenocysteine and pyrrolysine . however , the potential chemical diversity of amino acid sidechains is nearly infinite . exploiting this diversity by using non - natural sidechains to expand the building blocks of proteins and peptides 
 has recently found widespread applications in biochemistry , protein engineering and drug design . despite these applications , 
 there is currently no unified online bioinformatics resource for non - natural sidechains . with the swisssidechain database ( http://www.swisssidechain.ch ) , we offer a central and curated platform about non - natural sidechains for researchers in biochemistry , medicinal chemistry , protein engineering and molecular modeling . 
 swisssidechain provides biophysical , structural and molecular data for hundreds of commercially available non - natural amino acid sidechains , both in l- and d - configurations . 
 the database can be easily browsed by sidechain names , families or physico - chemical properties . 
 we also provide plugins to seamlessly insert non - natural sidechains into peptides and proteins using molecular visualization software , as well as topologies and parameters compatible with molecular mechanics software . 
INTRODUCTION
Content of the SwissSidechain database
Web interface
Browsing options
CONCLUSION AND OUTLOOK
FUNDING