nature 
 has evolved biominerals with complex morphologies and hierarchical 
 architectures that are hardly matched by synthetic materials so far . understanding the exquisite control exerted by 
 organisms over mineral properties might enable the exploitation of 
 natural design principles for the development of biomimetic functional 
 materials under physiological and environmentally friendly conditions . however , in many biomineralizing systems it is currently unclear 
 which of the many biological determinants are critical in controlling 
 particular material properties or steps in their formation such as 
 synthesis , nucleation , growth , and morphogenesis . in cases where the 
 molecular players are known , a typical 
 strategy for the identification of involved molecules 
 is their extraction from an organism and the characterization of biomolecules 
 that bind an isolated mineral phase . the 
 effect of the identified molecules on mineralization can then be studied 
 in vitro . examples have been reported for biogenic silica , magnetite , and calcium carbonate . as an alternative synthetic approach to 
 studying biomimetic molecular 
 structures that interact with solids , the biocombinatorial selection 
 of solid - binding peptides has developed into a powerful technique 
 to identify short peptides with specific affinities for a large range 
 of inorganic materials . recent examples are 
 selections for the binding of demosponge spicule silica , synthetic silica , zno , and gdo . because the selections can be performed under close - to - physiological 
 conditions , the question has arisen as to whether natural and synthetic 
 selection evolves molecules with similar characteristics and whether 
 the biomineralizing functionality might be encoded in homologue structures 
 for materials also found in organisms . here we investigated 
 the example of the iron oxide mineral magnetite 
 that is found in diverse organisms ( bacteria , mollusks , birds , and 
 fish ) and where it serves geonavigational or mechanical purposes . its biogenic formation is best studied in magnetotactic bacteria , 
 which form chains of magnetic nanoparticles termed magnetosomes . because of their size and high monodispersity , 
 magnetosomes are envisioned for mri contrast agents and cancer treatment 
 applications . furthermore , similarly 
 structured synthetic magnetic nanoparticle assemblies have recently 
 attracted much attention . simple magnetotactic organisms 
 have turned into a model system for iron oxide biomineralization because 
 the genomes of several strains have been sequenced and because molecular techniques have been developed for 
 their genetic manipulation . in particular , a whole 
 set of deletion mutants has been studied in magnetospirillum strains , with phenotypes ranging from size and morphology changes 
 to the complete disappearance of biomineralization . it has been shown that about 20 genes are sufficient to 
 restore magnetite formation in cells deficient of the whole magnetosome 
 island , the gene cluster responsible for magnetite biomineralization . the encoded mam , mms , and mtx proteins are therefore good potential 
 candidates for comparison with synthetically selected molecules and 
 subsequent in vitro mineralization studies . furthermore , biocombinatorial 
 peptide selection studies on magnetite have been reported earlier , 
 which provide a basis for such a comparison ( figure 1 ) . using the biocombinatorial techniques of cell surface and 
 phage display , brown et al . and barbas et al . had independently shown 
 that polycationic polypeptides attach to magnetite or possibly to 
 the very similar maghemite crystal surfaces . in this work , our idea is not to use phage display directly 
 for 
 the direct assessment of 12 amino acid sequences on mineralization 
 but rather to provide an alternative route toward the identification 
 of putative biomineralizing proteins without the need for in vivo 
 mutant generation . we thus combine biocombinatorial approaches with 
 a proteome homology search and assess in vitro the role of the identified 
 proteins and associated biomimetic polypeptides in the mineralization 
 of magnetite . a comparison of peptide 
 sequences 
 obtained by phage display and magnetosomal proteins affords proteins 
 and peptides of interest for further study in fe precipitation experiments . depending on the additive characteristics , mineralization can be influenced 
 to yield amorphous gels and magnetite in aggregates or self - assembled 
 particle chains . the ph.d.-12 phage 
 display peptide library ( new england biolabs ) with approximately 2.7 
  10 random 12-mer peptide sequences was used for 
 selections . two independent selections were performed on magnetite 
 powder . in a first experiment , 10 mg magnetite ( sigma - aldrich , 
 5 m particle size ) was exposed to 4  
 10 phages in 1 ml of tris - buffered saline ( tbs ) and 0.1% 
 tween-20 ( tbst , ph 7.5 ) for 30 min . the dispersed magnetite with bound 
 phages was trapped on a magnetic flow - through column ( macs , miltenyi 
 biotec ) and washed 10 times with 1 ml of tbst . bound phages were eluted 
 with a low - ph buffer ( 0.2 m glycine - hcl , ph 2.2 ) , neutralized with 
 1 m tris - hcl ( ph 9.1 ) , and amplified by the infection of e. 
 coli er2738 in liquid culture . after 4.5 h , growth phages 
 were isolated and concentrated by the removal of the bacterial cells 
 through centrifugation and repeated precipitation with an aqueous 
 peg-8000 ( 20% w / v)/nacl ( 2.5 m ) solution . the amplified phages were 
 used for the next round of selection on the mineral . in subsequent 
 rounds , individual 
 phage clones were picked for sequencing after three and six panning 
 rounds . phage dna was isolated with the m13 phage dna isolation kit 
 ( qiagen ) , sequenced ( eurofins mwg operon ) , and translated into the 
 encoded peptide . in a second experiment , the incubation time was reduced 
 to 10 min to select for binders with a faster binding rate kon . the surfactant concentration was kept constant 
 at 0.5% in both the incubation and washing phases to increase the 
 initial stringency . magnetite was kept in a 1.5 ml reaction tube , 
 washed repeatedly ( 10 times ) by precipitation with a magnet , resuspended 
 in 1 ml of tbst , and transferred to a new reaction tube to prevent 
 the potential selection of tube - material - binding peptides . individual 
 phages were picked and amplified for sequencing after four and six 
 rounds of panning . peptides were 
 compared to the m. magneticum ( amb-1 ) , m. 
 marinus ( mc-1 ) , m. magnetotacticum ( ms-1 ) , m. gryphiswaldense ( msr-1 ) , and d. magneticus ( rs-1 ) proteomes using the blastp 2.2.28 + tool on http://blast.ncbi.nlm.nih.gov/(30 ) using the default parameters ( word 
 size , 3 ; blosum62 matrix ; conditional compositional score matrix adjustment ; 
 gap costs , existence 11/extension 1 ; without low complexity filtering ) . proteins for precipitation experiments were chosen on the basis of 
 the lowest e values obtained for proteins unique to magnetotactic 
 bacteria ( msr-1 mamj , e = 1.1 ; msr-1 mtxa , e = 0.12 ) . genes mamj , mtxa , and mtxa124 were amplified from m. gryphiswaldense msr-1 genomic 
 dna by pcr ( oligonucleotides in table s1 , produced by mwg operon ) using kod polymerase ( novagen ) , purified , 
 and ligated into a pet-51b(+)ek / lic vector ( novagen ) . mtxa124 refers to the dna sequence encoding 
 for the mtxa124 protein without the leading 
 n - terminal 24 amino acids , which were identified as a signal peptide . mamj was expressed in e. coli rosetta 2 ( de3 ) ( novagen ) . mtxa and mtx124 were expressed in e. coli bl21 ( de3 ) ( novagen ) . precultures were grown in 
 5 ml of lb medium plus 100 g ml ampicillin 
 overnight at 37 c with 250 rpm stirring . culture batches of 
 250 ml of autoinducing zym-5052 medium supplied with 100 g 
 ml ampicillin were inoculated with 0.1% of the 
 preculture and grown for 24 h at 30 c with 250 rpm stirring . cells were pelleted by centrifugation ( 4 c , 
 4000 rpm , 15 min ) and stored at 80 c . thawed cells were 
 resuspended in 10 ml of strep - tactin wash buffer ( merck ) supplied 
 with 1 mg ml lysozyme and 1 mm pmsf . after 30 
 min of incubation on ice , cells were lysed by sonication ( 10  
 15 s burst with 15 s pauses ) . the cell lysate was cleared by centrifugation 
 ( 21  000 rpm , 4 c , 45 min ) and applied to a 5 ml bed strep - tactin 
 superflow agarose column ( iba ) . washing and elution at 4 c followed 
 the manufacturer s protocols using the respective buffers . fractions were analyzed by sds - page using coomassie - stained 420% 
 precast linear gradient polyacrylamide gels ( bio - rad ) . if the sds - page 
 indicated impurities , then pooled his - tagged proteins were subjected 
 to a second round of affinity chromatography on ni - ida matrix columns 
 ( macherey - nagel ) at 4 c . the protein identity was 
 verified by esi - ms fingerprinting after trypsine digestion . before 
 magnetite coprecipitation experiments , protein solutions were dialyzed 
 against milli - q water for 24 h with three solvent exchanges using 
 servapor dialysis tubing with a 1214 kda molecular weight 
 cutoff . protein concentrations were determined by uv absorption at 
 280 nm on an implen nanophotometer . molecular weights and extinction 
 coefficients were calculated on the basis of the sequence of the overexpressed 
 proteins using the protparam tool on the expasy server . mamj has a molecular weight of 48  481 
 da and absorbs 40  115 m cm . mtxa124 has a mass of 34  774 da 
 and absorbs 33  920 m cm . concentrations were adjusted to 1 mg ml by 
 dilution with milli - q water or concentration on u - tube concentrator 
 ( 30 kda molecular weight cutoff , novagen ) tubes by centrifugation 
 at 4 c . reactions 
 were performed with a computer - controlled titration system ( metrohm 
 ag ) consisting of a 776 dosimat dosing device with an 806 exchange 
 unit ( 1 ml dosing cylinder ) and a 719 titrino titration device with 
 an 806 exchange unit ( 5 ml dosing cylinder ) . reactions were performed 
 in a 50 ml vessel with a thermostated jacket kept at constant temperature 
 ( 25.0  0.1 c ) by a thermostat ( lauda m3 ) . ten milliliters 
 minus the additive volume of deionized water was initially set to 
 the ph of interest with naoh . in case of precipitation in the presence 
 of an additive , the additive magnetite precipitation was initiated by the addition of an iron chloride 
 solution ( fe / fe = 2/1 ) to the reactor at 
 a rate of 1 l min . the ph of the solution 
 in the reaction vessel was simultaneously kept constant ( ph 
 0.1 ) by the addition of sodium hydroxide . particles were adsorbed 
 from aqueous suspensions to carbon film cu 
 tem grids for 15 min . after the removal of the liquid with kimwipe 
 paper , grids were washed with a drop of milli - q water to remove residual 
 salt precipitates . standard imaging was performed on a zeiss em 912 
 omega at an acceleration voltage ( u ) of 120 kv . high - resolution 
 imaging was performed on ( i ) a jeol jem 4010 transmission electron 
 microscope ( u = 400 kv ) and on ( ii ) an fei titan 
 80/300 scanning transmission electron microscope ( u = 300 kv ) equipped with a probe corrector , an edx detector , and 
 an eels spectrometer . precipitates were 
 studied by synchrotron wide - angle x - ray diffraction at the -spot 
 beamline , bessy ii , berlin . samples were dried on a kapton thin film 
 ( breitlander gmbh ) clamped on a custom - made sample holder . the beam 
 size was set to 100 m with an energy of 15 kev (  = 0.82656 
  ) defined by a si(111 ) double - crystal monochromator . diffraction 
 data was acquired on a 3072 pixel  3072 pixel marmosaic 225 
 ccd camera ( mar usa ) with a 73.242 m pixel size . for data analysis , 
 the beam center and the detector tilt mean particle diameters were estimated 
 under the neglect of strain - induced broadening with the scherrer equation . we first compared 
 the published magnetite - adhering peptide sequences 
 ( rrtvkhhn , rrsrhh , rskrgr , rskkmr , and rfkrvrdr ) to the proteomes 
 of the well - studied magnetotactic bacterial strains magnetospirillum 
 magneticum amb-1 , magnetococcus marinus mc-1 , magnetospirillum magnetotacticum ms-1 , magnetospirillum 
 gryphiswaldense msr-1 , and desulfovibrio magneticus rs-1 . however , none of the sequences are similar 
 to mam- , mms- , or mtx - type proteins . we therefore performed new sequence 
 pannings on magnetite particles using a randomized 12-mer phage display 
 library to generate additional sequences ( table 
 s2 ) . in contrast to the earlier reported results , sequence 
 alignments revealed neither a common motif among the retrieved peptides 
 nor clear characteristics regarding amino acid composition , the isoelectric 
 point ( pi = 7.86  2.00 ) or the hydrophobicity ( average hydropathy 
 = 0.77  0.68 ) . we compared 
 the resulting sequences by alignment with the proteomes of the aforementioned 
 magnetotactic bacterial strains because our goal was to identify new 
 putative biomineralizing macromolecules within the proteome of magnetotactic 
 bacteria . because of the short length of the peptides as compared 
 to that of typical proteins , alignments yield a large number of random 
 hits when searching entire genomes . however , two sequences 
 ( out of 27 ) stood out because each one represented the best respective 
 hits ( lowest e values ) within the search space encompassing the complete 
 amb-1 , mc-1 , ms-1 , msr-1 , and rs-1 proteomes and could be attributed 
 to proteins mamj and mtxa of msr-1 with known or hypothesized magnetosome 
 or magnetotaxis functionality ( tables s3 and s4 ) . the leading 24 n - terminal amino acids of mtxa 
 represent a membrane translocation signal peptide ( figures s1s3 ) that renders the protein insoluble when 
 overexpressed in e. coli . we studied the influence of the selected proteins ( mamj and mtxa124 ) as well as the two peptide polymers poly - l - arginine ( polyr ) and poly - l - glutamic acid ( polye ) 
 on magnetite formation . apart from structural differences , the proteins / polymers 
 differ primarily in the availability of differently charged groups 
 that can interact with different iron or iron ( oxyhydr)oxide species . acid residues in mamj and polye provide binding moieties for cationic 
 fe , whereas the cationic guanidinium group of polyr 
 is able to interact electrostatically with ( in alkaline solution ) 
 negatively charged iron ( oxyhydr)oxide crystal surfaces . the presence 
 of the additives has a strong effect on the phase , crystallinity , 
 particle size , morphology , and aggregation of the precipitates , in 
 agreement with the interactions with soluble or solid iron species 
 that occur either prior to or after the nucleation of the magnetite 
 phase ( figures 24 ) . ( a  c ) magnetite particles formed in the presence 
 of poly - l - arginine . polyr serves as a proxy for a 
 potential polycationic biomolecular structure as inferred from the 
 earlier literature reports by barbas et al . and brown et al . coprecipitation of ferrous and ferric iron ( at fe / fe = 2/1 ) under alkaline conditions at ph 9 yields 
 crystalline magnetite with grain sizes dependent on the alkalinity . under these conditions , polyr affects the size , morphology , and 
 aggregation behavior of the formed magnetite nanoparticles : in its 
 presence , we obtained monodisperse , stable single - domain - sized nanoparticles 
 of 35  5 nm ( figure 3 ) that assemble 
 to chain structures in solution ( up to several micrometers ; figures 2a and s4s6 and 
 a video ) . despite their irregular morphology , particles are mostly 
 single - crystalline ( figure 2c ) . the nucleation 
 and colloidal stabilization effects of polyr , leading to particle 
 chain formation in vitro , are similar to the colloidal stabilization 
 by magnetosome compartimentalization in the bacteria in vivo . this 
 compartimentalization is provided by a lipid membrane containing diverse 
 transmembrane proteins of yet mostly unknown functions . such lipid layers therefore expose mainly positively 
 charged amines toward the intracellular magnetite crystals in line 
 with our observation of a polyr - induced colloidal stabilization effect . in contrast , mamj 
 and polye ( which resembles the polyanionic domain found in mamj ) strongly affect the phase of the formed precipitates by the inhibition 
 of magnetite nucleation with increasing additive concentration : in 
 both cases , we obtained either amorphous gels or crystalline phases 
 other than magnetite . only at low additive concentrations ( 0.01 mg 
 ml ) , ph 11 , in the presence 
 of 0.01 mg ml mamj , diffraction patterns are consistent 
 with an extremely small nanosized magnetite ( figure 5 , line features are extremely broadened magnetite peaks , intensity 
 increase toward low q ) . at ph 9 , we obtained unidentifiable 
 mixtures ( figure 4 , peaks at q = 19.13 , 26.66 , 32.57 , 34.03 , 41.16 , and 45.42 nm ; d = 0.33 , 0.24 , 0.19 , 0.18 , 0.15 , and 0.14 nm ) . 
 with 0.1 mg ml mamj , we obtained a poorly ordered 
 pattern at ph 9 ( figure 4 , possibly ferrihydrite ) 
 and at ph 11 ( figure 5 ) a pattern with two 
 distinct peaks at q = 15.51 nm ( d = 0.41 nm ) and q = 25.25 nm ( d = 0.25 nm ) , which are consistent 
 with goethite ( -feooh ) . accordingly , in the presence of 0.1 
 mg ml polye we obtained no crystalline material 
 within 1 h : at ph 9 , we obtained an orange amorphous gel - like precipitate 
 by centrifugation from a clear solution , whereas at ph 11 no pellet 
 could be formed even by centrifugation . at low concentration ( 0.01 
 mg ml ) , polye has no strong impact on the formed 
 phase ( figure 4 ) . mamj is known to be 
 involved in the magnetosome chain assembly and has been suggested 
 to serve as an anchor to the mamk filament in the cell . the anchoring function is provided exclusively 
 by the n- and c - terminal domains without the involvement of a repetitive 
 central sequence stretch rich in glutamic acid . the role of this polyanionic domain has remained unclear 
 but was initially suspected to take part in biomineralization through 
 the binding and accumulation of iron . the found consensus sequence 
 pva - lvnr can be located twice within this repetitive stretch of unknown 
 function ( table s3 ; mamj8390 and mamj171178 ) . however , the inhibitory function 
 with respect to magnetite mineralization in vitro is inconsistent 
 with the formation of the iron oxide both in and without the presence 
 of the protein in vivo , unless a yet unknown 
 regulatory function of mineral formation is required for magnetosome 
 chain formation in the bacteria . the strong effects of the synthetic 
 peptide polymer polye on magnetite nucleation inhibition suggest that 
 if such an inhibitor exists in the bacteria , it will likely act by 
 iron binding through acidic moieties . finally , mtxa124 had only a minor influence on the phase of the formed precipitates : 
 in the presence of 0.1 mg ml mtxa124 at ph 9 , we obtained only an amorphous / poorly crystalline material 
 with similar orange gel - like properties , whereas under all other conditions 
 we obtained mixtures of magnetite and amorphous gels ( figure 2 g ) . it has been 
 suggested to play a role in the magnetotaxis because of its gene location 
 in an operon encoding signal transduction genes . however , no experimental evidence has shown the implication 
 of the protein in such a mechanism so far . our in vitro assays suggest 
 that despite the possibility that the protein can bind to magnetite 
 it will likely have no effect on the crystallization of the mineral 
 in vivo . x - ray diffraction patterns of precipitations formed at ph 9 without 
 additive and in the presence of protein and peptide polymers . x - ray diffraction patterns of precipitates obtained 
 at ph 11 from 
 ferrous and ferric iron chloride solution mixtures without and in 
 the presence of protein or polypeptide additives . growth experiments over several hours at ph 
 9 , which without additives resembles the kinetics observed in the 
 bacteria in vivo , indicate that generally 
 the initial magnetite grain size becomes smaller with increasing additive 
 concentration ; however , the crystal growth rate after nucleation appears 
 to be largely unaffected ( figure 6 and table s5 ) . this is consistent with an effect 
 of ionic strength on the surface tension that in turn affects the 
 nucleus size at the formation threshold . furthermore , the nucleation is retarded in the presence of all investigated 
 additives except polyr , in line with an interaction of these proteins / polymers 
 with soluble iron species . growth kinetics of magnetite nanoparticles formed 
 in the presence 
 of ( a ) mamj , ( b ) mtxa124 , ( c ) polye , and 
 ( d ) polyr and control data . both thermodynamic and kinetic arguments can be used to explain our 
 observations of nucleation inhibition or phase stabilization . one 
 can argue thermodynamically that the interaction with a charged additive 
 will influence the surface energy of a nucleating particle . a positively 
 charged polymer in interaction with the negatively charged magnetite 
 particle will lower the surface tension and thereby facilitate nucleation . accordingly , the interaction between equally charged polymers and 
 particle surfaces would be energetically unfavorable with resulting 
 higher surface tension and eventually nucleation inhibition . kinetically , 
 one can argue that in the inhibited case the interaction between a 
 positively charged ionic iron precursor and an anionic polymer is 
 likely more rapid than the formation of the crystalline iron oxide 
 solid , therefore entrapping iron in a polymer - induced amorphous precursor 
 state . generally , synthetic polyelectrolytes 
 by virtue of their electrostatics find extensive use as nucleation 
 and crystal growth modifiers as well as in the stabilization or flocculation 
 of colloidal suspensions . equally , nature 
 has evolved highly charged proteins ( and other biomolecules ) that 
 can perform similar functions in vivo . in the context of biomineralization , 
 this applies to proteins involved in the phase selection and growth 
 modification of calcium carbonates and phosphates , silicate , and possibly 
 iron oxides as investigated here . similar to the effects observed 
 here for the iron precipitates , the charge of interacting polyelectrolytic 
 proteins determines the fate of the precursor species by stabilization 
 or destabilization . for example , polyanionic aspartic acid - rich proteins 
 play a presumably fundamental role in the stabilization of amorphous 
 calcium carbonate ( acc ) in mollusk shells . whereas early - stage calcium carbonate precipitates possess slightly 
 positive zeta potentials ( for equimolar calcium and carbonate mixtures ) 
 facilitating the binding of polyanionic proteins , silica possesses negative surface charge throughout physiologically 
 accessible ph ranges , favoring interaction with polycations . thus , polycationic biopolymers have been shown 
 to be involved in biomineralization by diatoms where they mediate 
 the formation of sio2 . although studying the mineralization role of proteins in vitro 
 represents a promising alternative because deletion mutants are difficult 
 to obtain and because their mechanistic role can be inferred more 
 easily , most of the studies have focused on only one such magnetosomal 
 protein ( mms6 ) in vitro so far . the mms6 protein and even its 26 amino acids c - terminal peptide 
 are supposed to impact the size of magnetite nanoparticles , although 
 controversial results have been obtained so far . mms6 was initially 
 chosen because it was shown to be tightly bound to the magnetite mineral . recently , the putative mechanistic role of mamp has also been inferred 
 from an in vitro study . although the 
 in vivo effect of mamp had been recognized earlier , only the recent combination of protein structural and chemical 
 reaction studies could assume its role of redox control in the oxidation 
 of fe(ii ) . although biocombinatorial approaches and in vitro mineralization 
 assays alone can not assume the role of proteins ( or other biomolecules ) 
 in biomineralization in vivo , they can point toward possible molecular 
 characteristics required by the involved biochemical machinery . our 
 experiments suggest that in the case of magnetite formation discussed 
 here , proteins , larger complexes , or membrane components promoting 
 the nucleation in vivo are likely to expose positively charged residues 
 to a negatively charged crystal surface . components with acidic ( negatively 
 charged ) functionality are likely inhibitors by the stabilization 
 of an amorphous structure through the coordination of iron .
biological materials typically display 
 complex morphologies and 
 hierarchical architectures , properties that are hardly matched by 
 synthetic materials . 
 understanding the biological control of mineral 
 properties will enable the development of new synthetic approaches 
 toward biomimetic functional materials . here , we combine biocombinatorial 
 approaches with a proteome homology search and in vitro mineralization 
 assays to assess the role of biological determinants in biomimetic 
 magnetite mineralization . 
 our results suggest that the identified 
 proteins and biomimetic polypeptides influence nucleation in vitro . 
 even though the in vivo role can not be directly determined from our 
 experiments 
 , we can rationalize the following design principles : proteins , 
 larger complexes , or membrane components that promote nucleation in 
 vivo are likely to expose positively charged residues to a negatively 
 charged crystal surface . in turn 
 , components with acidic ( negatively 
 charged ) functionality are nucleation inhibitors , which stabilize 
 an amorphous structure through the coordination of iron .