Conformation of the ATP binding peptide in actin revealed by proton NMR spectroscopy

The actin peptide 106-124 exists in a completely conserved region of the sequence and binds strongly to both ATP and tripolyphosphate. Binding particularly affects residues 116 and 118 and generally affects the two segments 115-118 and 121-124 [Barden, J. A., & Kemp, B. E. (1987) Biochemistry 26, 1471-1478]. One-dimensional nuclear Overhauser enhancement difference spectroscopy was used to detect molecular interactions between both adjacent and nonadjacent residues. The N-terminal segment 106-112 was found to be largely extended. A sharp bend was detected between Pro-112 and Lys-113. The triphosphate moiety binds to the strongly hydrophilic central segment of the peptide. Evidence was obtained for a reverse turn involving residues 121-124. Amide proton temperature coefficients and coupling constants provide evidence for a type I beta-turn. A model of the ATP binding site is proposed together with its relationship to other parts of the actin structure and to the phalloidin binding site.     

The roles of ATP4- and Mg2+ in control steps of phosphoglycerate kinase

1H-NMR measurements were made of solutions of yeast phosphoglycerate kinase containing the nucleotide substrate, ATP, and Mg2+ in varying concentrations in order to investigate the affect that the metal ion has on the mode of ATP binding to the enzyme. From the change in the chemical shifts of the 'basic-patch' histidine resonances (His62, His167 and His170) and the nucleotide C8H, C2H and C1'H resonances it is apparent that there are at least two ATP-binding sites on the enzyme. Downfield shifts observed for the above histidine resonances at low nucleotide/enzyme molar ratios indicates that the primary binding site involves electrostatic interactions between the nucleotide triphosphate chain and the basic-patch region of the N-terminal domain. The secondary binding site is shown to involve predominantly hydrophobic interactions between the adenosine moiety and the protein. Evidence from previous two-dimensional NMR experiments [Fairbrother et al. (1990) Eur. J. Biochem. 190, 161-169] suggests that the secondary site is equivalent to the crystallographically observed catalytic site. The affinity of the catalytic site is increased relative to the primary electrostatic site with increasing Mg2+ concentration. The possible importance of these observations in the regulation of this enzyme in vivo are discussed.     

Characterization of a cysteine-containing peptide after affinity labelling of Ca2+-ATPase of sarcoplasmic reticulum with the disulfide of 3'(2')-O-biotinyl-thioinosine triphosphate

3'(2')-O-Biotinyl-thioinosine triphosphate is a substrate of the Ca2+ pump of sarcoplasmic reticulum. Its disulfide inactivates the Ca2+-ATPase with two different velocities. The rapidly inactivated sulfhydryl group cannot be protected by ATP and is therefore considered to be outside the ATP binding site. The slowly reacting sulfhydryl group interacts with the disulfide of 3'(2')-O-biotinyl-thioinosine triphosphate with a dissociation constant of Kd = 137 microM and an inactivation velocity constant of 1.7 X 10(-3) s-1. It is protected by ATP with two different dissociation constants of the enzyme-ATP complex of Kd = 221 microM and 1130 microM. The slowly reacting sulfhydryl group is therefore considered to be part of the ATP binding site. Since it was impossible to isolate a tryptic peptide by affinity purification on matrix-bound avidin after affinity labelling with the disulfide of 3'(2')-O-biotinyl-thioinosine triphosphate, differential labelling with iodo[2-14C]acetic acid after affinity labelling with the disulfide of 3'(2')-O-biotinyl-thioinosine triphosphate was carried out. Tryptic digestion and FPLC purification led to the isolation of a radioactive carboxymethyl derivative of the cysteine-containing peptide ANACNSVIR. This peptide is equivalent to the cDNA-derived sequence 468-476 of Ca2+-ATPase [Brandl et al. (1986) Cell 44, 597-607] and is located between the phosphorylation site, Asp351, and Lys515, a part of the putative purine binding subsite of ATP. Although the carboxymethylation of Cys471 is hindered by (biotinyl-s6ITP)2, the strong dilution of the specific radioactivity of iodo[2-14C]acetic acid in the isolated peptide 468-476 argues against its direct interaction with the ATP analogue. It is therefore proposed that Cys471 undergoes ATP-dependent conformational changes.     

Structural characterization of the interactions between calmodulin and skeletal muscle myosin light chain kinase: effect of peptide (576-594)G binding on the Ca2+-binding domains

Calcium-containing calmodulin (CaM) and its complex with a peptide corresponding to the calmodulin-binding domain of skeletal muscle myosin light chain kinase [skMLCK(576-594)G] have been studied by one- and two-dimensional 1H NMR techniques. Resonances arising from the antiparallel beta-sheet structures associated with the calcium-binding domains of CaM and their counterparts in the CaM-skMLCK(576-594)G complex have been assigned. The assignments were initiated by application of the main chain directed assignment strategy. It is found that, despite significant changes in chemical shifts of resonances arising from amino acid residues in this region upon binding of the peptide, the beta-sheets have virtually the same structure in the complex as in CaM. Hydrogen exchange rates of amide NH within the beta-sheet structures are significantly slowed upon binding of peptide. These data, in conjunction with the observed nuclear Overhauser effect (NOE) patterns and relative intensities and the downfield shifts of associated amide and alpha resonances upon binding of peptide, show that the peptide stabilizes the Ca2+-bound state of calmodulin. The observed pattern of NOEs within the beta-sheets and their structural similarity correspond closely to those predicted by the crystal structure. These findings imply that the apparent inconsistency of the crystal structure with recently reported low-angle X-ray scattering profiles of CaM may lie within the putative central helix bridging the globular domains.     

Solution structure of the 45-residue MgATP-binding peptide of adenylate kinase as examined by 2-D NMR, FTIR, and CD spectroscopy

The structure of a synthetic peptide corresponding to residues 1-45 of rabbit muscle adenylate kinase has been studied in aqueous solution by two-dimensional NMR, FTIR, and CD spectroscopy. This peptide, which binds MgATP and is believed to represent most of the MgATP-binding site of the enzyme [Fry, D.C., Kuby, S.A., & Mildvan, A.S. (1985) Biochemistry 24, 4680-4694], appears to maintain a conformation similar to that of residues 1-45 in the X-ray structure of intact porcine adenylate kinase [Sachsenheimer, W., & Schulz, G.E. (1977) J. Mol. Biol. 114, 23-26], with 42% of the residues of the peptide showing NOEs indicative of phi and psi angles corresponding to those found in the protein. The NMR studies suggest that the peptide is composed of two helical regions of residues 4-7 and 23-29, and three stretches of beta-strand at residues 8-15, 30-32, and 35-40, yielding an overall secondary structure consisting of 24% alpha-helix, 38% beta-structure, and 38% aperiodic. Although the resolution-enhanced amide I band of the peptide FTIR spectrum is broad and rather featureless, possibly due to disorder, it can be fit by using methods developed on well-characterized globular proteins. On this basis, the peptide consists of 35 +/- 10% beta-structure, 60 +/- 12% turns and aperiodic structure, and not more than 10% alpha-helix. The CD spectrum is best fit by assuming the presence of at most 13% alpha-helix in the peptide, 24 +/- 2% beta-structure, and 66 +/- 4% aperiodic. The inability of the high-frequency FTIR and CD methods to detect helices in the amount found by NMR may result from the short helical lengths as well as from static and dynamic disorder in the peptide. Upon binding of MgATP, numerous conformational changes in the backbone of the peptide are detected by NMR, with smaller alterations in the overall secondary structure as assessed by CD. Detailed assignments of resonances in the peptide spectrum and intermolecular NOEs between protons of bound MgATP and those of the peptide, as well as chemical shifts of peptide resonances induced by the binding of MgATP, are consistent with the previously proposed binding site for MgATP on adenylate kinase.     

A semisynthetic bovine pancreatic ribonuclease containing a unique nitrotyrosine residue

A fully active semisynthetic ribonuclease, RNase 1-118:111-124, may be prepared by enzymatically removing six residues from the COOH terminus of the protein (positions 119-124) and then complementing the inactive RNase 1-118 with a chemically synthesized peptide containing the COOH-terminal 14 residues of the molecule (RNase 111-124) [M. C. Lin, B. Gutte, S. Moore, and R. B. Merrifield (1970) J. Biol. Chem. 245, 5169-5170]. Nitration of tyrosine-115 in the peptide followed by complex formation with RNase 1-118 affords a fully active enzyme containing a unique nitrotyrosine residue in a position which is known and which is very likely to be completely exterior to the active site region. The binding constant between the tetradecapeptide and RNase 1-118 (5 X 10(6) M-1 at pH 6.0) is not changed by the nitration. Crystals of the nitrated complex are isomorphous with those of RNase 1-118:111-124, for which a refined 1.8-A structure has recently been obtained.     

Extremely fast hydrogen exchange of ribonuclease-(1-118) as compared with native RNase A and its implication for the conformational energy state

RNase-(1-118) containing native disulfide bonds is similar in fold to native RNase A but not of lowest Gibbs energy as compared with the isomers containing non-native disulfide bonds. The present n.m.r. studies have indicated a dramatic increase in the exchange rate of all of the 'protected' amide protons of RNase-(1-118) over RNase A. A calculation shows a large increase in the rate of 'opening' of the structure. The exchange rate of the protected amide protons of RNase-(1-120) is slower than RNase-(1-118) but much faster than RNase A. Binding with a synthetic complementing fragment (114-124) markedly reduces the exchange rate of 20 to 25 amide protons of RNase-(1-118). It has previously been shown that binding with a complementing fragment of RNase-(1-118) generates a lowest Gibbs energy state. Thus, using available thermodynamic information for interpretation, we suggest that a) removal of six carboxy terminal residues of RNase A would disrupt coupling between these residues and those distant in the structure (loss of extra stabilizing energy), b) this would, in turn, alter the enthalpy-entropy compensation in such a way that the magnitude of Gibbs energy change favoring folding is significantly reduced without a large change of fold and c) in this activated state the molecule would be highly motile.     

Cis-trans isomerization of proline in the peptide (his 105-Val 124) of ribonuclease a containing the primary nucleation site

Conformational energy calculations have been carried out to determine the relative stabilities of the C-terminal sequence 105–124 of ribonuclease A, withcis andtrans forms, respectively, of Asn 113-Pro 114. Thecis form of Pro 114 is the one that occurs in the native protein. This peptide contains the sequence 106–118, which, on the basis of both theoretical and experimental studies, is thought to constitute the primary nucleation site for the folding of ribonuclease A. It is shown that both conformations of the isolated peptide (with Pro 114 in thecis andtrans forms, respectively) are of approximately equal stability. Both forms have similar conformations from residues 105–110 and 118–124, while they differ in the bend region involving residues 111–117. Calculations have also been carried out to deduce the possible low-energy paths for the interconversion between thecis andtrans forms of both Pro 114 and Pro 117. It is shown that there are two low-energy paths (with a minimum activation energy of 16.5 kcal/mole) for the interconversion of Pro 114. Attractive nonbonded interaction energies stabilize the transition state on these paths. Only one relatively low-energy path (with an activation energy of 18 kcal/mole) could be found for the isomerization of Pro 117, which occur in thetrans form in the native protein; in this case, allcis forms have significantly higher energy than thetrans form. These calculations thus show that native-like forms for the isolated peptide can exist with Pro 114 in either thecis or thetrans form and that these forms are readily interconvertible.     

Cardiac troponin I inhibitory peptide: location of interaction sites on troponin C

Cardiac troponin I(129-149) binds to the calcium saturated cardiac troponin C/troponin I(1-80) complex at two distinct sites. Binding of the first equivalent of troponin I(129-149) was found to primarily affect amide proton chemical shifts in the regulatory domain, while the second equivalent perturbed amide proton chemical shifts within the D/E linker region. Nitrogen-15 transverse relaxation rates showed that binding the first equivalent of inhibitory peptide to the regulatory domain decreased conformational exchange in defunct calcium binding site I and that addition of the second equivalent of inhibitory peptide decreased flexibility in the D/E linker region. No interactions between the inhibitory peptide and the C-domain of cardiac troponin C were detected by these methods demonstrating that the inhibitory peptide cannot displace cTnI(1-80) from the C-domain.     

Nonnative isomers of proline-93 and -114 predominate in heat-unfolded ribonuclease A

The peptide bonds preceding both Pro-93 and Pro-114, which are in the cis conformation in native RNase A, are predominantly in the trans conformation in the heat-unfolded protein. The percentages are estimated to be 60% and 63%, respectively, with a standard deviation of +/- 7% in each quantity. These ratios are close to those found for corresponding sequences in X-Pro-Y peptides. The concentration of the trans proline species was determined from the integrated intensities of resonance peaks of the C alpha H protons of Tyr-92 and Asn-113, which are well resolved in the 1D proton NMR spectrum of heat-unfolded RNase A. The assignments of the resonances were deduced from 2D NOESY and DQF-COSY spectra of unfolded RNase A in D2O. Furthermore, the C alpha H protons of both Tyr-92 and Asn-113 had an intense NOE cross-peak with the C delta H and C delta' H of the respective following prolines. For both Pro-93 and Pro-114, these NOE cross-peaks would arise only if the X-Pro peptide bond were in the trans conformation. It is generally believed that the rate of refolding of RNase A is considerably reduced by nonnative proline isomers, such as trans Pro-93. Two models for folding RNase A, that are consistent with these new results and the work of previous investigators, are presented here.     

Interaction of troponin I and troponin C: 19F NMR studies of the binding of the inhibitory troponin I peptide to turkey skeletal troponin C.

We have used 19F nuclear magnetic resonance spectroscopy to study the interaction of the inhibitory region of troponin (TnI) with apo- and calcium(II)-saturated turkey skeletal troponin C (TnC), using the synthetic TnI analogue N alpha-acetyl[19FPhe106]TnI(104-115)amide. Dissociation constants of Kd = (3.7 +/- 3.1) x 10(-5) M for the apo interaction and Kd = (4.8 +/- 1.8) x 10(-5) M for the calcium(II)-saturated interaction were obtained using a 1:1 binding model of peptide to protein. The 19F NMR chemical shifts for the F-phenylalanine of the bound peptide are different from the apo- and calcium-saturated protein, indicating a different environment for the bound peptide. The possibility of 2:1 binding of the peptide to Ca(II)-saturated TnC was tested by calculating the fit of the experimental titration data to a series of theoretical binding curves in which the dissociation constants for the two hypothetical binding sites were varied. We obtained the best fit for 0.056 mM less than or equal to Kd1 less than or equal to 0.071 mM and 0.5 mM less than or equal to Kd2 less than or equal to 2.0 mM. These results allow the possibility of a second peptide binding site on calcium(II)-saturated TnC with an affinity 10- to 20-fold weaker than that of the first site.     

Contribution of two conserved glycine residues to fibrillogenesis of the 106-126 prion protein fragment. Evidence that a soluble variant of the 106-126 peptide is neurotoxic

The fibrillogenic peptide corresponding to the residues 106-126 of the prion protein sequence (PrP 106-126) is largely used to explore the neurotoxic mechanisms underlying the prion disease. However, whether the neuronal toxicity of PrP 106-126 is caused by a soluble or fibrillar form of this peptide is still unknown. The aim of this study was to correlate the structural state of this peptide with its neurotoxicity. Here we show that the two conserved Gly114 and Gly119 residues, in force of their intrinsic flexibility, prevent the peptide assuming a structured conformation, favouring its aggregation in amyloid fibrils. The substitution of both Gly114 and Gly119 with alanine residues (PrP 106-126 AA mutated peptide) reduces the flexibility of this prion fragment and results in a soluble, beta-structured peptide. Moreover, PrP 106-126 AA fragment was highly toxic when incubated with neuroblastoma cells, likely behaving as a neurotoxic protofibrillar intermediate of the wild-type PrP 106-126. These data further confirm that the fibrillar aggregation is not necessary for the induction of the toxic effects of PrP 106-126.     

Application of peptide-mediated ring current shifts to the study of neurophysin-peptide interactions: a partial model of the neurophysin-peptide complex

Perdeuteriated peptides were synthesized that are capable of binding to the hormone binding site of neurophysin but that differ in the position of aromatic residues. The binding of these peptides to bovine neurophysin I and its des-1-8 derivative was studied by proton nuclear magnetic resonance spectroscopy in order to identify protein residues near the binding site through the observation of differential ring current effects on assignable protein resonances. Phenylalanine in position 3 of bound peptides was shown to induce significant ring current shifts in several resonances assignable to the 1-8 sequence, including those of Leu-3 and/or Leu-5, but was without effect on Tyr-49 ring protons. The magnitude of these shifts was dependent on the identity of peptide residue 1. By contrast, the sole demonstrable direct effect of an aromatic residue in position 1 was a downfield shift in Tyr-49 ring protons. Study of peptide binding to des-1-8-neurophysin demonstrated similar conformations of native and des-1-8 complexes except for the environment of Tyr-49, confirmed the peptide-induced ring current shift assignments in native neurophysin, and indicated an effect of binding on Thr-9. These observations are integrated with other results to provide a partial model of neurophysin-peptide complexes that places the ring of Tyr-49 at a distance 5-10 A from residue 1 of bound peptide and that places both the 1-8 sequence and the protein backbone region containing Tyr-49 proximal to each other and to peptide residue 3.(ABSTRACT TRUNCATED AT 250 WORDS)     

ATP effects on insulin-degrading enzyme are mediated primarily through its triphosphate moiety

It has been reported previously that ATP inhibits the insulysin reaction (Camberos, M. C., Perez, A. A., Udrisar, D. P., Wanderley, M. I., and Cresto, J. C. (2001) Exp. Biol. Med. 226, 334-341). We report here that with 2-aminobenzoyl-GGFLRKHGQ-ethylenediamine-2,4-dinitrophenyl as substrate, ATP and other nucleotides increase the rate >20-fold in Tris buffer. There is no specificity with respect to the nucleotide; however, ATP is more effective than ADP, which is more effective than AMP. Triphosphate itself was as effective as ATP, indicating it is this moiety that is responsible for activation. The binding of triphosphate was shown to be at a site distinct from the active site, thus acting as a noncompetitive activator. With the physiological substrates insulin and amyloid beta peptide, nucleotides and triphosphate were without effect. However, with small physiological peptides such as bradykinin and dynorphin B-9, ATP and triphosphate increased the rate of hydrolysis approximately 10-fold. Triphosphate and ATP shifted the oligomeric state of the enzyme from primarily dimer-tetramers to a monomer. These data suggest the presence of an allosteric regulatory site on insulysin that may shift its specificity toward small peptide substrates.     

NMR study of the phosphoryl binding loop in purine nucleotide proteins: evidence for strong hydrogen bonding in human N-ras p21

The structure of the phosphoryl binding region of human N-ras p21 was probed by using heteronuclear proton-observed NMR methods. Normal protein and a Gly-12----Asp-12 mutant protein were prepared with two amino acids labeled with 15N at their amide positions: valine and glycine, aspartic acid and glycine, and lysine and glycine. We completed the identification of amide 15NH resonances from Gly-12 and Asp-12 to the end of the phosphoryl binding domain consensus sequence (Lys-16) in protein complexed with GDP and have made tentative amide identifications from Val-9 to Ser-17. The methods used, together with initial identifications of the Gly-12 and -13 amide resonances, were described previously [Campbell-Burk, S. (1989) Biochemistry 28, 9478-9484]. The amide resonances of both Gly-13 and Lys-16 are shifted downfield below 10.4 ppm in both the normal and mutant proteins. These downfield shifts are presumed to be due to strong hydrogen bonds with the beta-phosphate oxygens of GDP.     

Immunochemical evidence for the binding of caldesmon to the NH2-terminal segment of actin

The binding of caldesmon and its actin-binding fragments to actin was studied by using peptide antibodies directed against two actin sites implicated in actomyosin interactions. Antibodies against residues 1-7 on skeletal alpha-actin strongly inhibited the binding of caldesmon to actin and perturbed to a smaller extent the interaction between actin and the actin binding fragments. Carbodiimide coupling of ethylenediamine to the NH2-terminal acidic residues on actin inhibited the binding of caldesmon and its fragments to actin to a similar extent as the (residues 1-7) antibodies. Antibodies against residues 18-28 showed only limited competition with caldesmon for the binding to actin. These results lead to the following conclusions. (i) The NH2-terminal residues on actin play an important role in the binding of caldesmon to actin, (ii) residues 18-28 on actin do not form a major caldesmon interaction site, and (iii) the actin-binding fragments do not contain the full actin-binding interface. These conclusions and other literature data suggest that caldesmon regulates the actomyosin ATPase by competing with myosin.ATP for the NH2-terminal segment on actin.     

Mapping the surface of Escherichia coli peptide deformylase by NMR with organic solvents

Identifying potential ligand binding sites on a protein surface is an important first step for targeted structure-based drug discovery. While performing control experiments with Escherichia coli peptide deformylase (PDF), we noted that the organic solvents used to solubilize some ligands perturbed many of the same resonances in PDF as the small molecule inhibitors. To further explore this observation, we recorded (15)N HSQC spectra of E. coli peptide deformylase (PDF) in the presence of trace quantities of several simple organic solvents (acetone, DMSO, ethanol, isopropanol) and identified their sites of interaction from local perturbation of amide chemical shifts. Analysis of the protein surface structure revealed that the ligand-induced shift perturbations map to the active site and one additional surface pocket. The correlation between sites of solvent and inhibitor binding highlights the utility of organic solvents to rapidly and effectively validate and characterize binding sites on proteins prior to designing a drug discovery screen. Further, the solvent-induced perturbations have implications for the use of organic solvents to dissolve candidate ligands in NMR-based screens.     

The environment of the high-affinity cation binding site on actin and the separation between cation and ATP sites as revealed by proton NMR and fluorescence spectroscopy

The lanthanide ions Lu3+ (diamagnetic) and Gd3+ (paramagnetic broadening probe) were used to displace Ca2+ from the high-affinity cation binding site on G-actin. The effects of these higher-affinity ions on the proton nuclear magnetic resonance spectrum of actin were recorded. The aliphatic proton envelope in the Gd-actin sample exhibited a complex array of changes due to the proximity of Gd to several aliphatic residues. No such changes were observed in the diamagnetic Lu-actin control spectrum. By contrast, the aromatic proton envelope remained largely unaffected in both Gd-actin and Lu-actin samples. However, the adenosine moiety on the actin-bound ATP became increasingly mobilized without the triphosphate chain being released from the ATP binding site. Maximum adenosine mobilization occurred with approximately 1 mol of lanthanide ion bound per mol of actin. The absence of changes in the aromatic proton envelope suggests that the high-affinity cation binding site is in a region well removed from the adenosine moiety of bound ATP as well as any aromatic side-chains. The separation of the ATP and cation sites was further explored using the fluorescent ATP analogues FTP and epsilon-ATP. Tb3+ bound to the high-affinity cation site was found to be separated by 16 A from the FTP chromophore bound to the nucleotide binding site on actin. Since this distance is greater than can be accommodated on a model of the Tb-ATP complex, we conclude that the sites are physically separate. This conclusion was further reinforced by experiments involving the quenching of epsilon-ATP fluorescence by Mn2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

15N and 1H NMR study of histidine containing protein (HPr) from Staphylococcus carnosus at high pressure

The pressure-induced changes in 15N enriched HPr from Staphylococcus carnosus were investigated by two-dimensional (2D) heteronuclear NMR spectroscopy at pressures ranging from atmospheric pressure up to 200 MPa. The NMR experiments allowed the simultaneous observation of the backbone and side-chain amide protons and nitrogens. Most of the resonances shift downfield with increasing pressure indicating generalized pressure-induced conformational changes. The average pressure-induced shifts for amide protons and nitrogens are 0.285 ppm GPa(-1) at 278 K and 2.20 ppm GPa(-1), respectively. At 298 K the corresponding values are 0.275 and 2.41 ppm GPa(-1). Proton and nitrogen pressure coefficients show a significant but rather small correlation (0.31) if determined for all amide resonances. When restricting the analysis to amide groups in the beta-pleated sheet, the correlation between these coefficients is with 0.59 significantly higher. As already described for other proteins, the amide proton pressure coefficients are strongly correlated to the corresponding hydrogen bond distances, and thus are indicators for the pressure-induced changes of the hydrogen bond lengths. The nitrogen shift changes appear to sense other physical phenomena such as changes of the local backbone conformation as well. Interpretation of the pressure-induced shifts in terms of structural changes in the HPr protein suggests the following picture: the four-stranded beta-pleated sheet of HPr protein is the least compressible part of the structure showing only small pressure effects. The two long helices a and c show intermediary effects that could be explained by a higher compressibility and a concomitant bending of the helices. The largest pressure coefficients are found in the active center region around His15 and in the regulatory helix b which includes the phosphorylation site Ser46 for the HPr kinase. This suggests that this part of the structure occurs in a number of different structural states whose equilibrium populations are shifted by pressure. In contrast to the surrounding residues of the active center loop that show large pressure effects, Ile14 has a very small proton and nitrogen pressure coefficient. It could represent some kind of anchoring point of the active center loop that holds it in the right place in space, whereas other parts of the loop adapt themselves to changing external conditions.