Crosstalk between the Protein Surface and Hydrophobic Core in a Core-swapped Fibronectin Type III Domain Two homologous fibronectin type III -LRB- fnIII -RRB- domains , FNfn10 -LRB- the 10th fnIII domain of human fibronectin -RRB- and TNfn3 -LRB- the third fnIII domain of human tenascin -RRB- , have essentially the same backbone structure , although they share only ∼ 24 % sequence identity . While they share a similar folding mechanism with a common core of key residues in the folding transition state , they differ in many other physical properties . We use a chimeric protein , FNoTNc , to investigate the molecular basis for these differences . FNoTNc is a core-swapped protein , containing the `` outside '' -LRB- surface and loops -RRB- of FNfn10 and the hydrophobic core of TNfn3 . Remarkably , FNoTNc retains the structure of the parent proteins despite the extent of redesign , allowing us to gain insight into which components of each parent protein are responsible for different aspects of its behaviour . Naively , one would expect properties that appear to depend principally on the core to be similar to TNfn3 , for example , the response to mutations , folding kinetics and side-chain dynamics , while properties apparently determined by differences in the surface and loops , such as backbone dynamics , would be more like FNfn10 . While this is broadly true , it is clear that there are also unexpected crosstalk effects between the core and the surface . For example , the anomalous response of FNfn10 to mutation is not solely a property of the core as we had previously suggested . Introduction Studies of the folding of structurally related proteins have been a powerful tool for investigating conservation of folding pathways ,1 identifying structurally important residues ,2 and examining the role of highly conserved residues .3 One of the most common folds in the SCOP database4 is the immunoglobulin -LRB- Ig -RRB- - like fold . Over 40,000 Ig-like domains have been identified in the current PFam database5 and the fold is found in a number of different superfamilies , where there is no apparent sequence identity between superfamilies .4 In this study , rather than seeking to understand what related proteins have in common , we ask a different question -- can differences between closely related proteins be explained ? Many members of the Ig-like fold have been well characterised , leading to : identification of the key residues essential for formation of the Greek key structure ,6,7 the observation of a correlation between stability and folding rate ,8 -- 11 the identification of a common folding pathway ,12 -- 16 and the identification of the role of conserved proline residues3 and the conserved tyrosine corner motif .2 However , when two Ig-like domains from the fibronectin type III -LRB- fnIII -RRB- superfamily , the 10th fnIII domain of human fibronectin -LRB- FNfn10 -RRB- and the third fnIII domain of human tenascin -LRB- TNfn3 -RRB- , were studied in detail and compared , they were found to differ markedly in several respects . FNfn10 and TNfn3 are essentially structurally identical -LRB- backbone RMSD is 1.2 Å -RRB- , but have low sequence identity -LRB- 24 % -RRB- . They differ in their stabilities17 ,18 and response to mutation19 ; they differ in their folding kinetics17 ,18,20 -LRB- although they share a common folding mechanism12 ,16 -RRB- ; they display different backbone and side-chain dynamics21 -- 23 ` ; and , finally , they differ in their response to mechanical force .24 -- 26 Why do proteins that are structurally almost identical behave so differently ? Which components of a protein are responsible for the various aspects of its behaviour ? How independent are the properties of the surface and hydrophobic core ? We have addressed these questions by making a chimera of these two fnIII domains . The chimera , FNoTNc , was created with the `` outside '' -LRB- surface and loops -RRB- of FNfn10 and the core of TNfn3 .26 Fifteen mutations were made in the core of FNfn10 so that all buried residues -LRB- with < 10 % solvent-accessible surface area -RRB- are identical with those residues in the core of TNfn3 -LRB- total number of core residues = 27 -RRB- -LRB- Fig. 1 -RRB- . Thus , we can test how transferable the properties of the core and surface of the respective parents are when combined in this way . FNoTNc is a stable , folded protein that is structurally almost identical with the parent proteins -LRB- Supplementary Fig. 1 -RRB- . FNoTNc has also retained the cell-adhesion activity of the FNfn10 parent protein mediated by specific integrin binding .26 We have previously used this chimera to demonstrate that resistance to mechanical force is a core property : FNoTNc has mechanical unfolding properties indistinguishable from TNfn3 and very different from FNfn10 .26 Here we investigate the stability , folding and dynamics of the new , chimeric protein . Most protein engineering analyses concentrate on the hydrophobic core of proteins , since it was shown in 1959 that the hydrophobic interaction is the major factor involved in protein folding27 and surface mutations rarely affect protein stability by more than ∼ 1 kcal mol − 1 . Our results suggest , however , that the surface and loops can play a key -LRB- and sometimes unexpected -RRB- role in determining the biophysical properties of a protein . Results Thermodynamic stability Wild-type FNoTNc FNoTNc has a free energy of unfolding -LRB- ΔGD -- N -RRB- of 7.5 kcal mol − 1 , intermediate between the stabilities of FNfn10 and TNfn3 -LRB- 9.4 and 6.7 kcal mol − 1 , respectively -RRB- at pH 5.0 . We infer that the surface interactions and loop and turn regions of FNfn10 make a significant contribution to the overall stability of FNoTNc . As was shown for FNfn10 , the ΔGD -- N of FNoTNc is independent of pH between pH 5.0 and pH 7.0 , whereas TNfn3 is less stable at pH 7 -LRB- 5.7 kcal mol − 1 -RRB- . The dependence of the stability of TNfn3 on pH has been shown to be the result of the presence of patches of acidic residues on the surface of TNfn3 , which are , of course , not present in FNoTNc .17 Anomalous response of certain peripheral mutations A number of core residues were mutated in FNoTNc to investigate the response of the protein to mutation . These were positions that had previously been investigated in the parent proteins FNfn10 and TNfn3 . The thermodynamic stability of the mutant proteins was determined by chemical denaturation in guanidinium chloride -LRB- GdmCl -RRB- . The mutations can clearly be divided into two categories . A few peripheral mutations -LRB- mutations in the A and G strands and the B -- C loop -RRB- have little effect on stability as was previously observed in FNfn10 -LRB- Fig. 2a -RRB- , whereas most other mutations were more typically destabilising . In the latter case , the ΔΔGD -- N was similar to -LRB- but in general slightly lower than -RRB- what had been observed previously in TNfn3 -LRB- Fig. 2b -RRB- . The residues in each category are mapped onto a backbone ribbon representation of FNoTNc in Fig. 2c . ΔΔGD -- N values for all mutations are compared to those of FNfn10 and TNfn3 in Supplementary Table 1 . Equilibrium hydrogen exchange The rates of hydrogen -- deuterium exchange were measured at pD 7.0 . Measured rate constants for exchange , kex , ranged between 9.3 × 10 − 2 and 1.6 × 10 − 4 min − 1 . Values of kex and the apparent free energies of exchange , ΔGexapp , are listed in Supplementary Table 2 . The ΔGexapp of FNoTNc , FNfn10 and TNfn3 are compared in Fig. 3 . The hydrogen exchange behaviour of FNoTNc clearly resembles that of FNfn10 : both have many residues that exchange in the experimental dead time , in particular in the edge strands -LRB- A , C ′ , E and G strands -RRB- that are more protected in TNfn3 . Note that this does not result from differences in the intrinsic stabilities of the domains -- TNfn3 is significantly less stable than either FNoTNc or FNfn10 under these experimental conditions . Folding kinetics Wild-type FNoTNc The rate constants of folding and unfolding were determined using stopped-flow measurements monitored by changes in fluorescence > 320 nm . The logarithm of the folding and unfolding rate constants was plotted against concentration of denaturant -LRB- Fig. 4 -RRB- . Both unfolding and refolding arms show a linear dependence on denaturant concentration . There is an additional refolding phase , which we attribute to proline isomerisation and do not consider further . -LRB- Both FNfn10 and TNfn3 have proline-isomerisation limited phases , and FNoTNc has eight Pro residues . -RRB- To compare the kinetics of FNoTNc with those of FNfn10 and TNfn3 -LRB- which have been studied using different denaturants due to large differences in stability -RRB- , the logarithm of the observed rate constants is plotted against stability in Fig. 4 . The free energy of unfolding calculated from the ratio of the folding and unfolding rate constants extrapolated to 0 M denaturant -LRB- 7.7 kcal mol − 1 -RRB- and the kinetic m value -LRB- 2.1 kcal mol − 1 M − 1 -RRB- are the same as the equilibrium ΔGD -- N -LRB- 7.5 kcal mol − 1 -RRB- and m value -LRB- 2.1 kcal mol − 1 M − 1 -RRB- within error -- consistent with the folding being a 2-state process , with no stable intermediates being populated . The relative compactness of the transition state can be determined from the folding and unfolding m values . FNoTNc has a βT value of 0.6 , similar to those of TNfn3 and FNfn10 . Φ-value analysis The folding kinetics of 18 variants of FNoTNc with nondisruptive deletion mutations were measured as for the wild type . Few of these mutants were in the A , B and G strands due to very small changes in stability with mutation in general . In a few of the mutants , the unfolding arm of the chevron plots shows negative curvature at high denaturant concentrations , which we attribute to the Hammond effect : the simplest model that fits the data .28 -- 30 All chevrons were fitted therefore to a two-state equation with a quadratic term in the unfolding arm . -LRB- All the chevron plots are shown in Supplementary Fig. 2 . -RRB- Note that the model chosen to fit the kinetic data does not affect our results because equilibrium values of ΔΔGD -- N were used to determine Φ . In Table 1 , the Φ values for each mutant are compared to those in TNfn3 and FNfn10 . The Φ values are generally low , closely resembling those of TNfn3 . Dynamics Backbone dynamics 15N T1 and T2 relaxation time constants and 1H15N nuclear Overhauser enhancement parameters were measured using 1H -- 15N correlation spectroscopy -LRB- Supplementary Table 3 -RRB- . The generalised order parameter , S2 , and a conformational exchange broadening parameter , Rex , were determined for each backbone amide -LRB- Fig. 5 and Supplementary Table 4 -RRB- . The S2 values are similar to those of both the parents . However , the high values of Rex that were seen in the A/B region of FNfn10 , have decreased in FNoTNc . Side-chain dynamics The relaxation of the operator terms IzCzDz , IzCzDy and IzCz was measured to give deuterium relaxation time constants T1 -LRB- D -RRB- and T2 -LRB- D -RRB- 31 -LRB- Supplementary Table 5 -RRB- . Order parameters , S2 , were determined for each methyl group using the standard model-free formalism as previously described22 -LRB- Supplementary Table 6 -RRB- . The dynamics data for those methyl groups that had overlapping peaks were treated with caution , as the contribution from the overlapped peaks can not be separated . Nevertheless , we have some confidence in these results due to the agreement of the order parameters for residues that have both overlapped and well-resolved methyl groups . S2 ranges from zero to unity , with higher values of S2 indicating greater conformational restriction . The methyl S2 values are shown projected onto the protein structure and compared to the parent proteins in Fig. 6 . Within the core of FNoTNc is a cluster of deeply buried residues , which have unusually low order parameters , as has previously been observed in TNfn3 . Discussion The core of FNoTNc is similar to that of TNfn3 , but apparently less closely packed . Evidence from mutations FNoTNc is close in structure to both the parent domains -LRB- backbone RMSD , excluding the mobile C -- C ′ and F -- G loops , is 0.95 and 0.89 Å compared to FNfn10 and TNfn3 , respectively -RRB- . From the crystal structure we would deduce that the core of FNoTNc is essentially the same in terms of core packing and number of core contacts as TNfn3 , the parent protein that donated the core residues -LRB- Fig. 7a -RRB- . However , the calculated free volume in the interior of FNoTNc is larger than for TNfn3 -LRB- 128.4 Å3 versus 118.1 Å3 , respectively -RRB- . When the response of FNoTNc to mutation is compared to TNfn3 , it is clear that the ΔΔGD -- N values of FNoTNc are slightly lower , on average ∼ 80 % of those in TNfn3 -LRB- Fig. 7b -RRB- . Since the loss of free energy on mutation is strongly correlated with the number of side-chain contacts deleted ,32,33 we infer that the less tight packing of the core of FNoTNc accounts for the difference in ΔΔGD -- N compared to TNfn3 . Evidence from side-chain dynamics The same conclusion can be drawn from the analysis of the side-chain dynamics . The core of TNfn3 has been shown to be exceptionally mobile , with several of the most buried residues falling several standard deviations below the expected order parameter for their residue type .22,23 FNfn10 has a core that is much more conformationally restricted , with order parameters within the usual range for buried residues . Our previous analysis of the side-chain dynamics of FNfn10 and TNfn3 led us to suggest that the differences in core dynamics between FNfn10 and TNfn3 could , at least in part , be ascribed to the slightly lower core packing in TNfn3 ; the residues in TNfn3 with unusually low order parameters are also found to have packing volumes that are larger than expected . The buried side chains of FNoTNc were found to have , on average , even lower order parameters than the same side chains in TNfn3 -LRB- Fig. 6d -RRB- . Behaviour of peripheral regions of the protein is modulated by the surface and loops Evidence from mutation A number of sites in FNfn10 were identified where upon mutation the ΔΔGD -- N is significantly lower than for the same -LRB- or an equivalent -RRB- mutation in TNfn3 .19 These sites are at the periphery of the protein and include residues Pro5 and Leu8 -LRB- A strand -RRB- , Pro25 -LRB- B -- C loop -RRB- and residues Ser85 and Phe92 -LRB- G strand -RRB- . Unexpectedly , in FNoTNc we see the same anomalous response to mutation for Pro5 , Pro25 and Ser85 , although Ile8A and Phe92A now have similar ΔΔGD -- N values to TNfn3 -LRB- Fig. 2a -RRB- . Pro5 , Pro25 and Ser85 are all found in the same region of the molecule -LRB- Fig. 2c -RRB- . The anomalous response to mutation in this peripheral region of the core of FNoTNc and FNfn10 is intriguing : apparently the `` plastic '' response of the protein to mutation of these three buried residues is modulated by the surface of the protein and is not determined by the core alone . The additional plasticity at this end of the hydrophobic core may be due to its proximity to the longer FG loop in FNfn10 and FNoTNc , which restricts motion less than the corresponding loop in TNfn3 . Evidence from hydrogen exchange FNoTNc , FNfn10 and TNfn3 have very similar structures and hydrogen bonding patterns . Further evidence for `` plasticity '' of FNfn10 compared to TNfn3 came from the observation that the peripheral A , C ′ , E and G strands were significantly less well protected against amide exchange in FNfn10 than in TNfn3 , despite the fact that FNfn10 is considerably more stable than TNfn3 .19 Under the experimental conditions , hydrogen exchange is in the EX2 regime ,34,35 meaning that exchange reflects local stability ; that is , these peripheral regions of FNfn10 have lower local stabilities than TNfn3 . Our experiments clearly show that the exchange behaviour of FNoTNc is similar to FNfn10 -- with low protection of residues in the same peripheral strands -LRB- Fig. 3 -RRB- . Again surprisingly , local instability appears to be a function of the surface of the protein and not a property of the core ; however , the plasticity inferred from response to mutation and local instability still appear to be related , as was previously inferred . This is not , however , related to slow exchange motions of the backbone , as we had previously suggested , since the millisecond time scale motions observed in the A/B region of FNfn10 are not found in FNoTNc . The stability of FNoTNc is modulated by both the core and the surface In summary , despite the evidence for FNoTNc having a less well packed core than TNfn3 , with the mutation of buried side chains having less effect on the overall stability -LRB- Fig. 7b -RRB- , FNoTNc is significantly more stable than TNfn3 , suggesting again that the surface and loops of FNfn10 contribute significantly to the stability of FNoTNc . This is not a new observation ; it has been previously demonstrated that surface interactions can play a significant role in the stabilisation of proteins .36 Interestingly , however , the `` local stability '' of the peripheral regions of FNoTNc is lower than the local stability of TNfn3 , as manifested in the hydrogen exchange experiments . The surface residues of FNfn10 are modulating the stability of these peripheral regions . A concurrent study by Siggers et al. 37 investigated the stability and dynamics of a series of loop-swap mutants of FNfn10 and TNfn3 : essentially these were mutants where the C -- C ′ and F -- G loops were exchanged . Interestingly , both TNfn3 domains had lower thermal stabilities than the wild-type protein , whereas the thermal stability of FNfn10 was unaffected by the exchange . We infer that in our chimeric protein , interactions of the surface residues are most important in the increase in stabilisation of FNoTNc beyond that of TNfn3 , and not the new C -- C ′ and F -- G loops . The TNfn3 core governs the folding kinetics Wild-type kinetics In order to compare the kinetics of the three proteins directly , the logarithm of the rate constant has been plotted against stability -LRB- Fig. 4 -RRB- . This unusual scale is used as each protein was characterised using a different denaturant . FNoTNc folds at a rate intermediate between FNfn10 and TNfn3 . In broad terms , the chevron of FNoTNc resembles that of TNfn3 . It has previously been pointed out that the folding rates of Ig-like domains in water do not correlate with contact order -LRB- the average sequence separation of native contacts -RRB- .8,38 For the present three proteins , which have almost identical contact order -LRB- absolute contact order 15 -- 16 % , relative contact order 16 -- 17 % -RRB- , the rate constants at the folding midpoint span 2.5 orders of magnitude . Thus , the variation in the folding rate can not be explained only by differences in contact order -- protein stability plays a key role .8,39 FNfn10 shows clear rollover in both the folding and unfolding arms . The rollover in the folding arm has previously been ascribed to the presence of a populated folding intermediate .18 Neither FNoTNc nor TNfn3 show any evidence for population of a folding intermediate -- the presence of a stable folding intermediate in FNfn10 appears to be the result of core interactions . Curvature in the unfolding arm of a chevron plot has been ascribed to the presence of a high-energy intermediate , to Hammond effects , or to population of an unfolding intermediate .30,40,41 This has not been investigated for any of these fnIII domains ; indeed , it is often not possible to distinguish between the first two cases .42 Both FNfn10 and some mutants of FNoTNc display curvature in the unfolding arm that is not seen in wild-type TNfn3 . It should be noted , however , that unfolding curvature has been seen in a less stable form of TNfn3 -LRB- missing the final two C-terminal residues -RRB- and may simply not be observed in this case because the unfolding arm in the stable form of TNfn3 used here is relatively short . In summary , FNoTNc has similar folding characteristics to TNfn3 . This suggests that the core of these proteins plays the major role in determining the folding behaviour . However , FNoTNc is more stable than TNfn3 . When we compare the folding and unfolding rate constants in water we find that FNoTNc folds some 10 times faster than TNfn3 -LRB- kfH2O = 60 and 6 s − 1 , respectively -RRB- but unfolds at approximately the same rate as TNfn3 -LRB- kuH2O = 2 × 10 − 4 and 5 × 10 − 4 s − 1 , respectively -RRB- . Thus , the stabilising surface interactions apparently stabilise the transition state of FNoTNc as much as the native state , while still not causing a folding intermediate to be populated . This , perhaps , allows us to pinpoint further which surface interactions are responsible for the added stability of FNoTNc -LRB- over TNfn3 -RRB- . At the transition state , the loops and the peripheral A and G strands are largely unstructured , but there is structure in the B , C , C ′ , E and F strands , particularly towards the centre of the core -LRB- see Φ value section below -RRB- . Interactions between surface residues in these strands are therefore the most likely candidates for providing additional stability to the FNoTNc protein -LRB- both native and transition states -RRB- , above those interactions between residues that are packing in the core . In contrast , FNoTNc folds some three times more slowly than FNfn10 and the unfolding rate constant of FNfn10 in water is approximately an order of magnitude lower -LRB- ∼ 2 × 10 − 5 s − 1 -RRB- . Φ-value analysis The Φ-value analysis reveals the extent of formation of structure in the transition state .43 The pattern of Φ values in FNfn10 and TNfn3 are similar , suggesting that they have a common folding mechanism . There are differences , however . The Φ values of TNfn3 tend to be much lower than those of FNfn10 : TNfn3 has five Φ values greater than 0.4,12 while FNfn10 has eight16 ; in FNfn10 the folding nucleus appears to be more extensive than in TNfn3 , with more than one residue in the central C , E and F strands having high Φ values . FNoTNc has even lower average Φ values than TNfn3 with only two Φ values greater than 0.4 -LRB- Table 1 -RRB- Both FNoTNc and TNfn3 have less extensive formation of structure in the transition state than FNfn10 . However , Φ-value analysis is at its most powerful when patterns of Φ values are compared in different proteins rather than by direct comparison of Φ values . Previous analysis of TNfn3 has identified a ring of interacting residues in the B , C , E and F strands as the folding nucleus , with residues in the C ′ strand packing onto these .12 The Φ values of the peripheral A and G strands are all close to 0 , suggesting that they are unformed at the transition state . A similar pattern of Φ values was seen in FNfn10 , although this Φ-value analysis was less complete .16 The Φ values for FNoTNc were divided into three categories of low -LRB- Φ < 0.25 -RRB- , medium -LRB- 0.25 < Φ < 0.35 -RRB- and high -LRB- Φ > 0.35 -RRB- -LRB- Fig. 8 -RRB- and compared to the pattern of Φ values for the identical residues in TNfn3 . The pattern of Φ values is very similar to that of TNfn3 , although there are slight qualitative differences . Again , the highest Φ values are found in the B , C , C ′ , E and Φ strands and the Φ values in the A and G strands are ∼ 0 . The folding mechanism of FNoTNc is unperturbed and the same as both the parents , although from the magnitude and extent of the residues with higher Φ values the transition-state structure appears to be closer to that of TNfn3 than FNfn10 . Dynamics from NMR is determined by local interactions Side-chain dynamics The side-chain dynamics appear to reflect core packing , as was discussed above , and so FNoTNc resembles TNfn3 more closely than FNfn10 . Backbone dynamics The S2 values of FNoTNc are generally very similar to those of both the parents -LRB- Fig. 5 -RRB- . It is interesting to compare our study with that of the loop swap mutants of FNfn10 and TNfn3 from Palmer and coworkers ,37 where the F -- G loop of FNfn10 was grafted into TNfn3 . In that case , the S2 values in the loop region were more similar to those in FNfn10 , than those in the equivalent positions in TNfn3 . A similar result was seen in the protein where the C -- C ′ loop was grafted in . It is difficult to compare our results directly to those of Siggers et al. 37 : their method of analysis leads to greater differences between the S2 values of FNfn10 and TNfn3 , resulting in more visible effects on the S2 values when the loops are swapped than we see in FNoTNc compared to the parent proteins . However , there is a general agreement that the behaviour of the loops is a local property , rather than being a direct effect of either core or surface interactions . Conclusion We have grafted the core of one fnIII domain -LRB- TNfn3 -RRB- into the homologue FNfn10 , creating a chimera , FNoTNc , which has retained the structure of the parent proteins . Using several different probes , we have shown that FNoTNc does not behave like either one of the parent proteins alone . Instead , it has retained a number of properties of each . We find that each property investigated clearly resembles the behaviour of one of the parents , enabling us to separate the contribution of the core and the surface of the protein in determining the behaviour of the domain . Some of these are unsurprising , such as the pH dependence of stability , the core side-chain dynamics and the dependence of folding on the composition of the core . However , the surface of the protein confers significant stability not only on the native state , but also on the transition state for folding . Others properties are less predictable . The surface of the protein confers `` plasticity '' in peripheral regions of the proteins as detected by the anomalous response of some regions of the core to mutation and hydrogen exchange protection patterns . This suggests that the surface of a domain may have a more significant coupling with the core than we had previously considered . Since most biophysical studies tend to focus on the core of a protein , this coupling is a relatively unexplored area of research . Materials and Methods Chemicals GdmCl was purchased from MP Biomedicals Inc. , guanidine isothiocyanate from Gibco-BRL and urea from BDH Laboratory Supplies . Protein expression and purification Site-directed mutagenesis reactions were performed with the QuickChange kit from Stratagene using the FNoTNc plasmid . The identity of the mutants was confirmed by DNA sequencing . The mutants studied in this work are listed in Table 1 and Supplementary Table 1 . The nature of the mutation is indicated with the wild-type residue first -LRB- single-letter code -RRB- , the position of the mutation second and the mutant residue third . Expression and purification of FNoTNc and mutants was performed as described earlier for TNfn3 .20 Measurements of protein stability All biophysical measurements were performed in 50 mM sodium acetate buffer , pH 5.0 at 25 °C unless otherwise stated . The stability of FNoTNc and FNoTNc mutants were determined by equilibrium denaturation experiments using GdmCl and by standard methods using 1 μM protein .20 Kinetic measurements Kinetics were measured using fluorescence stopped-flow measurements in 50 mM sodium acetate buffer , pH 5.0 , at 25 °C and were monitored by changes in fluorescence above 320 nm . Refolding measurements for FNoTNc were made in 0 M denaturant by stopped-flow fluorescence using pH jumps from pH 12.4 to pH 5.0 as previously described .12 -LRB- FNoTNc is acid stable . -RRB- NaOH -LRB- 25 mM , pH 12.4 -RRB- unfolds FNoTNc completely -LRB- data not shown -RRB- . Φ-value analysis Φ values were determined from refolding data at 1.0 M GdmCl , to avoid the errors associated with long extrapolation , from Eq . -LRB- 1 -RRB- : 43whereand kfWT and kfmut are the rate constants for folding of wild-type and mutant proteins , respectively . NMR sample preparation FNoTNc was expressed and purified by affinity chromatography as previously described .17 Uniformly 15N labelled and 13C and 15N labelled samples were expressed in M9 minimal media containing 15NH4Cl and -LSB- U-13C -RSB- 6-glucose as the sole nitrogen and carbon sources . Samples for side-chain dynamics were expressed as previously described .22 Chemical shift assignments Backbone assignment experiments were carried out on a double-labelled -LRB- 13C , 15N -RRB- sample of FNoTNc , at an approximate concentration of 1 -- 2 mM , in 50 mM imidazole buffer at pH 7.0 in 10 % D2O . Sodium azide -LRB- 0.05 % -RRB- was added to prevent microbial growth . The sample was centrifuged to remove insoluble protein and degassed . Spectra were acquired at 298 K on a Bruker DRX500 spectrometer with an inverse triple-resonance cryogenic probe . Backbone assignments were based on HNCACB , HNCO and CBCA -LRB- CO -RRB- NH experiments together with the 1H -- 15N heteronuclear single quantum coherence -LRB- HSQC -RRB- spectrum . Side-chain 1H and 13C resonance assignments were obtained from 3-D HCCH -- total correlated spectroscopy -LRB- TOCSY -RRB- H -LRB- CCCO -RRB- NH and -LRB- H -RRB- CC -LRB- CO -RRB- NH preceding TOCSY spectra . The spectra were processed and analysed using NMRpipe and Sparky .44,45 The 1H -- 15N HSQC has excellent resolution , although resonances from 12 residues in loop regions can not be detected at either pH 5 or pH 7 -LRB- Supplementary Fig. 3 and Supplementary Table 7 -RRB- . All samples for side-chain methyl assignment were prepared in 50 mM sodium acetate buffer at pH 5.0 in 10 % D2O , at a concentration of 1 -- 2 mM . Sodium azide was added to prevent microbial growth . The sample was centrifuged to remove insoluble protein and degassed . All experiments were carried out as previously described .22,46,47 Chemical shift assignments of the side chains were made using standard triple-resonance experiments . Many of the signals in the 1H -- 13C HSQC are overlapped , meaning that 5 of the 64 methyl groups in the 1H -- 13C HSQC could not be assigned with confidence . Assignment of the leucine and valine methyl groups was made stereospecifically , based upon the phase of peaks in a 1H -- 13C HSQC acquired for a sample with 10 % 13C enrichment -LRB- Supplementary Fig. 4 and Supplementary Table 7 -RRB- . Hydrogen exchange Hydrogen exchange experiments were carried out under EX2 conditions on a 15N-labelled sample of FNoTNc , at an approximate concentration of 1 -- 2 mM , in 50 mM imidazole buffer at pD 7.0 in 10 % D2O . Sodium azide -LRB- 0.05 % -RRB- was added to prevent microbial growth . The exchange of amide protons was followed by the decay of intensity of peaks in HSQC spectra .48 The apparent free energy of exchange , ΔGexapp , was determined from the rate constant of exchange , kex , and the intrinsic rate constant , kint , determined from peptide data to take account of the primary sequence of the protein and exchange conditions49 ,50 using Eq . -LRB- 2 -RRB- and intrinsic rate constants determined using the software Sphere † .51 Backbone 15N relaxation measurements Backbone dynamics were determined from 15N T1 and T2 relaxation times and the steady-state heteronuclear 1H15N nuclear Overhauser enhancement at 500 MHz as previously described .22 The data were analysed using standard protocols for backbone dynamics with the program TENSOR2 . Side-chain methyl 2H relaxation measurements Side-chain deuterium relaxation times T1 -LRB- D -RRB- and T1ρ -LRB- D -RRB- were determined by measuring the relaxation of the two - and three-spin operator terms , IzCz , IzCzDz and IzCzDy and analysed as previously described .22,31

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

fnfn10, 10th fniii domain of human fibronectin

tnfn3, third fniii domain of human tenascin

fnotnc, a core-swapped protein with the “outside” (surface and loops) of fnfn10 and the core of tnfn3

hsqc, heteronuclear single quantum coherence


Treatment of migraine attacks based on the interaction with the trigemino-cerebrovascular system Primary headaches such as migraine are among the most prevalent neurological disorders , affecting up to one-fifth of the adult population . The scientific work in the last decade has unraveled much of the pathophysiological background of migraine , which is now considered to be a neurovascular disorder . It has been discovered that the trigemino-cerebrovascular system plays a key role in migraine headache pathophysiology by releasing the potent vasodilator calcitonin gene-related peptide -LRB- CGRP -RRB- . This neuropeptide is released in parallel with the pain and its concentration correlates well with the intensity of the headache . The development of drugs of the triptan class has provided relief for the acute attacks but at the cost of , mainly cardiovascular , side effects . Thus , the intention to improve treatment led to the development of small CGRP receptor antagonists such as olcegepant -LRB- BIBN4096BS -RRB- and MK-0974 that alleviate the acute migraine attack without acute side events . The purpose of this review is to give a short overview of the pathological background of migraine headache and to illustrate the mechanisms behind the actions of triptans and the promising CGRP receptor blockers . The trigemino-cerebrovascular system Pain-sensitive structures such as the intracranial vessels and the meninges , especially the dura mater , are supplied with sensory nerve fibers . In fact they are innervated by the ophthalmic ramus of the first branch of the trigeminal nerve that arises from pseudounipolar neurons located in the trigeminal ganglion . Those neurons project onto second order sensory neurons in the trigmenial nucleus caudalis -LRB- TNC -RRB- in the brain stem and its related extensions down to the C2-level called the trigeminocervical complex -LSB- 1 -RSB- . From this region a signal is transmitted to the ventroposterior thalamus leading to activation in cortical areas , including frontal cortex , insulae and cingulate cortex . This results in the experience of pain -LRB- Fig. 1 -RRB- . In addition , a migraine active region has been pointed out in the brainstem by using positron emission tomography -LRB- PET -RRB- -LSB- 2 -- 4 -RSB- . Thus , an involvement of the trigemino-cerebrovascular system in the transmission of nociceptive information to the central nervous system is a viable position . Fig. 1In migraine attacks , sensory nerves , which innervate the cerebral blood vessels , are activated to antidromically release calcitonin gene-related peptide . They originate from the first branch of the trigeminal nerve that arises from pseudounipolar neurons located in the trigeminal ganglion -LRB- TG -RRB- . Those neurons project onto second order sensory neurons in the trigeminal nucleus caudalis -LRB- TNC -RRB- from where a signal is transmitted to the cortex The trigemino-cerebrovascular reflex There is a dense supply of CGRP-containing nerve fibers originating in the first division of the trigeminal ganglion that innervate intracranial vessels . Experimental studies have shown that induction of vasoconstriction provoked rapid normalization of the vascular tone due to -- -- in part -- -- reflex activation of the trigeminal nerve . Vasoconstriction triggers an antidromic release of sensory trigeminal neuropeptides , mainly CGRP , being a strong intracranial dilatator -LSB- 5 , 6 -RSB- . After denervation there is no alteration in the contractile response of the vessel but the time to attain initial basal tone is markedly prolonged . This is of importance as headache may be initiated by a spreading wave of depression of cortical neurons resulting in contraction of some parts of the cerebral circulation . This phenomenon has now been shown in man -LSB- 7 -RSB- and could activate the trigeminal vascular system to release CGRP to reestablish the vascular tone . Thus , the trigemino-cerebrovascular reflex is probably part of a counter-balancing system , which is able to mediate dilatation of brain vessels and sustain cerebral blood flow -LSB- 8 -RSB- . This may assure the maintenance of local brain blood flow within normal limits as the cerebral circulation requires high and constant flow . Activation of the neurons in the trigeminal ganglion leads on the other hand to an activation of neurons in the trigeminocervical complex that project to the central nervous system -LRB- CNS -RRB- and mediate the central aspects of pain -LSB- 1 , 9 -RSB- . Calcitonin gene-related peptide CGRP is a neuropeptide consisting of 37 amino acids and is predominantly expressed in the nervous system . It originates from the calcitonin gene encoding calcitonin and CGRP depending upon alternative splicing . Histochemical studies of cerebral perivascular nerve fibers and human trigeminal ganglia revealed that almost half of the cell bodies contain CGRP -LSB- 10 , 11 -RSB- . Although many other signal molecules are located in the trigeminal ganglion , CGRP is the most abundant in man -LSB- 12 -RSB- and can be seen as a marker for trigeminal activity . Furthermore it is the only transmitter reliably released in acute migraine attacks at present . There was a marked increase in CGRP levels during migraine attacks , but no changes could be observed for other neuropeptides such as vasoactive intestinal peptide -LRB- a marker for parasympathetic activity -RRB- , neuropeptide Y -LRB- sympathetic activity -RRB- and substance P -LRB- sensory activity -RRB- when their levels were analyzed in the cranial venous outflow during migraine attacks -LSB- 8 , 13 -RSB- . In migraine attacks elicited within clinical studies by administration of nitroglycerin a good correlation has been described between increased levels of CGRP and the intensity of the headache -LSB- 14 , 15 -RSB- . But it is noteworthy to mention that nitroglycerin elicits head pain , but not always genuine migraine attacks . There may be a need for the subject to be in a `` prone phase '' , where the trigemino-cerebrovascular system might be hyper-reactive as it was demonstrated in cluster headache -LSB- 16 -RSB- . Triptans The increased cranial venous levels of CGRP observed in conjunction with migraine attacks thus indicate activation of the trigeminal system . Administration of sumatriptan -- -- belonging to the highly effective anti-migraine drugs called triptans or 5-HT1B / 1D agonists-results in alleviation of the headache and normalization of the cranial venous CGRP levels -LSB- 8 -RSB- . They are thought to act mainly as powerful vasoconstrictors of cerebral vessels via 5-HT1B receptors , which have been shown to be expressed in the medial smooth muscle cell layer of the human middle meningeal -LSB- 17 -RSB- and cerebral arteries -LSB- 18 -RSB- . In addition , triptans may bind to presynaptic 5-HT1B / 1D receptors on trigeminal afferents to inhibit nerve activity and hence reduce CGRP release . 5-HT1B / 1D receptors were found on trigeminal sensory fibers -LSB- 19 -RSB- , trigeminal ganglion cells -LSB- 20 -RSB- and in the trigemino-cerebrovascular system in man -LSB- 21 -RSB- . Furthermore , the 5-HT1B / 1D receptors were co-localized with CGRP in the human trigeminal ganglion -LSB- 20 -RSB- . Autoradiographic studies using labeled zolmitriptan confirmed the presence of 5-HT1B / 1D receptors in certain brain stem nuclei that are involved in cranial nociceptive processing -LSB- 22 -RSB- and are likely to be activated during migraine . Thus , it can be presumed that a potential relaxation of intracranial blood vessels during migraine attacks can be normalized by triptans , which activate the cerebrovascular 5-HT1B receptors and thereby cause vasoconstriction . By stimulating 5-HT1B / 1D receptors , triptans inhibit the release of CGRP from the perivascular nerve terminals and the trigeminal ganglion cells . Activation of 5-HT1B / 1D receptors at the level of the CNS -LRB- trigeminal nucleus caudalis -RRB- may interrupt central aspects of the headache process . Recently the relationship between pharmacokinetic and pharmacodynamic aspects of triptans in migraine was investigated . It has been observed that subcutaneous administration of sumatriptan is more effective than oral application . The difference in efficacy is most likely due to the rapid initial rise in plasma level after subcutaneous administration -LSB- 23 -RSB- . Furthermore migraine attacks may influence gastric stasis leading to delayed absorption of drugs -LSB- 24 , 25 -RSB- . Thus , to increase efficacy of treatment , attention should be paid to the development of migraine drugs , which can be quickly absorbed . Triptans cause sometimes cardiovascular adverse effects as they also act as constrictors in coronary arteries . But triptans are considered as more powerful vasoconstrictors in human isolated cerebral arteries than in coronary arteries . They are at least threefold more potent -LSB- 26 -RSB- . The reason for the higher potency and efficiency of triptans in intracranial arteries could be due to at least three differences . First there is a higher number of 5-HT1B receptors in those vessels compared to peripheral arteries -LSB- 18 , 27 -RSB- . Secondly there are few , if any , 5-HT2A receptors in intracranial vessels whereas the 5-HT2A receptor population dominates in coronary arteries -LSB- 27 -RSB- . Thirdly , there might be potential differences in coupling of the 5-HT1B receptors between cerebral and coronary arteries . In consideration of the fact that triptans are weak constrictors in coronary arteries -LSB- 28 , 29 -RSB- and have little impact on coronary blood flow , the clinical consequences in healthy subjects are minor . However , small contractions may anyhow have proportionally greater impact in diseased artery with luminal narrowing . Triptans are hence contra-indicated in patients with known cardiovascular disease . Sensitization in migraine Treatment with triptans achieves good results in patients suffering from sensitization . Sensitization is defined as an increased afferent activity in response to an unchanged stimulus -LSB- 30 -RSB- and both peripheral and central sensitization may play a role in migraine attacks . Peripheral sensitization of meningeal perivascular nociceptors arising from trigeminal neurons can clinically explain phenomena like intracranial hypersensitivity -LRB- worsening pain during physical activity -RRB- and the throbbing element in migraine pain . This may be due to normally innocuous meningeal nociceptors becoming hyper-responsive to rhythmic fluctuation in intracranial pressure that is produced by normal arterial pulsations -LSB- 31 , 32 -RSB- . Central sensitization of nociceptive second order neurons in the trigeminocervical complex can be the reason for extracranial tenderness and cutaneous allodynia . The latter is an abnormal sensory state in which otherwise innocuous stimuli are sensed as painful -LSB- 31 -RSB- . Two-thirds of patients develop allodynia during or even after a migraine attack -LSB- 33 -RSB- . Based on clinical studies new guidelines for triptan treatment in migraine has been established . They include that allodynia-free patients can receive good results from triptan treatment at any time during the migraine attack . Furthermore , patients suffering from allodynia can expect excellent results from triptans before allodynia gets established . Therefore they should take triptans as early as possible during the attack . After the onset of allodynia , migraineurs can still benefit from triptans , but pain relief may be incomplete -LSB- 34 -- 36 -RSB- . However , a slightly different view has been expressed in a recent study -LSB- 37 -RSB- . After subcutaneous administration of sumatriptan , there was no difference in alleviation of migraine pain irrespective if the drug was given early or late . Therefore , it was suggested that the sensitization is of minor importance and that the relatively poor effect of sumatriptan late in migraine attacks is primarily due to gastric stasis -LSB- 37 -RSB- . CGRP receptors Local vasodilatation and release of CGRP from the vascular nerve fiber endings -- -- caused by electrical field stimulation or capsaicin treatment -LSB- 38 , 39 -RSB- -- -- are attenuated by administration of triptans , but also by application of CGRP receptor blockers -LSB- 40 -RSB- . The proof that CGRP plays a key role in migraine was provided by demonstrating that the potent CGRP receptor antagonist olcegepant -LRB- BIBN4096 -RRB- could abort acute migraine attacks to a comparable degree as sumatriptan -LSB- 41 , 42 -RSB- . In addition , CGRP receptors are located at several sites , which play an important role during migraine , including the cerebrovasculature -LSB- 43 -- 45 -RSB- , the trigeminocervical complex within the brainstem -LSB- 46 -RSB- and the trigeminal ganglion -LSB- 47 -RSB- . Functional CGRP receptors consist of three components , namely a seven-transmembrane domain G-protein coupled receptor called calcitonin receptor-like receptor -LRB- CLR -RRB- , a receptor activity-modifying protein -LRB- RAMP -RRB- and a receptor component protein -LRB- RCP -RRB- . CLR shares 55 % sequence identity with the calcitonin receptor . Co-expression of CLR with RAMP1 composes the CGRP receptor , while co-expression of CLR with either RAMP2 or RAMP3 represents the adrenomedullin -LRB- AM -RRB- receptor or possibly a combined receptor -LRB- CGRP and AM -RRB- . Apart from contributing to the receptor specificity , RAMP1 is required for glycosylation and transport of CLR to the plasma membrane -LSB- 48 -- 50 -RSB- . In addition , RAMP1 seems to be functionally rate limiting for CGRP receptor activity in the trigeminovascular system . It was proposed that elevated RAMP1 expression could sensitize the trigeminal ganglion of individuals to CGRP actions such as CGRP release and neurogenic inflammation -LSB- 47 -RSB- . The third component of the CGRP receptor , the accessory protein RCP , is required for proper biological function as it is involved in coupling the receptor to downstream signaling pathways like the protein kinase A -LRB- PKA -RRB- pathway -LSB- 51 -RSB- . Activation of CGRP receptors in cultured trigeminal ganglion cells increased the cAMP levels , which in turn led to elevated CGRP promoter activity and increased CGRP mRNA levels . These results point to an autoactivation of CGRP expression and indicate that elevated release of CGRP in the trigeminal system may create a self-sustaining feedback loop -LSB- 47 -RSB- . CGRP receptor antagonists olcegepant and MK-0974 The excellent correlation between CGRP release and migraine headache has long pointed towards the potential usefulness of a specific CGRP receptor antagonist in the treatment of primary headaches -LSB- 9 , 52 -RSB- . Thus , a series of small molecule CGRP receptor blockers were developed . Among those is olcegepant , which demonstrates extremely high affinity for human CGRP receptors , even higher affinity than the endogenous ligand -LSB- 53 -RSB- . Therefore it was selected for further pharmacological investigations . Olcegepant was shown to be a pure antagonist for the human CGRP receptor and to be selective against a broad panel of enzymes and receptors such as calcitonin , amylin or adrenomedullin receptors -LSB- 54 -RSB- . In isolated human temporal , middle meningeal and cerebral arteries olcegepant effectively inhibits CGRP induced dilatation -LSB- 44 , 55 -RSB- . That may be due to the block of the CGRP receptor as mRNA for CLR and RAMP1 -- 3 has been found in human middle meningeal arteries , cerebral arteries as well as in microvessels -LSB- 56 -RSB- and CGRP receptor components could be detected in the human cerebral vasculature -LSB- 43 -RSB- . In addition , olcegepant dose-dependently inhibited vasodilatation induced by electrical stimulation of the trigeminal ganglion in primates -LSB- 54 -RSB- . One major advantage of a CGRP receptor blocker is the lack of vasoconstrictor ability . But blockade of the receptor of a strong vasodilatation involves a theoretical risk of causing both peripheral and cerebral vasoconstriction , especially as high affinity binding sites for CGRP have been reported in the heart , liver , spleen , intestine , urogenital system , skeletal muscles , and the skin -LSB- 57 -- 59 -RSB- . But olcegepant in doses up to 10 mg did not cause any clinical relevant changes of vital signs like blood pressure and pulse rate , in the ECG or in forearm blood flow in healthy volunteers -LSB- 60 -RSB- . Detailed studies of peripheral arteries such as mesenteric and coronary arteries -LSB- 44 , 61 -RSB- revealed that those arteries show weaker responses to CGRP and lower degree of antagonism by olcegepant . This is possibly related to a lower number of CGRP receptors . In addition , a study with seven healthy volunteers disclosed that olcegepant had no influence on cerebral blood flow or on the blood flow velocity in the middle cerebral artery -LSB- 62 -RSB- . It was concluded that CGRP receptor blockade has no effect on the cerebral circulation in man . However , it is still not clear where olcegepant acts as a CGRP antagonist . It has been reported to inhibit -LRB- 1 -RRB- dilatation of dural arteries initiated by CGRP -LSB- 63 -RSB- , -LRB- 2 -RRB- CGRP-induced hypotension -LSB- 40 -RSB- , and -LRB- 3 -RRB- increase in facial blood flow after trigeminal ganglion stimulation -LSB- 54 -RSB- . In contrast , olcegepant did not significantly inhibit changes in tone of cerebral arterioles or of local cortical cerebral blood flow -LSB- 40 -RSB- . Those results indicate that olcegepant poorly penetrates the blood-brain barrier , but is very effective in preventing vasodilatation of vessels without this feature -LSB- 64 -RSB- . Thus , this CGRP receptor blocker was suggested to exert its antagonistic effects mainly extracerebral , which correlates with results from a study of healthy volunteers -LSB- 65 -RSB- . In contrast , it was recently shown that CGRP-induced relaxation of the rat middle cerebral artery can only be inhibited by abluminally administered olcegepant as olcegepant was prevented from reaching the CGRP receptors by the arterial endothelium -LSB- 66 -RSB- . These results suggest that the anti-migraine effect of this CGRP receptor blocker is on the abluminal side of the blood-brain barrier . This is supported by the fact that olcegepant only has an antimigraine effect in high doses . Anyhow , olcegepant was effective in a dose of 2.5 mg in the treatment of acute migraine attacks -LSB- 41 -RSB- . The overall treatment effect revealed a responder rate -LRB- a moderate to severe headache going to mild or no headache -RRB- of 60 % at 2 h after the start of the infusion compared with 27 % in the placebo group -LRB- Fig. 2 -RRB- . Thus , intravenous olcegepant was found to be as efficacious as oral sumatriptan in the relief of acute attacks of migraine -LSB- 42 -RSB- . Fig. 2The anti-migraine effect of the CGRP receptor antagonists MK-0974 and olcegepant and the triptans rizatriptan and sumatriptan compared to placebo at 2-h pain relief . The overall treatment effect of MK-0974 -LRB- p = 0.015 -RRB- and rizatriptan -LRB- p = 0.010 -RRB- showed significance versus placebo -LRB- left part -RRB- -LSB- 69 -RSB- . A dose of 2.5 mg of olcegepant also showed significant superiority over placebo -LRB- p = 0.001 -RRB- -LSB- 41 -RSB- . Data of sumatriptan from another study -LSB- 42 -RSB- were added to demonstrate the similar efficacy of olcegepant compared to triptans Significant superiority over placebo was seen regarding the pain-free rate at 2 h , the sustained pain relief at 24 h and the improvement of nausea , photophobia and phonophobia -LRB- Fig. 3 -RRB- . The response was still rising at 4 h pointing at a long duration of action . At 24 h the pain-free rate was better than with triptans , suggesting a lower grade of rebound . Fig. 3Comparison of the sustained pain free-rate at 24 h of the CGRP receptor antagonists MK-0974 and olcegepant , the triptans rizatriptan and sumatriptan , and placebo . MK-0974 displayed superior efficacy versus placebo -LRB- p < 0.001 -RRB- -LSB- 69 -RSB- . In order to compare the efficacy of olcegepant to a triptan , data of sumatriptan from another study -LSB- 42 -RSB- were added The rate of recurrence was 19 % as compared to a placebo rate of 46 % . This is clearly lower than the recurrence rate of triptans which usually is around 28 % -LSB- 42 -RSB- . Safety and tolerability studies also showed that olcegepant is well tolerated . Adverse events were , in general , minor and dose-dependent . Transient mild paresthesia was the single most frequent adverse event -LSB- 60 -RSB- . One major disadvantage of olcegepant remains in its structure . It is a hydrophilic molecule , has poor penetration across the blood-brain barrier and is not suitable as an oral drug . Considering the crucial advantages of an oral formulation of a drug , a small-molecule , non-peptide CGRP antagonist called MK-0974 was recently developed for the acute treatment of migraine attacks -LSB- 67 -RSB- . MK-0974 was , among others , tested in a noninvasive pharmacodynamic model in rhesus monkeys , in which endogenous CGRP release and a vasodilatatory response are achieved by topical application of capsaicin to the forearm of the rhesus monkey -LSB- 68 -RSB- . Infusion of different CGRP receptor antagonists inhibited the increase in local dermal blood flow and showed effectiveness with MK-0974 being the most potent among them -LRB- > 3.5-fold -RRB- -LSB- 67 -RSB- . In addition , MK-0974 displayed a low clearance -LRB- 7.0 mL min − 1 kg − 1 -RRB- and a good i.v. half-life -LRB- 2.8 h -RRB- . The compound was finally chosen as a clinical candidate , when it showed > 10,000-fold selectivity in assays including more than 160 receptors , transporters , and enzymes -LSB- 67 -RSB- . Recently , MK-0974 has been tested in a randomized , double-blind , placebo-and active-controlled , outpatient study with a two-stage , dose-ranging design to test safety and effectiveness -LSB- 69 -RSB- . Three hundred and thirty patients suffering from migraine with and without aura were treated with MK-0974 -LRB- in doses of 25 -- 600 mg -RRB- , rizatriptan -LRB- 10 mg -RRB- or placebo . As doses below 300 mg were insufficient , they were discontinued . Thus , the overall treatment effect was defined as the average of the 300 , 400 , and 600 mg doses and showed significance vs. placebo on the primary endpoint -LRB- p = 0.015 -RRB- , which was defined as pain relief at 2 h after treatment -LRB- Fig. 2 -RRB- . MK-0974 displayed also superior efficacy vs. placebo for secondary endpoints such as pain freedom at 2 h -LRB- overall treatment effect p < 0.001 -RRB- , sustained pain relief at 24 h -LRB- overall treatment effect p < 0.001 -RRB- or sustained pain freedom at 24 h -LRB- overall treatment effect p < 0.001 -RRB- -LRB- Fig. 3 -RRB- . In addition , MK-0974 provided relief of migraine-associated symptoms such as photophobia , phonophobia and nausea . Rizatriptan -LRB- 10 mg -RRB- was used as an active control and demonstrated effectiveness vs. placebo -LRB- p = 0.010 -RRB- on the primary endpoint defined as pain relief at 2 h. By comparison , 300 and 600 mg doses of MK-0974 appeared to work as well as rizatriptan or showed even numerical superiority regarding the sustained pain freedom at 24 h and the sustained pain relief at 24 h. Concerning tolerability and safety , MK-0974 was generally well-tolerated . The reported side effects were mainly nausea , dizziness and somnolence , but the incidence of adverse experiences seemed to be comparable to the placebo group . In addition , there was no increase in adverse events with increasing dose . Thus , MK-0974 showed only mild side effects , but significant relief of migraine pain 2 h after treatment compared to placebo and the relief was sustained through 24 h -LSB- 69 -RSB- . Conclusion The novel CGRP receptor antagonists appear to be effective in the treatment of moderate and severe migraine attacks . They do not only provide pain relief at 2 h , but also show efficacy regarding the secondary endpoints such as pain freedom , improvement of associated symptoms and functional disability -LSB- 41 , 69 -RSB- . In addition , they show a prolonged effect of action compared to the triptans . That may suggest not only less rebound headache but perhaps also a prophylactic possibility . Furthermore , only minor adverse effects were reported and no cardiovascular side effects could be seen so far . This is probably due to the absence of vasoconstrictor properties and it also suggests that vasoconstriction is not necessary to abort acute migraine attacks . This lack of direct vasoconstrictor activity may represent an important advantage over the triptans . Further development of drugs against the CGRP receptor will offer a new promising way of treatment .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O B O O B O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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