bacterial strains and growth of bacteria  strains used in 
  this work are presented in table 
  1 .
e. coli strains were grown in lysogeny broth or on 
  lysogeny broth plates , and b. subtilis strains were grown in nutrient 
  sporulation medium with phosphate 
  ( 15 ) with appropriate 
  antibiotics added as follows : ampicillin , 100 g / ml ; kanamycin , 10 g / ml 
  ( for b. subtilis ) or 50 g / ml ( for e. coli ) ; and 
  chloramphenicol , 3 or 4 g / ml ( for b. subtilis ) and 15 g / ml 
  ( for e. coli ) .
liquid cultures were grown in baffled e - flasks on a 
  rotary shaker ( 200 rpm ) at 37 c .
table 1strains and plasmids used in this workstrain or plasmidgenotype and/or relevant 
  propertiesaorigin or ref.e .
coli bl21 
   fompt hsdsb(rb 
  mb ) gal dcm novagen 
   b834(de3 ) 
   fompt hsdsb(rb 
  mb ) gal dcm met ( de3 ) 
   novagen 
   mm294 
  thi , pro , hsdr supe4 45 
 
  top10 
 
  fmcra [mrr - hsdrms - mcrbc ] 
  f80laczm15 lacx74 reca1 arad139 
  [ara - leu]7697 galu galk rpsl enda1 nupg 
  invitrogen 
  b. subtilis 1a1 
  trpc2bacillus genetic stock center , columbus , oh 
   lul20 
  trpc2 stoaplle39 ; cm 2 
 
  lul30 
  trpc2 (ykvu - stoa)::tet ; tc 
  2 
  plasmids pbluescript sk(+ ) 
   cloning vector ; ap stratagene 
   pcr - blunt - ii - topo 
   cloning vector ; km invitrogen 
   pdg148 
   expression vector ; em km 46 
   pgex4t1 
   gst fusion expression vector ; ap ge healthcare 
   plle83 
   pdg148 derivative containing the stoa gene ; km 2 
   plmc19 
   pbadmyc - hisc derivative encoding sstoa ; ap this work 
   plym001 
   pbluescript sk(+ ) derivative containing stoa on a 2-kb fragment ; 
  ap this work 
   plym004 
   plym001 derivative encoding c68a stoa ; ap this work 
   plym006 
   plym001 derivative encoding e71q stoa ; ap this work 
   plym009 
   plym001 derivative encoding c65a stoa ; ap this work 
   plym012 
   plle83 variant encoding e71q stoa ; km this work 
   plym013 
   plle83 variant encoding c68a stoa ; km this work 
   plym015 
   plle83 variant encoding c65a stoa ; km this work 
   plym025 
   pcr - blunt - ii - topo containing stoa ; km this work 
   plym028 
   pgex4t1 derivative encoding gst - sstoa fusion protein ; ap this work 
   plym031 
   plym028 variant encoding gst - sstoa with c65a mutation ; ap this work 
   plym032 
   plym028 variant encoding gst - sstoa with c68a mutation ; ap this work 
 
  plym033 
 
  plym028 variant encoding gst - sstoa with e71q mutation ; ap 
  this work 
  aap , cm , em , km , and 
  tc indicate resistance to ampicillin , chloramphenicol , 
  erythromycin , kanamycin , and tetracycline , respectively .
strains and plasmids used in this work ap , cm , em , km , and 
  tc indicate resistance to ampicillin , chloramphenicol , 
  erythromycin , kanamycin , and tetracycline , respectively .
construction of plasmids encoding sstoa  for production of 
  sstoa plasmid plmc19 was constructed by amplifying part of the stoa 
  gene using oligonucleotides le051 and le052 ( supplemental table s1 ) , phusion 
  polymerase ( finnzymes ) , and b. subtilis 1a1 chromosomal dna as 
  template .
the pcr product was cloned into pcr-blunt - ii - topo 
  ( invitrogen ) .
the insert was cut out from the plasmid using psti and hindiii 
  and ligated into pbadmyc - hisc cut with the same enzymes resulting in plasmid 
  plmc19 .
the cloned stoa fragment was verified by dna sequence 
  analysis . for production of a thrombin - cleavable gst - sstoa fusion protein ,
a fragment 
  of the stoa gene encoding residues 21 - 165 of stoa was first amplified 
  by pcr as above using oligonucleotides ly001 and ly002 as primers and 
  subsequently cloned into pcr - blunt - ii - topo generating plym025 , which was 
  verified by sequencing .
plym025 , propagated in e. coli strain mm294 , 
  was digested by bamhi and sali , and the stoa fragment was cloned into 
  pgex4t1 , resulting in plym028 . for the construction of plasmids encoding mutant stoa variants ,
plle83 was 
  digested by hindiii and bamhi , and the 2-kb fragment containing stoa 
  was cloned in pbluescript sk(+ ) , resulting in plym001 .
site - directed 
  mutagenesis was carried out with the quikchange ii kit and protocol 
  ( stratagene ) using plym001 and primers ly003-ly008 to generate plasmids 
  plym009 ( c65a ) , plym004 ( c68a ) , and plym006 ( e71q ) , respectively , which were 
  verified by sequencing .
the hindiii / bamhi fragment of each of these three 
  plasmids was subsequently cloned into pdg148 , generating , respectively , 
  plym015 , plym013 , and plym012 , which were used for expression of full - length 
  mutant stoa genes in b. subtilis .
plasmids encoding 
  gst - sstoa fusion protein with c65a or c68a in sstoa were obtained by first 
  amplifying stoa as above using primers ly001 and ly002 and plym009 or 
  plym004 plasmid dna , respectively , as template .
pcr products were then cloned 
  into pgex4t1 as described above for the wild - type variant , generating plasmids 
  plym031 and plym032 , which were verified by sequencing .
purification of sstoa  non - tagged sstoa , which was utilized 
  in initial crystallization trials and to generate a stoa antiserum , was 
  purified from e. coli top10/plmc19 as described in the supplemental 
  data .
for the production of gst - sstoa fusion protein , e. coli 
  bl21/plym028 was grown in 1-liter portions in 5-liter e - flasks . at 
  a600
= 0.6 - 0.8 expression was induced by addition of 1 
  mm isopropyl -d - thiogalactoside ( final 
  concentration ) .
after incubation for 5 h , cells were collected by 
  centrifugation , washed in pbs ( 140 mm nacl , 2.7 mm kcl , 
  10 mm na2hpo4 , 1.8 mm 
  kh2po4 , ph 7.3 ) , and stored as pellets at -20 c 
  until required . the cell pellet from 1 liter of culture was suspended in 20 ml 
  of ice - cold pbs containing 1 mm dtt and lysed by passage ( three 
  times ) through a french pressure cell at 18,000 p.s.i .
the lysate was 
  centrifuged at 48,000  g for 40 min at 4 c , and the 
  supernatant was centrifuged at 100,000  g for 1 h at 4 c . 
 
the final supernatant was mixed with 2 ml of 50% slurry of 
  glutathione - sepharose 4b ( ge healthcare ) , and the gst - sstoa fusion protein was 
  purified according to the resin manufacturer 's instructions .
affinity - purified 
  gst - sstoa fusion protein was cleaved by 50 units of thrombin ( ge healthcare ) 
  at room temperature for 5 h and then loaded onto a sephacryl s-100 hr gel 
  filtration column .
protein was eluted using 20 mm tris - hcl , ph 8.0 , 
  containing 100 mm nacl and 1 mm dtt .
fractions 
  containing sstoa were identified using sds - page and western blot with stoa 
  antiserum , pooled , and concentrated .
the n - terminal amino acid residue 
  sequence of the purified protein was verified by edman degradation ( see 
  fig .
cysteine variants of 
  sstoa were produced in e. coli bl21 containing plym031 or plym032 and 
  purified as described for wild - type sstoa .
for the production of selenomethionine - labeled sstoa , e. coli 
  b834(de3)/plym028 was grown overnight in 10 ml of selenomet medium 
  ( athenaes ) supplemented with 50 g / ml methionine .
the overnight culture was 
  used to inoculate 1 liter of selenomet medium containing 50 g/l 
  methionine to an a600 of 0.1 , and the culture was grown 
  until a600 was 0.8 .
cells were harvested by 
  centrifugation for 10 min at 10,000  g at 4 c , and the 
  pellet was resuspended in 1 liter of non - supplemented selenomet medium and 
  incubated for 2 h. selenomethionine was then added to a final concentration of 
  50 g / ml , and the culture was incubated for a further 30 min when 
  production of gst - sstoa was induced by the addition of 1 mm 
  isopropyl -d - thiogalactoside ( final concentration ) .
four 
  hours after induction the culture was harvested by centrifugation for 10 min 
  at 10,000  g at 4 c .
the cells were washed in cold pbs 
  and stored as a pellet at -20 c .
a 2-l 
  sitting drop was formed by mixing equal volumes of protein solution ( 12 mg / ml 
  sstoa in 25 mm mops , ph 7.0 ) and crystallization reagent ( 27% ( w / v ) 
  peg 2000 , 0.2 m ammonium acetate , 100 mm sodium acetate , 
  ph 4.8 ) over an 800-l reservoir of the reagent alone .
crystals grew over a 
  period of 1 - 2 days and were cryoprotected in a solution of 30% ( w / v ) peg 2000 , 
  100 mm sodium acetate , ph 4.8 , 20% ( v / v ) ethylene glycol before 
  flash freezing in liquid nitrogen .
x - ray data sets for native and 
  selenomethionine - labeled sstoa were collected on beam line id23 - 1 of the 
  european synchrotron radiation facility ( grenoble , france ) .
structure 
  determination utilized programs of the ccp4 
  ( 16 ) and phenix 
  ( 17 ) software suites . 
 
diffraction patterns were indexed and integrated with mosflm 
  ( 18 ) and scaled with scala 
  ( 19 ) .
selenium sites were 
  identified using a combination of automated methods implemented in phenix.hyss 
  and manual inspection of anomalous difference maps produced with fft .
an 
  initial electron density map was obtained using sad phases calculated with 
  mlphare and subsequent density modification with dm .
a key factor in producing 
  an interpretable electron density map was the identification of the correct 
  non - crystallographic symmetry relating each sstoa molecule in the asymmetric 
  unit .
non - crystallographic symmetry averaging in the phase improvement 
  procedure benefited from the use of a predefined protein mask derived from a 
  monomer of resa ( 12 ) .
further 
  phase improvement was obtained by cross - crystal averaging with a second 
  sad - phased selenomethionine data set composed of merged data from two 
  individual sstoa crystals .
manual model building was conducted in coot , and 
  initial phased refinement of the model was conducted with refmac 
  ( 20 ) .
further refinement of 
  the model ( against a single selenomethionine data set with a  low  
 
twin fraction (  = 0.36 ) ) utilized phenix.refine , which was essential 
  for proper refinement of the twinned data .
the final model of sstoa is 
  composed of seven ordered protein chains and 99 water molecules .
a small 
  amount of residual density located at a coordinate of ( 42.34 , 64.52 , 19.53 ) 
  may indicate the presence of an additional stoa monomer of low occupancy and 
  high mobility that is insufficiently well resolved to enable further model 
  building . the coordinates and structure factors
reduction potential determination  sstoa ( 0.2 
  m ) in 50 mm potassium phosphate , ph 7 , was added to 
  5 mm oxidized dtt in the same buffer to obtain the fully oxidized 
  protein .
the protein was subsequently titrated with reduced dtt in the same 
  buffer , allowing 10 min for the protein to equilibrate to each new potential . 
 
the transition from oxidized to reduced protein was monitored by the increase 
  in tryptophan fluorescence emission at 344 nm ( excitation at 280 nm ) measured 
  at 25 c using a perkinelmer life sciences ls-55 fluorescence spectrometer 
  with 10-nm excitation and emission slits .
midpoint reduction potentials were 
  determined as described previously 
  ( 11 , 
  21 ) ; further details are given 
  in the supplemental data .
ph stability and cysteine pka 
  measurements  reduced wild - type and variant sstoa protein stocks 
  were prepared in 10 mm mops , ph 7 , 2 mm tcep ( pierce ) 
  and subsequently diluted ( 30-fold to a final protein concentration of 0.15 
  m ) with pctc ( potassium phosphate , sodium citrate , tris , and 
  ches , all at 50 mm ) buffer ( pre - prepared at the appropriate ph ) and 
  equilibrated in a sealed cuvette for 1 h before measurement of tryptophan 
  fluorescence spectra as above . for pka measurements , 
  reduced protein solutions ( wild - type sstoa and single cysteine variants ) were 
  prepared in 10 mm mops , ph 7 , with 2 mm tcep as 
  reductant .
reaction with 6-bromoacetyl-2-dimethylaminonaphthalene was carried 
  out under pseudo - first order conditions , and pka values 
  were determined as described previously 
  ( 13 ) .
modification of sstoa with mal - peg  wild - type sstoa and c65a 
  and c68a variant proteins in 20 mm tris - hcl , 100 mm 
  nacl , ph 8.0 were treated with 1 mm tcep at room temperature for 30 
  min .
excess tcep was removed using a ym10 column ( millipore ) , and each reduced 
  sstoa sample ( 10 g ) was incubated with 0.1 mm or 1 
  mm monomethyl polyethylene glycol 5000 2-maleimidoethyl ether 
  ( mal - peg ) ( 90% ; fluka ) at room temperature for 30 min .
non - tagged sstoa was used 
  to immunize rabbits ( custom polyclonal antibody production service ; medprobe , 
  oslo , norway ) . for western blot analysis ,
proteins were separated by sds - page 
  using the schgger and von jagow 
  ( 22 ) system and subsequently 
  electroblotted to a polyvinylidene difluoride membrane ( millipore ) by wet blot 
  using 20 mm tris , 150 mm glycine , 20% ( v / v ) methanol . 
 
the membrane was 
  blocked using 5% ( w / v ) nonfat dry milk in 0.1% ( v / v ) tween 20 in pbs .
bound primary antibodies were 
  detected using horseradish peroxidase - linked anti - rabbit antiserum from donkey 
  ( ge healthcare ) diluted 3000-fold .
immunodetection was carried out by 
  chemiluminescence using supersignal west pico substrate ( pierce ) and an 
  eastman kodak co. image station .
preparation of cell - free extracts from b. subtilis 
  strains  samples of 200 ml were taken from a 1.5-liter culture at 
  time points spanning from 1 h before entry into postexponential ( t = 
  -1 ) to 5 h into stationary growth phase ( t = 5 ) .
cells were harvested 
  by centrifugation ; immediately washed in 50 mm potassium phosphate , 
  ph 8.0 ; and frozen as a pellet at -20 c . when required , cell pellets were 
  thawed and suspended in 0.8 ml of phosphate buffer containing 0.7 mg / ml 
  lysozyme , 25 g / ml dnase , 25 g / ml rnase , 4 mm 
  mgso4 , and complete protease inhibitor ( roche applied science ; one 
  tablet/50 ml of buffer ) ) .
after incubation at 37 c for 45 min , an aliquot 
  was frozen for subsequent analysis of total cell lysate .
the remaining main 
  part of the lysate was centrifuged at 48,000  g for 60 min at 
  4 c : the supernatant was used for the analysis of the soluble cell 
  fraction , whereas the pellet , after washing in phosphate buffer , was suspended 
  in 0.3 ml of the buffer and used for the analysis of the membrane 
  fraction .
other methods  chromosomal dna from b. subtilis was 
  isolated according to marmur 
  ( 23 ) .
plasmid dna was isolated 
  using quantum miniprep ( bio - rad ) or by cscl density gradient centrifugation . 
 
sds - page was carried out using the nupage system ( invitrogen ) or schgger 
  and von jagow ( 22 ) system . 
 
protein concentrations were determined by measuring the absorbance at 280 nm 
  using an extinction coefficient of 15,460  100 
 
m cm determined as described previously 
  ( 25 ) or using the bca reagent 
  ( bio - rad ) with bovine albumin as reference .
n - terminal sequencing was carried out by edman degradation ( protein 
  analysis center , karolinska institutet , sweden ) on proteins separated by 
  sds - page and electroblotted onto a polyvinylidene difluoride membrane as 
  described for western blotting .
the membrane was stained with 0.1% ( w / v ) 
  coomassie brilliant blue r-250 in 2% ( v / v ) acetic acid , 45% ( v / v ) methanol . 
 
mass spectrometry analysis was performed using an ultraflex - maldi - tof / tof mass 
  spectrometer ( bruker daltonics , coventry , uk ) on samples prepared by mixing 
  sstoa in a 1:1 ratio with a saturated solution of sinapinic acid matrix in 30% 
  acetonitrile , 0.05% trifluoroacetic acid .
0.5 l of this combined mixture 
  was spotted onto a polished stainless steel target and allowed to crystallize 
  prior to analysis .
the spectrometer was externally calibrated using a 
  two - point linear calibration through the singly and doubly charged ions of 
  trypsinogen .
the molecular mass of sstoa was determined using high performance liquid 
  chromatography with an ultraspherogel sec 3000 column ( beckman ) .
1.2 nmol of 
  sstoa in 10 l was applied to the column equilibrated with 20 mm 
  tris - hcl , ph 8.0 , 0.15 m nacl , 1 mm dtt and eluted in 
  the same buffer at a flow rate of 1 ml min .
mass was calculated 
  based on a calibration curve obtained using catalase ( 230 kda ) , bovine serum 
  albumin ( 67 kda ) , ovalbumin ( 46 kda ) , carboanhydrase ( 26 kda ) , myoglobin ( 17.2 
  kda ) , and horse heart cytochrome c ( 12.3 kda ) .
stoa is a membrane - bound protein  from its amino acid 
  sequence ( fig .
1 ) , stoa was 
  predicted to be a ditopic membrane - bound protein with an n - terminal 
  30-residue segment that anchors the 135-residue tdor domain to the 
  membrane . as shown previously by the use of a stoa - phoa ( alkaline phosphatase ) 
  fusion protein in e. coli cells , the transmembrane segment of stoa 
  functions as a signal sequence to direct translocation and to membrane anchor 
  the c - terminal tdor domain ( 2 ) . 
 
experiments in b. subtilis indicated that the function of stoa is not 
  dependent on the membrane anchor 
  ( 3 ) , and prediction programs 
  suggested that the n - terminal segment might be cleaved off after the tdor 
  domain has been translocated across the membrane . to establish whether stoa is 
  a membrane - bound protein , b. subtilis strain 1a1 was grown in 
  nutrient sporulation medium with phosphate for sporulation , and samples taken 
  at different time points during growth were analyzed for stoa by using western 
  blot with polyclonal antiserum directed against the tdor domain of stoa .
no 
  stoa antigen could be detected in cell - free extracts , although bdbd , a protein 
  very similar to stoa , was readily detected in all samples ( not shown ) .
to 
  facilitate detection of stoa by increasing the level of the protein in cells , 
  plasmid plle83 containing stoa under control of the pspac promoter 
  was used .
stoa was found in the membrane fraction of 1a1/plle83 cells from 
  early stationary phase cultures but not in late stationary phase cultures 
  ( fig .
the results indicate 
  that stoa is membrane - associated but is present in very low amounts and is 
  degraded or trapped in maturing endospores so that it is not detectable by the 
  western blot procedure used .
figure 1.amino acid residue sequence alignment of b. subtilis sstoa used 
  in structural and biochemical analyses , full - length stoa , and full - length resa 
  proteins .
stars 
  indicate the active site residues cys-65 and cys-68 and conserved residue 
  glu-71 of stoa .
amino acid residue sequence alignment of b. subtilis sstoa used 
  in structural and biochemical analyses , full - length stoa , and full - length resa 
  proteins .
stars 
  indicate the active site residues cys-65 and cys-68 and conserved residue 
  glu-71 of stoa .
western blot analysis of total 
  cell - free lysates , membrane fractions , and soluble fractions of b. 
  subtilis 1a1/plle83 for stoa antigen is shown .
cells were harvested at 
  different time points during growth in nutrient sporulation medium with 
  phosphate .
time point 0 is at the entry of stationary growth phase , and 
  subsequent numbers indicate hours into stationary phase . 
 
sstoa indicates a sample of purified sstoa loaded on the gel as a 
  reference .
approximately 20 g of cell protein was loaded in each lane 
  except for purified sstoa where 20 ng was loaded .
western blot analysis of total 
  cell - free lysates , membrane fractions , and soluble fractions of b. 
  subtilis 1a1/plle83 for stoa antigen is shown .
cells were harvested at 
  different time points during growth in nutrient sporulation medium with 
  phosphate .
time point 0 is at the entry of stationary growth phase , and 
  subsequent numbers indicate hours into stationary phase . 
 
sstoa indicates a sample of purified sstoa loaded on the gel as a 
  reference .
approximately 20 g of cell protein was loaded in each lane 
  except for purified sstoa where 20 ng was loaded . in vivo
functional analysis of active site variants of 
  stoa  to establish that the function of stoa in endospore biogenesis 
  is dependent on the cysteine residues of the protein , b. subtilis 
  strain lul20 in which the stoa gene is inactivated and strain lul30 
  in which stoa is deleted from the chromosome were used 
  ( 2 ) .
these two strains 
  containing a plasmid encoding wild - type stoa ( plle83 ) , c65a stoa ( plym015 ) , 
  c68a stoa ( plym013 ) , or empty vector ( pdg148 ) were grown for sporulation and 
  tested for production of heat - resistant cells .
western blot analysis showed 
  that stoa proteins were present in membranes of strains containing plym015 and 
  plym013 ( see supplemental fig .
, the presence of 
  either stoa variant resulted in a 100-fold reduction in sporulation 
  efficiency ( table 2 ) .
cells 
  completely lacking stoa , however , showed more than a 1000-fold reduction in 
  sporulation efficiency indicating some residual activity of stoa even when one 
  of the two cysteine residues is missing .
table 2efficiency of b. subtilis strains in producing heat - resistant 
  cells after growth for 2 days at 37 c in nutrient sporulation medium with 
  phosphate supplemented with 1 mm isopropyl 
  -d - thiogalactosidepresented are typical results obtained from at least two independent 
  experiments with each strain , including analysis of two sister clones .
b. 
  subtilis lul20 and lul30 are stoa - deficient , and 1a1 is the parental 
  strain ( table 1).strainstoa variant encoded by plasmidviable count before heatingviable count after 15 min at 80 csporulation 
  efficiencya % 
   1a1 
   4.0  10 3.4  10 85 
   lul20 
   5.5  10 2.0  10 < 0.05 
   lul20/pdg148 
   4.7  10 1.2  10 < 0.05 
   lul20/plle83 
   wild type 
   2.6  10 1.3  10 50 
   lul20/plym015 
   c65a 
   4.9  10 3.7  10 0.7 
   lul20/plym013 
   c68a 
   7.4  10 5.0  10 0.7 
   lul20/plym012 
  
e71q 
   4.1  10 2.1  10 5.1 
   lul30 
   1.9  10 1.4  10 < 0.05 
   lul30/pdg148 
   2.1  10 3.7  10 < 0.05 
   lul30/plle83 
   wild type 
   1.4  10 8.6  10 61 
   lul30/plym015 
   c65a 
   4.9  10 9.2  10 0.2 
   lul30/plym013 
   c68a 
   4.6  10 6.2  10 0.1 
 
  lul30/plym012 
 
  e71q 
 
  3.9  10 
  1.6  10 
  0.4 
  aviable count after heat treatment divided by that before heating .
efficiency of b. subtilis strains in producing heat - resistant 
  cells after growth for 2 days at 37 c in nutrient sporulation medium with 
  phosphate supplemented with 1 mm isopropyl 
  -d - thiogalactoside presented are typical results obtained from at least two independent 
  experiments with each strain , including analysis of two sister clones .
b. 
  subtilis lul20 and lul30 are stoa - deficient , and 1a1 is the parental 
  strain ( table 1 ) .
the high resolution structure of the soluble domain of stoa in a 
  mixture of oxidized and reduced states
 sstoa ( residues 22 - 165 ) was 
  produced in e. coli and purified .
gel filtration analysis indicated a 
  molecular mass of 17  3 kda ( data not shown ) consistent with the 
  protein being monomeric in solution .
the crystal structure of sstoa was solved 
  using the selenomethionine sad method of phase determination .
the crystals 
  used in structure determination belong to space group p31 and 
  contained seven molecules of sstoa per asymmetric unit ( see supplemental fig . 
 
although structure determination was hampered by the fact that sstoa 
  crystals were merohedrally twinned , it was nonetheless possible to refine the 
  structure to acceptable rwork and 
  rfree values using twin refinement against x - ray data 
  obtained from a single sstoa crystal with a twin fraction (  ) of 0.36 . 
 
data collection and refinement statistics for the sstoa structure are given in 
  table 3 .
table 3x - ray data collection and refinement statistics for sstoase - met , selenomethionine ; r.m.s . , root mean square
. values in parentheses 
  represent the highest resolution shell.two-crystal merged se - met data sethires se - met data set space group 
   p31 p31 cell parameters (  ) 
  a = b = 133.74 , c = 64.82 
  a = b = 133.72 , c = 64.82 
   energy ( ev ) 
   12,656.6 
  
12,656.6 
  f 
   7.79 
   7.79 
  f 
   6.38 
   6.38 
   twinning operator 
   -k , -h , -l -k , -h , -l twinning fraction 
   0.38 
   0.36 
   resolution (  ) 
   43.19 - 2.60 ( 2.74 - 2.60 ) 
   36.27 - 2.50 ( 2.64 - 2.50 ) 
  rsym 0.154 ( 0.565 ) 
   0.083 ( 0.293 ) 
  i/ 
   18.9 ( 4.0 ) 
   16.1 ( 3.3 ) 
   anomalous completeness ( % ) 
   99.4 ( 96.0 ) 
   97.2 ( 83.8 ) 
   anomalous multiplicity 
   5.0 ( 3.6 ) 
   2.5 ( 1.7 ) 
   unique observations 
   44,319 ( 6,023 ) 
  r 0.1787 
  rfree 0.2011 
   r.m.s .
angle (  ) 
 
  2.191 
   x - ray data collection and refinement statistics for sstoa se - met , selenomethionine ; r.m.s . , root mean square .
values in parentheses 
  represent the highest resolution shell . overall the structure of each sstoa monomer 
  ( fig .
3a ) can be 
  described as a modified thioredoxin - like fold that is highly reminiscent of 
  b. subtilis resa 
  ( 12 ) , ccmg ( from e. 
  coli and bradyrhizobium japonicum ) 
  ( 26 , 
  27 ) , and mycobacterium 
  tuberculosis dsbe ( 28 ) , 
  which are all extracytoplasmic tdors ( see 
  fig .
3b ) . like these 
  proteins , the classical thioredoxin - like motif of stoa
is embellished by a 
  central  insertion and an n - terminal -hairpin ( in addition 
  to the transmembrane helix predicted from primary sequence analysis ) . unlike 
  other extracytoplasmic tdors
, stoa also possesses a short insertion of 
  residues between strand 4 and helix 2 that forms an ordered loop 
  at the surface of the protein .
figure 3.the three - dimensional structure of the soluble domain of stoa . 
 a , three - dimensional structure of sstoa showing that the protein 
  exhibits a classical thioredoxin - like fold with two significant insertions : 
  the n - terminal region contains a two - stranded , antiparallel hairpin , whereas 
  the central insert , located after the 3-1-4 motif of the 
  thioredoxin fold , comprises one helix ( 2 ) and one strand ( 5 ) . 
 
secondary structure elements are labeled from the n terminus ( with the 
  n - terminal transmembrane helix being 0 ) , and the n and c termini of sstoa are 
  indicated .
b , overlay of the stoa ( gray ) and reduced resa 
  ( yellow ) peptide backbones ( in ribbon representation ) .
c , 
  the active site region of stoa showing the cppc motif , surrounding residues , 
  and a buried water molecule ( red sphere ) .
all structural figures were 
  prepared with pymol ( 44 ) and 
  annotated with gimp .
the three - dimensional structure of the soluble domain of stoa . 
 a , three - dimensional structure of sstoa showing that the protein 
  exhibits a classical thioredoxin - like fold with two significant insertions : 
  the n - terminal region contains a two - stranded , antiparallel hairpin , whereas 
  the central insert , located after the 3-1-4 motif of the 
  thioredoxin fold , comprises one helix ( 2 ) and one strand ( 5 ) . 
 
secondary structure elements are labeled from the n terminus ( with the 
  n - terminal transmembrane helix being 0 ) , and the n and c termini of sstoa are 
  indicated .
b , overlay of the stoa ( gray ) and reduced resa 
  ( yellow ) peptide backbones ( in ribbon representation ) .
c , 
  the active site region of stoa showing the cppc motif , surrounding residues , 
  and a buried water molecule ( red sphere ) . all structural figures were 
  prepared with pymol ( 44 ) and 
  annotated with gimp . in the structure determined here , the active site cysteines of sstoa appear 
  as a mixture of oxidized and reduced redox states ( in each monomer ) . 
 
crystallization of sstoa utilized solely the oxidized form of the protein , and 
  thus it is likely that partial reduction of the disulfide bond was induced by 
  photoreduction in the x - ray beam . the electron density associated with the 
  partially broken disulfide
4a along with separated models of the oxidized and 
  reduced conformations shown in fig . 4 , 
  b and c , respectively .
figure 4.the active site of stoa in oxidized and reduced states . a , 
  electron density ( contoured at 1.2  ) of the active site cppc motif of 
  stoa reveals a mixture of oxidized and reduced states .
b and 
  c , separated representations of the active site region in oxidized 
  and reduced states , respectively .
the active site of stoa in oxidized and reduced states . a , 
  electron density ( contoured at 1.2  ) of the active site cppc motif of 
  stoa
b and 
  c , separated representations of the active site region in oxidized 
  and reduced states , respectively .
a , fluorescence spectra of sstoa 
  in 50 mm potassium phosphate , 5 mm oxidized dtt , ph 7.0 
  following incubation with increasing concentrations of reduced dtt at 25 
  c .
b , plot of fraction of reduced sstoa ( calculated from the 
  fluorescence ( fluor . )
intensity at 344 nm as described in the 
  supplemental data ) as a function of the cell potential .
a , fluorescence spectra of sstoa 
  in 50 mm potassium phosphate , 5 mm oxidized dtt , ph 7.0 
  following incubation with increasing concentrations of reduced dtt at 25 
  c .
b , plot of fraction of reduced sstoa ( calculated from the 
  fluorescence ( fluor . )
intensity at 344 nm as described in the 
  supplemental data ) as a function of the cell potential .
the oxidized ( disulfide - bonded ) conformation of the active site dominates 
  the electron density and is best described as adopting a classical 
  right - handed hook conformation with a 3 angle of 73.5  1.8 
  formed between the two cysteine residues .
the sulfur - to - sulfur bond distance 
  for the oxidized conformation is 2.06  , whereas in the reduced 
  conformation an average sulfur - to - sulfur distance of 3.4  separates the 
  cysteine residues .
this distance is considerably shorter than that observed 
  for the structure of resa that has an exceptionally long sulfur - to - sulfur 
  distance of 4.5  in the reduced state 
  ( 12 ) .
partial reduction of the 
  disulfide does not seem to cause significant rearrangement of the local 
  protein structure , and thus , unlike resa , there is no evidence for any 
  redox - linked conformational changes due to reduction of the cysteines in 
  stoa . in
almost all known natural thioredoxin - like proteins , at least one of the 
  two residues that intervene between the active site cysteines residues 
  ( i.e. within the cxxc motif ) is a proline . in stoa , 
  both of these intervening residues are proline .
pro-66 and pro-67 
  both adopt the trans conformations and have backbone - 
  angles that are consistent with an -helical conformation .
like all 
  other thioredoxin - like tdors , the cxxc motif of stoa is found at the 
  n terminus of a reasonably long -helix ( 1 in stoa ) , and the 
  macrodipole arising from this helix is often invoked as a primary cause of the 
  lowered pka values associated with the more n - terminal 
  cysteine residue of the cxxc motif in most tdors 
  ( 29 ) .
the presence of proline 
  residues at the cap of this active site helix in stoa is therefore likely to 
  have important consequences for the distribution of the electrostatic field 
  near the cysteines as proline does not possess a standard peptide group
. 
  furthermore the limited conformational freedom of proline ( in comparison with 
  other residues ) may be an important factor in maintaining rigidity of the cppc 
  motif and may be one of the reasons that the reduced form of sstoa is so 
  similar to its oxidized form . a further proline residue ( pro-135 ) , which is in the cis 
  conformation and is conserved in all thioredoxin - like proteins , is found in 
  van der waals contact with two buried polar residues , a histidine and a 
  glutamate , which are located immediately behind the second cysteine of the 
  active site motif .
his-59 is located on strand 3 , whereas glu-71 arises 
  from helix 1 directly opposite .
a buried water molecule is observed in 
  the space between these two residues within hydrogen - bonding distance of the 
  cys-68 sulfur ( see fig . 
 
the arrangement of these two buried polar residues and the intervening 
  water molecule ( in stoa ) is very similar to that observed in resa where the 
  glutamate is conserved ( glu-80 ) and an asparagine ( asn-68 ) residue takes an 
  equivalent position to that of his-59 .
substitution of glu-80 in resa has been 
  shown previously to have a significant effect on the active site properties of 
  the enzyme ; for example , the pka values of both active 
  site cysteines were significantly lowered in an e80q variant 
  ( 13 ) , and a b. 
  subtilis strain containing e80q resa was also severely impaired in its 
  ability to mature c - type cytochromes 
  ( 14 ) .
thus , it may be that 
  these buried polar residues are also important in stoa function . to analyze 
  the functional role of residue glu-71 in stoa ,
the stoa - deficient b. 
  subtilis strains lul20 and lul30 containing plasmid plym012 encoding e71q 
  stoa were studied .
the presence of the variant stoa protein in membranes was 
  confirmed by western blot ( supplemental fig .
s1 ) , and the efficiency in 
  production of heat - resistant endospores was found to be 10 - 150-fold ( depending 
  on strain ) lower in these strains compared with wild - type controls 
  ( table 2 ) .
stoa is a low potential tdor  the reduction potential of the 
  active site cysteines of sstoa was measured using the difference in tryptophan 
  fluorescence intensity of oxidized and reduced sstoa to follow oxidation state 
  as a function of reduction potential ( see 
  fig .
the data fitted well 
  to the nernst equation , giving a midpoint reduction potential of -248  
  2 mv versus normal hydrogen electrode at ph 7 with n = 2.18 
   0.16 , as expected for a two - electron reduction process .
this value is 
  similar to that measured for b. subtilis resa ( -256 mv at ph 7 ) 
  ( 21 ) and e. coli 
  thioredoxin ( -270 mv at ph 7 ) 
  ( 30 , 
  31 ) and is entirely consistent 
  with the structural similarity between these proteins and a role for stoa in 
  the reduction of ( as yet unidentified ) proteins involved in endospore cortex 
  synthesis ( 2 , 
  9 ) .
sds - page of 
  purified reduced wild - type ( wt ) sstoa and c65a and c68a sstoa 
  following reaction with mal - peg is shown .
the protein variants were incubated 
  with 0 , 0.1 , and 1 mm mal - peg before electrophoresis as indicated , 
  and the gel was stained for protein .
sds - page of 
  purified reduced wild - type ( wt ) sstoa and c65a and c68a sstoa 
  following reaction with mal - peg is shown .
the protein variants were incubated 
  with 0 , 0.1 , and 1 mm mal - peg before electrophoresis as indicated , 
  and the gel was stained for protein .
both active site cysteines can be modified by alkylating 
  agents  the solvent accessibility of the active site cysteines of 
  wild - type sstoa and two variants , c65a and c68a sstoa , was investigated using 
  mal - peg , a high molecular mass , cysteine - specific alkylating agent .
sstoa 
  proteins were incubated with either 0 , 0.1 , or 1 mm mal - peg as 
  described under 
experimental procedures  and analyzed by 
  sds - page . samples with exposed thiols are able to react with the mal - peg to 
  form covalent complexes of significantly greater molecular mass relative to 
  non - alkylated samples and thus are retarded during subsequent migration in the 
  electrophoretic gel ( see fig . 
 
unmodified wild - type , c65a , and c68a sstoa variants migrated 
  with an apparent molecular mass of 18 kda , which is in reasonable 
  agreement with the actual mass ( 16.4 kda ) . in the presence of mal - peg , both 
  single cysteine variants reacted to give a single species with a significantly 
  lower mobility .
protein molecular mass standards can not be used to judge the 
  mass of mal - peg - modified proteins , but the significant retardation of sstoa 
  variants is consistent with the alkylation of a single cysteine residue in 
  each .
in contrast , wild - type sstoa gave rise to two bands when incubated with 
  0.1 mm mal - peg .
the lower ( faster running ) band corresponded to the 
  singly modified single cysteine variants , and we conclude that under these 
  conditions sstoa is present as a mixture of singly and doubly modified 
  molecules .
this was confirmed by incubating sstoa with a higher ( 1 
  mm ) concentration of mal - peg that resulted in the observation of 
  only the larger , slower running band corresponding to the doubly modified 
  protein .
in addition to these major bands , other much fainter bands were 
  observed on the gel ; these most likely arise from a small degree of 
  heterogeneity in the size of the peg adducts in the mal - peg reagent and 
  therefore do not represent additional ( non - cysteine ) alkylation events or 
  protein heterogeneity .
certainly no protein heterogeneity was observed in any 
  of the untreated sstoa samples . from the structure it is apparent that the more n - terminal of the two 
  active site cysteines is exposed to the solvent , whereas the other is not . 
 
solvent - accessible surface area calculations ( using a solvent probe of 1.2 
   ) on the structure of reduced sstoa showed that the cys-65 sulfur has 
  an accessible surface area of 5.43  , whereas the sulfur 
  atom of cys-68 is inaccessible from the bulk solvent .
a similar arrangement 
  exists in all structurally characterized thioredoxin - like proteins , and in 
  virtually all of them , the second , buried cysteine thiol does not react with 
  modifying reagents in solution 
  ( 32 , 
  33 ) .
this is not the case for 
  b. subtilis resa in which we showed previously that both cysteines 
  are readily modified by alkylating reagents 
  ( 13 ) . to our knowledge ,
resa 
  and now stoa are the only examples where this behavior has been demonstrated . 
  for stoa ,
one possibility is that alkylation of the solvent - exposed cys-65 
  might cause a structural rearrangement that allows subsequent modification of 
  cys-68 . alternatively
the reduced protein in solution may undergo dynamic 
  motion that would allow occasional access to the sulfur of cys-68 .
pka values of stoa active site cysteines  the ph 
  stability profiles of the wild - type and single cysteine variant sstoa proteins 
  were first determined to verify the range of values over which the acid - base 
  properties of each protein could be investigated .
the intrinsic tryptophan 
  fluorescence was measured as a function of ph for each protein under reducing 
  conditions .
significant changes in the character of the tryptophan 
  fluorescence emission spectrum , resulting from unfolding of the proteins , were 
  observed at extremes of ph .
both the emission wavelength maxima and 
  fluorescence intensity maxima were affected by ph - induced unfolding .
the 
  former has the advantage of being independent of protein concentration and was 
  thus used preferentially in monitoring ph stability ( see 
  fig .
the data showed that 
  wild - type sstoa is stable between ph 3.5 and 9.3 , whereas c65a and c68a sstoa 
  variants are stable between 4.4 and 9.6 and between 3.7 and 9.3 , 
  respectively .
the acid - base properties of the active site cysteines of wild - type sstoa 
  and the c65a and c68a variants were investigated by measuring rates of 
  reaction with the fluorescent probe 6-bromoacetyl-2-dimethylaminonaphthalene 
  as described under  experimental procedures  ( see 
  fig .
the 
  fluorescence is sensitive to the environment of the modified cysteine with 
  emission occurring in the range of 440 - 550 nm , depending on the solvent 
  exposure of the modified cysteine 
  ( 34 ) . here
the emission maxima 
  for cys-65 and cys-68 were 510 nm , indicating that the fluorescent group of 
  both modified residues is located in a relatively solvent - exposed position . 
 
for the single cysteine variants of sstoa , data fitted well to an equation 
  describing a single protonation / deprotonation event , giving 
  pka values of 7.0  0.1 and 7.1  0.1 for 
  cys-65 and cys-68 , respectively ( see fig . 
  8 , c and d ) . for the wild - type protein
in which 
  both cysteine residues are intact , the data fitted well to two independent 
  protonation / deprotonation events , giving pka values of 5.5 
   0.4 and 7.8  0.2 ( fig . 
 
both of these values are lower than the 
  typical value of 8.5 - 9.0 observed for cysteine , and the data are 
  consistent with the reactivity of both residues toward alkylating reagents 
  ( which react with the deprotonated form only ) .
a low pka 
  value is normally observed for the n - terminal active site cysteine of 
  thioredoxin - like proteins , which in some cases exhibit pka 
  values as low as 3.5 ( 35 ) . 
 
however , the pka of the second cysteine is normally 
  estimated to be > 9 ( 32 , 
  36 , 
  37 ) . in this respect , stoa is 
  similar to resa in that the c - terminal cysteinyl has a pka 
  value low enough to be measurable in the stable ph range of the protein .
figure 7.ph stability of wild - type and variant sstoa proteins monitored by 
  fluorescence .
plots of tryptophan fluorescence emission maxima as a 
  function of ph for solutions of wild - type ( wt ) sstoa and c65a and 
  c68a sstoa as indicated ( all at 0.15 m in pctc buffer ) as a 
  function of ph are shown .
plots of tryptophan fluorescence emission maxima as a 
  function of ph for solutions of wild - type ( wt ) sstoa and c65a and 
  c68a sstoa as indicated ( all at 0.15 m in pctc buffer ) as a 
  function of ph are shown .
this large separation of pka values is consistent with 
  the close proximity of the two thiol groups , indicating that the ionization of 
  one significantly influences that of the other .
the wide separation of active 
  site thiol pka values appears to be a general feature of 
  tdors that act with low specificity 
  ( 33 , 
  38 ) . for sstoa
, we also 
  observed an interdependence of the cysteine acid - base properties : the sstoa 
  single cysteine variants have pka values that are very 
  similar , but in the wild - type protein they differ by more than 2 ph units . 
 
this suggests that in the wild - type protein cys-65 and cys-68 have a 
  significant effect on one another such that the presence of both cysteines 
  causes the pka of the n - terminal cysteine to drop , whereas 
  that of c - terminal cysteine rises ( relative to the respective single cysteine 
  variants )
. such interdependence of pka values was not 
  observed for resa for which respective cysteines in single cysteine variants 
  showed acid - base properties similar to those for the wild - type protein
. the 
  stronger interdependence of the cysteine pka values in 
  stoa may well be linked to the significantly shorter sulfur - to - sulfur distance 
  observed in reduced stoa ( 3.4  ) compared with that of resa 
  ( 4.5  ) ( 12 ) .
specificity determinants of stoa and resa  here we have 
  demonstrated that stoa and resa have many features in common .
the 
  three - dimensional structure , redox properties , and acid - base properties of 
  active site cysteine residues are similar in these proteins .
furthermore 
  in vivo , the two proteins are believed to interact with the same 
  integral membrane protein , ccda , which functions to supply electrons from 
  thioredoxin ( trxa ) in the cytoplasm to the extracytoplasmic compartment 
  ( 10 , 
  39 - 41 ) ; 
  thus , structural / physical features important for this interaction are expected 
  to be shared by stoa and resa . despite the similarities ,
so how do stoa and resa 
  achieve specificity for their particular substrates ? to try to answer this 
  question , it is important to identify regions of the proteins that do show 
  differences .
first , the two proteins differ in the active site sequence motif : cppc in 
  stoa and cepc in resa .
recently we reported the effects of altering the 
  dipeptide intervening sequence on the properties of resa , and this included a 
  resa cppc variant ( 21 ) . 
 
significant effects were observed : the redox potential increased by 25 
  mv , and the pka values of the two cysteines decreased by 
  1.8 and 1.6 ph units , respectively .
these findings are consistent with the 
  midpoint reduction potential and pka values measured here 
  for stoa and those previously reported for resa 
  ( 13 ) .
alteration of the 
  dipeptide sequence was also shown to impair the in vivo activity of 
  resa ( 21 ) . beyond their effect 
  on the biophysical properties of the active site cysteines ( i.e. 
  redox potential and pka values )
, the intervening two 
  residues may also be important for interaction with potential substrates .
the 
  close proximity of these residues to the active site cysteines and the fact 
  that both are exposed on the surface of the protein make it highly likely that 
  they contact partner proteins , at least transiently , and thus affect 
  specificity of interaction .
second , the protein surfaces close to the active site are subtly different 
  in stoa and resa . the structures of oxidized and reduced resa previously 
  revealed redox - linked conformational changes , the most significant of which 
  was the opening up of a hydrophobic cavity close to the active site upon 
  reduction ( 12 ) . despite the 
  conservation or conservative substitution of several of the residues that line 
  the resa cavity , reduction of stoa does not lead to the opening up of an 
  equivalent cavity ( see fig .
. 
  the lack of a cavity in stoa may be the result of the substitution of thr-159 
  ( in resa ) with pro-153 in stoa ; thr-159 undergoes one of the biggest 
  conformational movements upon formation of the hydrophobic cavity in resa , and 
  thus its replacement by a proline ( pro-153 ) in stoa might well restrict 
  conformational change in this region of the protein . alternatively the lack of 
  a cavity in stoa might be linked to the much smaller conformational change in 
  the cxxc motif itself , which is likely to be the driving force for 
  the larger conformational changes around the active site motif in resa .
a , time - dependent increases in 
  fluorescence at 510 nm upon reaction of wild - type ( wt ) sstoa ( 1 
  m ) with 6-bromoacetyl-2-dimethylaminonaphthalene ( 15 
  m ) in pctc buffer system at ph values from 5 to 9 as indicated 
  at 25 c .
plots were fitted ( solid lines ) to obtain an observed , 
  pseudo - first order rate constant , ko .
b , plot of 
  ko as a function of ph for wild - type sstoa .
c and d , 
  plots of ko as a function of ph obtained from similar 
  experiments with c65a and c68a sstoa , respectively .
pka plots for wild - type and single 
  cysteine sstoa variants . a , time - dependent increases in 
  fluorescence at 510 nm upon reaction of wild - type ( wt ) sstoa ( 1 
  m ) with 6-bromoacetyl-2-dimethylaminonaphthalene ( 15 
  m ) in pctc buffer system at ph values from 5 to 9 as indicated 
  at 25 c .
plots were fitted ( solid lines ) to obtain an observed , 
  pseudo - first order rate constant , ko .
b , plot of 
  ko as a function of ph for wild - type sstoa .
c and d , 
  plots of ko as a function of ph obtained from similar 
  experiments with c65a and c68a sstoa , respectively .
figure 9.a structural comparison between stoa and resa . a and 
  c , three - dimensional structures of sstoa and sresa , respectively , in 
  schematic representation .
regions colored red indicate areas 
  of high negative electrostatic potential , whereas blue areas indicate 
  areas of high positive potential .
the main 
  differences between sstoa and sresa are indicated ; see the main text for 
  details . a structural comparison between stoa and resa .
a and 
  c , three - dimensional structures of sstoa and sresa , respectively , in 
  schematic representation .
regions colored red indicate areas 
  of high negative electrostatic potential , whereas blue areas indicate 
  areas of high positive potential .
the main 
  differences between sstoa and sresa are indicated ; see the main text for 
  details .
it has been demonstrated that glu-80 of resa plays a key role in 
  vivo ( 14 ) , and in 
  vitro studies showed that it is important for the elevated 
  pka values of the active site cysteines and that it is 
  capable of hydrogen bonding to amino acid side chain residues bound in the 
  cavity ( 13 , 
  21 ) .
this led us to propose 
  that glu-80 is important for the binding of apocytochrome substrates to resa 
  ( 12 - 14 , 
  42 ) .
this residue is conserved 
  in stoa ( glu-71 ) , and we have shown here that it is also functionally 
  important in stoa ( table 2 ) . 
 
sequence alignments showed that it is conserved in many extracytoplasmic tdors 
  that are proposed to have a reductive function 
  ( 13 ) .
the data presented here 
  suggest that it fulfils a similar role in resa and stoa and also in other 
  tdors
. this could be in controlling the acid - base properties of the active 
  site cysteines or through direct participation in the reduction mechanism . 
 
therefore , it is highly unlikely that this glutamate is itself an important 
  determinant for specificity . it remains a possibility that , in resa , it 
  interacts directly with substrates , but this would be an additional role 
  facilitated by the formation of the hydrophobic cavity upon reduction of the 
  protein .
interaction of glu-71 with substrate is not favored in stoa because 
  the residue remains buried in both oxidation states .
third , with the exception of the aforementioned hydrophobic cavity in 
  ( reduced ) resa , the only major difference in the electrostatic surfaces of 
  each protein is in helix 3 ( stoa numbering ) , which is positively 
  charged in stoa and negatively charged in resa 
  ( fig . 9 ) .
however , this helix 
  is quite distant from the active site , and it seems unlikely that this feature 
  is responsible for differences in substrate recognition by resa and stoa . the final major difference is the presence in stoa of an extended loop 
  between strand 4 and helix 2 . in the primary sequence alignment 
  ( fig .
1 ) , this can be clearly 
  seen as an apparent insertion / deletion of several residues that are present in 
  the stoa but not in resa .
the sequence alignment also shows that there is 
  little similarity between the two proteins in this region .
the structure shows 
  that the extended 4-2 loop ( composed of ser-97 , glu-98 , gln-99 , 
  and asn-100 ) lies close to the active site of stoa 
  ( fig .
9 ) and represents the 
  most significant difference in the surface shape of stoa compared with resa 
  and is , therefore , likely to be important for the differential substrate 
  selectivity / specificity of these proteins . for example , this loop could be 
  involved in specific binding interactions with stoa substrate(s ) or could 
  serve to sterically hinder interactions with non - substrate molecules . concluding remarks  the structural , biochemical , and in 
  vivo characterization of b. subtilis stoa reported here provides 
  new knowledge about this unprecedented endospore biogenesis factor whose 
  physiological function is not completely understood 
  ( 9 ) . furthermore the data 
  reveal that this low potential extracytoplasmic tdor is remarkably similar to 
  resa , another well characterized extracytoplasmic tdor from the same organism 
  that is required for cytochrome c maturation .
it is thought that both 
  proteins interact with the same integral membrane protein , ccda , which 
  supplies them with electrons from the cytoplasm .
the high structural 
  similarity of resa and stoa is no doubt connected with their shared need to 
  interact with this protein . despite the large extent of their similarity ( in 
  both sequence and structure ) , the proteins can not functionally substitute for 
  one another in vivo .
bacteria usually contain several 
  thioredoxin - like proteins , soluble in the cytoplasm as well as membrane - bound . 
 
b. subtilis contains at least 10 such proteins , and none of these are 
  essential for growth , indicating a narrow substrate specificity for each 
  protein ( 43 ) .
the results of 
  this work raise important general questions about how tdors achieve substrate 
  specificity : resa can recognize at least four different apo - c - type 
  cytochrome polypeptides as substrates , whereas stoa recognizes a different but 
  as yet unknown substrate(s ) critical for endospore cortex biosynthesis . from 
  the structures of stoa and resa , we have identified four principal structural 
  differences between the two proteins that we believe provide the basis of 
  substrate specificity / selectivity .
the work demonstrates that 
  protein - substrate specificity / selectivity can apparently be achieved through 
  remarkably subtle variations in amino acid sequence and three - dimensional 
  structure .
 bacillus subtilis stoa is an extracytoplasmic thiol - disulfide 
  oxidoreductase ( tdor ) important for the synthesis of the endospore 
  peptidoglycan cortex protective layer . here 
 we demonstrate that stoa is 
  membrane - associated in b. subtilis and report the crystal structure 
  of the soluble protein lacking its membrane anchor . 
 this showed that stoa 
  adopts a thioredoxin - like fold with n - terminal and internal additions that are 
  characteristic of extracytoplasmic tdors . 
 the cxxc active site of the 
  crystallized protein was found to be in a mixture of oxidized and reduced 
  states , illustrating that there is little conformational variation between 
  redox states . 
 the midpoint reduction potential was determined as -248 mv 
  versus normal hydrogen electrode at ph 7 consistent with stoa 
  fulfilling a reductive role in endospore biogenesis . 
 pka 
  values of the active site cysteines , cys-65 and cys-68 , were determined to be 
  5.5 and 7.8 . 
 although cys-68 is buried within the structure , both cysteines 
  were found to be accessible to cysteine - specific alkylating reagents . in 
  vivo studies of site - directed variants of stoa revealed that the active 
  site cysteines are functionally important , as is glu-71 , which lies close to 
  the active site and is conserved in many reducing extracytoplasmic tdors . 
 the 
  structure and biophysical properties of stoa are very similar to those of 
  resa , a b. subtilis extracytoplasmic tdor involved in cytochrome 
  c maturation , raising important general questions about how these 
  similar but non - redundant proteins achieve specificity . 
 a detailed comparison 
  of the two proteins demonstrates that relatively subtle differences , largely 
  located around the active sites of the proteins , are sufficient to confer 
  specificity . 
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
Supplementary Material