equimolar amounts of each of the   naturally occurring amino acids was used to a total of 10-fold molar excess .
cysteine was not included in this mix and arginine was   used at 1.5 molar concentration to compensate for previously   observed low incorporation of this amino acid ( 4 ) .
the randomness of the peptide mixture was then analyzed by hplc and laser - desorption time of flight mass spectrometry .  
hla - b53   and hla - b8 were produced using vectors pgmt7b53his ( 5 )   and pgmt7b8 , respectively .
hla - a2 and 2 m were produced   using the vectors phn1a2 and phn12 m , respectively ( a gift   from d. garboczi , harvard university , cambridge , ma ) . hlab53 and hla - a2 were refolded with a random mix of peptides   using a dilutional method as previously described ( 6 ) . in brief ,   30 mg of random peptide pool was dissolved in a small volume   of 8 m urea and added to a solution of 1 m heavy chain , 2 m   2 m in refolding buffer ( 0.4 m l - arginine , 0.1 m tris [ ph =   7.5 ] , 2 m edta , 5 mm reduced glutathione , 0.5 mm oxidized   glutathione , 0.5 mm pmsf ) .
after incubation for 3648 h at   4c , the solution was concentrated and the refolded complex purified by gel filtration .
hla - b8 was refolded using a dialysis method of refolding . in   brief ,
peptide , 2 m , and heavy chain were mixed with 20 mm   tris ( ph = 7.5 ) and 150 mm nacl and then dialysed against refolding buffer .
additional heavy chain was added during incubation at 4c for 48 h , then the solution was concentrated and the   refolded complex purified by gel filtration . in both methods , random peptides were used at a final concentration of 200-fold   molar excess over heavy chain in the refolding reaction .
self - peptides from hla - b53 were purified using a w6 - 32 affinity column as previously described ( 7 ) .
bound peptides were eluted   from the mhc class i molecules as previously described ( 7 ) . in   brief , mhc class i  peptide complexes were first concentrated on   a centricon-3 microconcentrator ( amicon , beverly , ma ) before   the addition of 0.1% trifluoroacetic acid .
the eluate was collected   and separated by hplc before sequencing with an applied biosystems 473a protein sequencer ( applied biosystems , foster   city , ca ) .
individual self - peptides eluted from affinity - purified   hla - b53 were sequenced as previously described ( 8) .
equimolar amounts of each of the   naturally occurring amino acids was used to a total of 10-fold molar excess .
cysteine was not included in this mix and arginine was   used at 1.5 molar concentration to compensate for previously   observed low incorporation of this amino acid ( 4 ) .
the randomness of the peptide mixture was then analyzed by hplc and laser - desorption time of flight mass spectrometry .
  hla - b53   and hla - b8 were produced using vectors pgmt7b53his ( 5 )   and pgmt7b8 , respectively .
hla - a2 and 2 m were produced   using the vectors phn1a2 and phn12 m , respectively ( a gift   from d. garboczi , harvard university , cambridge , ma ) . hlab53 and hla - a2 were refolded with a random mix of peptides   using a dilutional method as previously described ( 6 ) . in brief ,   30 mg of random peptide pool was dissolved in a small volume   of 8 m urea and added to a solution of 1 m heavy chain , 2 m   2 m in refolding buffer ( 0.4 m l - arginine , 0.1 m tris [ ph =   7.5 ] , 2 m edta , 5 mm reduced glutathione , 0.5 mm oxidized   glutathione , 0.5 mm pmsf ) .
after incubation for 3648 h at   4c , the solution was concentrated and the refolded complex purified by gel filtration .
hla - b8 was refolded using a dialysis method of refolding . in   brief ,
peptide , 2 m , and heavy chain were mixed with 20 mm   tris ( ph = 7.5 ) and 150 mm nacl and then dialysed against refolding buffer .
additional heavy chain was added during incubation at 4c for 48 h , then the solution was concentrated and the   refolded complex purified by gel filtration . in both methods , random peptides were used at a final concentration of 200-fold   molar excess over heavy chain in the refolding reaction .
self - peptides from hla - b53 were purified using a w6 - 32 affinity column as previously described ( 7 ) .
bound peptides were eluted   from the mhc class i molecules as previously described ( 7 ) . in   brief , mhc class i  peptide complexes were first concentrated on   a centricon-3 microconcentrator ( amicon , beverly , ma ) before   the addition of 0.1% trifluoroacetic acid .
the eluate was collected   and separated by hplc before sequencing with an applied biosystems 473a protein sequencer ( applied biosystems , foster   city , ca ) .
individual self - peptides eluted from affinity - purified   hla - b53 were sequenced as previously described ( 8) .
analysis of the random peptide library by hplc and laser desorption - mass spectrometry showed that the library   was a homogeneous mix of peptides without any obvious   peaks of abundant peptides and conformed to the mass   range expected for nonamer peptides .
refolding of mhc   class i molecules with the random peptide mixture resulted   in the formation of stable complexes of heavy chain , peptide , and 2 m , which migrated at the correct mass using gel   filtration . a final concentration and wash step before peptide elution ensured that only peptides that formed complexes stable enough to remain associated during these procedures were analyzed .
peptides were eluted from 500   g of mhc class i. the results of pool sequencing of these   peptides and of the random peptide library showed that   while there were no significant enrichments for any amino   acids in the random library , such enrichments were found   in the mhc class i  associated pool .
positions where the   yield of a particular amino acid was increased to 150% or   more of the previous cycle were considered significant   ( fig .
the motif identified using this method could   be compared with that identified by previous studies ( fig .   2 ) .
it can be seen that although the motifs were not identical ,   the same anchor residues were observed by both methods   with the exception of the cooh - terminal hydrophobic   anchor previously observed in studies of hla - a2 and   hla - b8 .
in addition , in some cases in which enrichment   for anchor residues did not reach the 150% level , the same   patterns of enrichment were seen for both the naturally   bound peptides and the peptides bound by the recombinant class i. it is also interesting that the strength of motifs   for different mhc class i alleles is conserved using both   methods , so that , for example , whereas hla - a2 shows   strong enrichment for several possible minor anchors in   both pool sequences , hla - b53 shows a relatively weak   proline anchor at position 2 using both methods ( fig .
2 ) . in   addition , whereas analysis of four individual self - peptides   eluted from hla - b53 and three t cell epitopes suggests a   preference for hydrophobic cooh termini ( fig .
3 ) , this   was not observed in the results from pool sequencing . thus   the amino acid preferences observed from pool sequencing   of hla - b53 seem somehow less strong than those of , for   example , hla - a2 .
previous studies of the peptide binding motifs of mhc   class i molecules have indicated that most hla class i alleles   show a preference for a hydrophobic cooh - terminal   amino acid , with a small number favoring a positive charge   at this position ( reviewed in reference 3 ) .
the majority of   these motifs are derived from analysis of the peptides bound   to mhc class i on the surface of b cells and thus reflect   both the specificity of the mhc class i molecule itself and   the effects of processing and transport .
direct binding of   synthetic peptides to mhc class i has also been used to   study the peptide binding specificity of these molecules .   although a number of methods have been used ( reviewed   in reference 3 )
, it is difficult to reflect the dynamic interactions of mhc and peptide using single peptides
. the number of mhc peptide complexes on the cell surface will be   determined both by the ability of peptides to compete for   binding and also by the stability of the subsequent complex .  
parker and colleagues ( 9 ) have described experiments in   which they measured the stability of complexes formed between recombinant hla - a2 and synthetic peptides .
interestingly , when the requirements of the peptide cooh   terminus were analyzed , these did not conform with the   expected restrictions .
the motifs observed in this study differ from those previously reported because they show no preference for a   cooh - terminal hydrophobic or positive charge . a trivial   explanation for this would be that the solutions and the   temperature at which binding was carried out may interfere   somehow with peptide  mhc interactions .
however , since   preferences for both hydrophobic and other peptide anchors are conserved elsewhere in the motifs , this seems unlikely .
one simple mechanism to explain the observed differences in peptide cooh termini is that naturally processed   peptides are subject to selection for transport into the endoplasmic reticulum by the tap transporter , whereas our   random mixture is not .
studies of the substrate specificity   of the tap protein in human , mouse , and rat have suggested that whereas the rat tap2 and murine tap were   highly selective for hydrophobic or aromatic cooh termini , the human tap and the rat tap2 transporters were   only weakly selective for hydrophobic or positive charge   and against negative charge ( 10 ) .
therefore , this bias would   tend to favor hydrophobic cooh termini in most mhc   class i alleles .
those mhc class i molecules that appear to   exhibit a preference for positively charged cooh termini   instead may simply be those which are less able to tolerate   hydrophobic cooh termini and thus bind the next most   abundant species , those with positively charged cooh termini . however ,
analysis of hla - a2-associated peptides in   a mutant cell line lacking the tap transporter suggests that   the preference for a hydrophobic cooh terminus is still   observed ( 11 ) .
therefore , it is likely that other mechanisms   of peptide processing may act independently of or synergistically with the tap transporter to affect the cooh terminus   of the peptides .
experiments to determine the alterations in   substrate specificity conferred by the mhc - encoded lmp2   and lmp7 subunits of the proteasome have suggested that   the presence of these subunits enhances proteolysis after   hydrophobic and positively charged amino acids and decreases proteolysis after negatively charged amino acids ( 12 ,   13 ) .
in addition , we can not exclude the possibility that   other processes involved in the generation and assembly of   mhc  peptide complexes , such as the involvement of molecular chaperones , may also act to bias the repertoire of   peptides available for binding to mhc class i. the preference of both the putative proteolytic enzymes   and transporter molecules involved in class i processing for   hydrophobic or positively charged cooh termini of peptides appears to accord with the observation that most   mhc class i motifs have hydrophobic cooh termini ,   and the few that do not have positively charged coohtermini .
this has been interpreted as suggesting that the antigen processing machinery has evolved to supply the   mhc class i molecule with peptides optimal for binding .  
however , because most mhc class i motifs are derived from   the analysis of peptides eluted from mhc class i molecules   on the cell surface , these motifs include the effects of both   antigen processing and mhc binding .
therefore , we favor   an alternate explanation for the observed preference for hydrophobic or positively charged cooh termini in mhc   class i motifs , namely that this is largely an effect of processing and that in the absence of such processing this preference is not found . this observation does not affect the use of currently   available mhc class i motifs in the prediction of t cell   epitopes , since a hydrophobic cooh terminus will still be   required for natural processing and presentation of antigens .  
on the other hand , these observations necessitate a reevaluation of the role of processing in the selection of peptides   for binding to mhc class i. a preference for cooh - terminal hydrophobic residues for both the processing machinery and class i molecules would imply that the system   had evolved to optimize peptide delivery to class i. if class i   does not select for hydrophobic cooh termini in the absence of processing , then it would seem that the selection   for cooh - terminal hydrophobic anchors , in fact , acts to   restrict the repertoire of peptides available to bind mhc   class i. although this may seem counterproductive , a precedent for such a limitation of peptides is already known .  
the tap transporter of human , mouse , and rat appears to   be a poor transporter of peptides with proline at positions 2   and 3 ( 14 ) .
however , a number of hla - b alleles ( including hla - b53 ) appear to bind a proline anchor at position 2   of the peptide .
thus , it would seem that either the selectivity of the transporter is acting to reduce the number of   peptides available to bind these , or that there is some advantage in longer peptides being transported and then   cleaved to generate the mhc binding peptide within the   endoplasmic reticulum . if the former is the case one can   only speculate as to the possible benefits to the host of reducing the repertoire of endogenous peptides presented on   mhc class i. however , it is possible that this acts to decrease the effective hole in the t cell repertoire caused by   negative selection of t cells in the thymus .
the results of this work demonstrate that recombinant   mhc class i molecules produced in e. coli retain their peptide specificity during refolding with peptide and 2 m molecules in vitro .
in addition , the observed differences between the peptide binding motifs derived from naturally   processed peptides and peptides bound to mhc class i in vitro suggest that peptide processing and transport play a dominant role in determining which peptides will ultimately be   presented in association with mhc class i.
the yield of each amino acid ( indicated by single letter   code ) for each position of the peptide is shown for hla - a2 , hla - b8 , and hla - b53 .
positions where the yield of amino acid is 150% greater compared   with the previous cycle are underlined .
comparison of the peptide binding motifs for hla - a2 ,   hla - b8 , and hla - b53 derived by different methods .
( 2 ) , namely ,   dominant or strong anchor residues are indicated in bold type , and listed   vertically in order of descending importance .
it should be noted that different criteria were used to assign anchors in some studies , particularly   when individual peptide as opposed to pool sequences were analyzed .  
sequences of individual self - peptides eluted from hla - b53   and sequenced using tandem mass spectrometry .
the isobaric residues   isoleucine and leucine could not be distinguished in the unknown peptide .
 recombinant hla - a2 , hla - b8 , or hla - b53 heavy chain produced in escherichia coli was   combined with recombinant 2-microglobulin ( 2 m ) and a pool of randomly synthesised nonamer peptides . 
 this mixture was allowed to refold to form stable major histocompatability   complex ( mhc ) class i complexes , which were then purified by gel filtration chromatography .   
 the peptides bound to the mhc class i molecules were subsequently eluted and sequenced as   a pool . 
 peptide binding motifs for these three mhc class i molecules were derived and compared with previously described motifs derived from analysis of naturally processed peptides   eluted from the surface of cells . 
 this comparison indicated that the peptides bound by the recombinant mhc class i molecules showed a similar motif to naturally processed and presented   peptides , with the exception of the peptide cooh terminus . 
 whereas the motifs derived   from naturally processed peptides eluted from hla - a2 and hla - b8 indicated a strong preference for hydrophobic amino acids at the cooh terminus , this preference was not observed in   our studies . 
 we propose that this difference reflects the effects of processing or transport on the   peptide repertoire available for binding to mhc class i molecules in vivo . 
Materials and Methods
Random Peptide Library.
Assembly and Purification of MHC Class I Complexes.
Peptide Elution and Sequencing.
Results and Discussion
Figures and Tables