a significant proportion of chemical reactions involving proteins are mediated through electrostatic interactions of their ionizable residues ( 1 ) .
such residues greatly influence the conformation of a protein and therefore its function ( 2,3 ) , as demonstrated by their folding mechanisms ( 46 ) , enzyme catalysis and protein  protein interactions ( 7 ) .
with respect to enzyme catalysis , residues can act as proton donors and acceptors within the catalytic site and help stabilize transition states , with a concomitant influence on the rate of reaction ( 8,9 ) .
the dissociation constant ( ka ) is a measure of the acidity of a compound , i.e. its ability to donate a proton .
ka values range widely from 10 for the strongest acids , such as sulphuric , to 10 for the weakest , such as methane . therefore a negative logarithmic scale is usually applied ( pka = log10 ka ) , whereby ka values for sulphuric acid and methane would become pka values of 10 and 50 , respectively . generally , more negative pka values correspond to stronger acids .
the pka values of individual amino acid residues in proteins are determined by the ionization of their side - chain groups .
for the 20 natural amino acids , pka values range from 4.0 for the side - chain carboxyl of aspartate to 12.0 for the side - chain guanididium group of arginine .
main - chain groups are not ionizable , although two additional ionizable groups exist at the n- and c - termini .
residues within proteins have pka values that are moderated by their micro - environments , the nature of their near neighbours , the extent of hydrogen bonding and so on and can take on a range of values different from that of a model residue .
nmr spectroscopy is the most widely used method for determining the pka values of individual residues , with an accuracy of 0.1 ph units .
although many nmr methods are available , most entries in the protein pka database ( ppd ) are derived using h , c and n experiments .
inaccuracies in nmr experiments stem from the range of ph values tested , variations in ionic strength and the reversibility of the titration ( 10 ) . in light of this ,
new combination methods are being used based on nmr spectroscopy coupled with site - directed mutagenesis , which leads to more accurate pka values ( 10,11 ) .
the functional importance of ionizable residues has led to numerous attempts to predict individual residue - specific pka values ( 1216 ) .
molecular dynamic simulations have also been used for such predictions , although this only gives rise to a marginal increase in accuracy ( 17 ) . as only a small handful of reviews
have attempted to compile residue - specific protein pka values ( 10,18,19 ) , it was decided to develop a database that would serve as a standard compendium against which to compare new experimental or theoretical results .
the ppd v1.0 contains > 1400 amino acid pka values , sourced from experimental data .
cross - references to several external databases  the protein data bank ( pdb ) ( 20 ) , the enzyme nomenclature and classification database ( 21 ) and the national center for biotechnology information ( ncbi ) entrez - protein  have also been incorporated into the database .
ppd v1.0 has been implemented using a postgresql relational database , which provides an appropriate infrastructure for all foreseeable future developments of the archive .
the data were initially compiled in a microsoft access database after exhaustive searching of the primary literature , which included using keyword searches of the ncbi pubmed database ( ) . the postgresql database is structured into seven normalized tables , populated from a flat - file export of the access database using perl scripts integrated with sql . as data are continually accumulating , archiving data is an on - going process : automatic , periodic updates will be made to the postgresql database .
these forms target either a perl / sql script or a cgi script which in turn queries the database .
the bespoke search engine facilitates fast , efficient and flexible data retrieval ( searching the database ) .
the data within ppd was sourced from the primary literature to give > 1400 entries , containing pka values for > 160 proteins ( table 1 ) .
the database contains pka values for amino acid side - chains , as well as the n- and c - termini .
however most entries focus on glutamate , lysine , histidine and aspartate , which together account for > 75% of the data . as these four
are all key ionizable residues , the apparent bias is not driven by our selection , but by the available experimental data .
very little data are currently available for arginine : its pka value ( 12 ) essentially precludes measurement by titration as proteins will denature at such a high basic ph .
these provide links to the protein sequence , using ncbi entrez - protein , and any relevant protein structure in the pdb ( 20 ) .
if applicable , the enzyme classification is also given , with links to the enzyme nomenclature and classification database , developed in line with the international union of biochemistry and molecular biology ( 21 ) , providing details of the enzyme reactions .
in addition , a link is given to the original literature reference via the ncbi pubmed journals database .
the ability to carry out accurate predictions of pka values depends on having access to a high quality source of data ; a principal aim of ppd is to provide such a source .
only experimentally determined pka values are cited in ppd ; predicted pka values are not included .
the quality of data contained in ppd v1.0 is largely dependent upon the accuracy of each experimental determination , thus it contains only values from certain selected techniques : nmr spectroscopy , raman difference spectroscopy and uv spectroscopy .
intrinsic factors include invariant properties of the protein investigated , such as sequence and structure .
extrinsic factors include the experimental conditions used , such as the temperature , the range of ph tested , protein concentrations as well as the experimental method .
as logistic considerations preclude us from undertaking independent verification of the data , we are obliged to trust the values reported in the literature .
it should be noted that the phenomenon of cooperative deprotonation can create circumstances under which pka values can not be used as a parameter that describes the ionization behaviour of the corresponding group ( 2224 ) .
two methods to search ppd are available : an amino acid query - based interface ( figure 1 ) and a blast ( 25 ) interface .
the implementation of a bespoke search system allows the user to perform extensive or focussed searches from a single user interface .
the simplest search , using the amino acid query interface , would specify one amino acid residue only .
a complex search would accommodate up to four amino acids and pka ranges , along with experimental method , protein name and species .
the default option returns amino acids and their associated properties ( figure 1b ) ; while the second option returns proteins which contain the specified amino acids ( figure 1c ) .
a local database of protein sequences found in ppd was compiled from swissprot ( 26 ) and an additional postgresql table was created to hold this data .
the local database is searched using the ncbi blastp and blastx programs ( 25 ) , allowing input of either protein or nucleotide sequences .
the html front - end connects to a web server - based pl / cgi script which interacts with the blastp or blastx programs .
the output contains links to ppd entries , which are created using swissprot ( 26 ) accession codes .
there is an obvious need to extend the number of entries through continuous addition of data from new , and newly - identified , publications .
the database also needs to be maintained , ensuring links to external databases remain current .
initially , as with all databases , random errors will occur owing to human error during data acquisition or will be extant within the original experimental data .
the database will be assessed for errors and inconsistencies , thus maintaining , as far as possible , the overall veracity of our data . as mentioned ,
we have tried to maintain a high degree of accuracy , through rigorous data selection ; however , user feedback will foment improvements .
moreover , feedback focussing on the search interfaces and the general infrastructure will allow us to develop appropriately both the database and its interface in an efficient and ergonomic manner .
the ppd is a unique compilation of protein pka values sourced from experimental data only .
ppd is novel : no database of its kind currently exists . compared with other post - genomic databases ,
the size of ppd is limited , but this reflects its highly focused nature : the burgeoning of such focussed databases is a continuing trend in modern bioinformatics ( 27,28 ) .
access to ppd data is given through an interface available via the world wide web and includes both a blast search and an amino acid query search system .
the blast search , which is linked to pka entries and external databases , allows ppd to be a cohesive and integrated source of protein information .
ppd facilitates data - driven in silico prediction methods addressing the relationship between ionizable groups and protein function , be that protein  protein interaction , protein folding or enzyme catalysis . a brief summary of pka data for each amino acid is shown in table 3 , which also includes both the mean and sd of the corresponding measured pka values . from the ppd data , we have shown the distribution of pka values for the six most frequent residues : glutamic acid , lysine , tyrosine , aspartic acid , histidine and cysteine ( figure 2 ) .
certain residues ( aspartate , glutamate , lysine and histidine ) have pka values which show relatively narrow distributions , while other residues ( cysteine and tyrosine ) show a wider dispersion of values ; however , this may only be a reflection of the amount of data available for these residues . while it is clear that mean values approximate closely model values , the corresponding sds are high , reflecting the wide distribution of ionization states in actual proteins .
aspartate , for example , has a mean pka of 3.6 versus a model value of 4.0 , yet the sd is 1.4 .
as the data for each residue increases , trends in residue - specific pka data will become more evident and more certain . in recent years
, there has been an impetus to accumulate data on all scales from the atomic to the genomic ; this has led to a rapid increase in the number of databases .
databases are increasingly forming the backbone of science in general and post - genomic biology in particular .
ppd v1.0 was developed to provide an easily accessible compilation of protein pka values . despite the small size of ppd , the data it contains has utility throughout many different disciplines and
, we may hope , the database will grow , through time , into a comprehensive protein pka resource .
( b ) shows the default result presentation , from which the pka data ( d ) for the specified residues can be accessed .
( c ) shows the alternative presentation , with the display of proteins containing the nominated amino acid(s ) .
each column represents a count of pka values for the specified amino acid and pka .
 the protein pka database ( ppd ) v1.0 provides a compendium of protein residue - specific ionization equilibria ( pka values ) , as collated from the primary literature , in the form of a web - accessible postgresql relational database . 
 ionizable residues play key roles in the molecular mechanisms that underlie many biological phenomena , including protein folding and enzyme catalysis . 
 the ppd serves as a general protein pka archive and as a source of data that allows for the development and improvement of pka prediction systems . 
 the database is accessed through an html interface , which offers two fast , efficient search methods : an amino acid - based query and a basic local alignment search tool search . 
 entries also give details of experimental techniques and links to other key databases , such as national center for biotechnology information and the protein data bank , providing the user with considerable background information . 
 the database can be found at the following url : . 
INTRODUCTION
DATABASE DEVELOPMENT
DATABASE CONTENT
SEARCHING THE DATABASE
FUTURE WORK
DISCUSSION AND CONCLUSIONS
Figures and Tables