Hydrogen exchange of primary amide protons in basic pancreatic trypsin inhibitor: evidence for NH2 group rotation in buried asparagine side chains

Hydrogen-deuterium exchange is measured for the buried primary amide groups of Asn-43 and Asn-44 in bovine pancreatic trypsin inhibitor. Amide protons trans and cis to the amide carbonyl oxygen (HE and HZ, respectively) exchange at indistinguishable rates. Uncorrelated exchange of HE and HZ is established for both residues by following the nuclear Overhauser enhancement from HE to HZ during the deuterium exchange. The exchange of Asn-43 and Asn-44 side-chain protons differs qualitatively from exchange of primary amide groups in fully solvated model compounds, for which HE generally exchanges faster than HZ. The equal rates for the buried primary amide HE and HZ in BPTI are not a consequence of coupled exchange. The data indicate rapid rotation around the CO-NH2 bond for both Asn-43 and Asn-44 and suggest considerable lability of intramolecular hydrogen bonds. The side chain of Asn-43 has all of its polar atoms integrated into the very stable hydrogen-bonded structure of the protein. Asn-44 is hydrogen-bonded to side chains and to a buried water molecule. Solvent isotope exchange is several orders of magnitude more restricted by protein secondary and tertiary structure than the CO-NH2 rotation, indicating that N delta H2 groups flip many times before hydrogen isotope exchange occurs.     

Hydrogen exchange kinetics of bovine pancreatic trypsin inhibitor beta-sheet protons in trypsin-bovine pancreatic trypsin inhibitor, trypsinogen-bovine pancreatic trypsin inhibitor, and trypsinogen-isoleucylvaline-bovine pancreatic trypsin inhibitor

Hydrogen exchange rates of six beta-sheet peptide amide protons in bovine pancreatic trypsin inhibitor (BPTI) have been measured in free BPTI and in the complexes trypsinogen-BPTI, trypsinogen-Ile-Val-BPTI, bovine trypsin-BPTI, and porcine trypsin-BPTI. Exchange rates in the complexes are slower for Ile-18, Arg-20, Gln-31, Phe-33, Tyr-35, and Phe-45 NH, but the magnitude of the effect is highly variable. The ratio of the exchange rate constant in free BPTI to the exchange rate constant in the complex, k/kcpIx, ranges from 3 to much greater than 10(3). Gln-31, Phe-45, and Phe-33 NH exchange rate constants are the same in each of the complexes. For Ile-18 and Tyr-35, k/kcpIx is much greater than 10(3) for the trypsin complexes but is in the range 14-43 for the trypsinogen complexes. Only the Arg-20 NH exchange rate shows significant differences between trypsinogen-BPTI and trypsinogen-Ile-Val-BPTI and between porcine and bovine trypsin-BPTI.     

Hydrogen exchange kinetics changes upon formation of the soybean trypsin inhibitor-trypsin complex.

The hydrogen exchange kinetics of the complex of trypsin-soybean trypsin inhibitor (Kunitz) have been compared to the calculated sum of the exchange kinetics for the inhibitor and trypsin measured separately. The exchange rates observed for the complex are substantially less than the sum of the exchange rates in the two individual proteins. These results cannot be accounted for by changes in intermolecular or intramolecular hydrogen bonding. The decrease in exchange rates in the complex are ascribed to changes in solvent accessibility in the component proteins.     

The pH dependence of hydrogen exchange in proteins

The static accessibility modified discrete charge model for electrostatic interactions in proteins is extended to the prediction of the pH dependence of hydrogen exchange reactions. The exchange rate profiles of buried amide protons are shown to follow the calculated pH dependence of the electrostatic component of protein stability. Rate profiles are calculated for individual buried amide protons in ribonuclease S and bovine pancreatic trypsin inhibitor. The electrostatic free energy of stabilization of the protein and the energy required to bring the catalytic ion to an exchange site are expressed as an apparent, pH-dependent contribution to the activation energy. Changes in the electrostatic stabilization of the proteins affect the calculated exchange rate for buried amide protons by more than 1000, while local field effects raise or lower the predicted exchange rates by less than 100. The pH dependence of exchangeable protons at the protein surface, such as the C-2 imidazole protons, is shown to follow the estimated energy required to introduce the catalytic ion at the exchange site. These calculations are discussed in terms of current models for proton exchange which incorporate the dynamic nature of the structure to explain exchange data from the interior of a protein.     

Correlation between the amide proton exchange rates and the denaturation temperatures in globular proteins related to the basic pancreatic trypsin inhibitor.

Nuclear magnetic resonance was used to measure the hydrogen-deuterium exchange rates for individual interior amide protons in a group of small globular proteins related to the basic pancreatic trypsin inhibitor (BPTI). These proteins include two homologous proteins and seven chemical modifications of BPTI. It was previously shown that the spatial structure of BPTI is preserved in all these related proteins. The exchange rates for corresponding amide protons in the different proteins were found to vary by a factor of as much as 5 X 104. The proton exchange is correlated with the thermal stability of the proteins, i.e. the lower the denaturation temperature, the faster the NH exchange. Further evidence that the exchange of interior amide protons is promoted by global fluctuations of the protein structures comes from the observation that the order of the relative exchange rates for the individual protons is the same in all the different species. This is the third in a series of three papers on nuclear magnetic resonance studies of labile protons in BPTI-related proteins. A detailed interpretation of the data will be given in a forthcoming paper.     

Amide-proton exchange studies by two-dimensional correlated 1H NMR in two chemically modified analogs of the basic pancreatic trypsin inhibitor

The backbone amide proton exchange with the solvent was investigated in 2H2O solutions of the basic pancreatic trypsin inhibitor and two chemical modifications thereof, which were obtained by transamination of the N-terminus and by cleavage of the disulfide bond 14-38, respectively. The three proteins have nearly identical conformations, but the stability with respect to thermal denaturation is markedly different. Exchange rates for a large number of individually assigned amide protons located both in central and peripheral parts of the protein structures were measured by two-dimensional correlated spectroscopy (COSY). From analysis of the individual proton exchange rates in the three proteins at different temperatures, an interplay of global and local structure fluctuations was characterized, which promote hydrogen exchange in distinct regions of the molecules. The exchange of particular amide protons may be governed by different motional processes at different temperatures. As a general trend, global fluctuations involving breakage of numerous hydrophilic secondary bonds appear to be dominant at higher temperatures, whereas at lower temperatures the influence of local fluctuations in hydrophobic regions of the protein structures is also clearly noticeable.     

Hydrogen exchange kinetics of surface peptide amides in bovine pancreatic trypsin inhibitor

The acid and base catalytic rate constants, kH, obs and kOH, obs and the pH at the minimum rate, pHmin, of 25 rapidly exchanging protons in bovine pancreatic trypsin inhibitor have been determined. Here we report the labeling procedure giving 1H nuclear magnetic resonance spectral resolution of seven additional rapidly exchanging NH protons and the pH dependence of their chemical shifts. Values of kH,obs kOH,obs and pHmin are given for Ala16, Gly28 and Arg53 NH groups, the only backbone amide protons with static accessibility of more than zero in the crystal structure not previously reports, and for Gly56 NH, buried at the C terminus of an alpha-helix. All four protons reported here have pH min greater than or equal to 3. Conclusions of the previous study predict that peptide protons with pHmin higher than those of model compounds have greater static accessibility of the peptide O than of the peptide N atom. The locations in the crystal structure of the four NH groups whose exchange rates are reported here are in qualitative agreement with these predictions. The ionic strength dependence of Ala16 at pH 5.5 shows a sharp increase in the exchange rate with decreasing salt concentration, as expected for base-catalyzed exchange in a positive electrostatic field.     

Raman studies of the conformation of the basic pancreatic trypsin inhibitor.

The vibrational Raman spectra of the basic pancreatic trypsin inhibitor in aqueous solution, as lyophilized powder and in a single crystal and presented. The thermal stability of this protein is demonstrated by the fact that minor alterations in the spectrum, mainly in the amide III band near 1260 cm-1, occur in the solution spectrum only at temperatures above 75 degrees C. No significant spectral changes appear when the pH value of the solution is varied in the range from 1.5 to 8.7. The distinct differences of the powder spectrum compared to that of the solution, show that lyophilization causes appreciable conformational changes both in the main-chain and in the side-chains. A difference in main chain conformation of the basic pancreatic trypsin inhibitor in single crystal and in solution is suggested by different amide III frequencies.     

Crystal structure of a Kunitz-type trypsin inhibitor from Erythrina caffra seeds

The trypsin inhibitor DE-3 from Erythrina caffra (ETI) belongs to the Kunitz-type soybean trypsin inhibitor (STI) family and consists of 172 amino acid residues with two disulphide bridges. The amino acid sequence of ETI shows high homology to other trypsin inhibitors from the same family but ETI has the unique ability to bind and inhibit tissue plasminogen activator. The crystal structure of ETI has been determined using the method of isomorphous replacement and refined using a combination of simulated annealing and conventional restrained least-squares crystallographic refinement. The refined model includes 60 water molecules and 166 amino acid residues, with a root-mean-square deviation in bond lengths from ideal values of 0.016 A. The crystallographic R-factor is 20.8% for 7770 independent reflections between 10.0 and 2.5 A. The three-dimensional structure of ETI consists of 12 antiparallel beta-strands joined by long loops. Six of the strands form a short antiparallel beta-barrel that is closed at one end by a "lid" consisting of the other six strands coupled in pairs. The molecule shows approximate 3-fold symmetry about the axis of the barrel, with the repeating unit consisting of four sequential beta-strands and the connecting loops. Although there is no sequence homology, this same fold is present in the structure of interleukin-1 alpha and interleukin-1 beta. When the structure of ETI and interleukin-1 beta are superposed, the close agreement between the alpha-carbon positions for the beta-strands is striking. The scissile bond (Arg63-Ser64) is located on an external loop that protrudes from the surface of the molecule and whose architecture is not constrained by secondary structure elements, disulphide bridges or strong electrostatic interactions. The hydrogen bonds made by the side-chain amide group of Asn12 play a key role in maintaining the three-dimensional structure of the loop. This residue is in a position corresponding to that of a conserved asparagine in the Kazal inhibitor family. Although the overall structure of ETI is similar to the partial structure of STI, the scissile bond loop is displaced by about 4 A. This displacement probably arises from the fact that the structure of STI has been determined in a complex with trypsin but could possibly be a consequence of the close molecular contact between Arg63 and an adjacent molecule in the crystal lattice.     

Carbonyl 13C NMR spectrum of basic pancreatic trypsin inhibitor: resonance assignments by selective amide hydrogen isotope labeling and detection of isotope effects on 13C nuclear shielding

The carbonyl region of the natural abundance 13C nuclear magnetic resonance (NMR) spectrum of basic pancreatic trypsin inhibitor is examined, and 65 of the 66 expected signals are characterized at varying pH and temperature. Assignments are reported for over two-thirds of the signals, including those of all buried backbone amide groups with slow proton exchange and all side-chain carbonyl groups. This is the first extensively assigned carbonyl spectrum for any protein. A method for carbonyl resonance assignments utilizing amide proton exchange and isotope effects on nuclear shielding is described in detail. The assignments are made by establishing kinetic correlation between effects of amide proton exchange observed in the carbonyl 13C region with development of isotope effects and in the amide proton region with disappearance of preassigned resonances. Several aspects of protein structure and dynamics in solution may be investigated by carbonyl 13C NMR spectroscopy. Some effects of side-chain primary amide group hydrolysis are described. The main interest is on information about intramolecular hydrogen-bond energies and changes in the protein due to amino acid replacements by chemical modification or genetic engineering.     

Comparison of hydrogen exchange rates for bovine pancreatic trypsin inhibitor in crystals and in solution.

Hydrogen exchange rate constants for the 17 slowest exchanging amide NH groups in bovine pancreatic trypsin inhibitor (BPTI) were measured in solution and in form II and form III crystals. All 17 amide hydrogens are buried and intramolecularly hydrogen bonded in the crystal structure, except Lys 41 which is buried and hydrogen bonded to a buried water. Large-scale crystallization procedures were developed for these experiments, and rate constants for both crystal and solution exchange were measured by 1H NMR spectroscopy of exchange-quenched samples in solution. Two conditions of pH and temperature, pH 9.8 and 35 degrees C, and pH 9.4 and 25 degrees C, bring two groups of hydrogens into the experimental time window (minutes to weeks). One consists of the 10 slowest exchanging hydrogens, all of which are associated with the central beta-sheet of BPTI. The second group consists of seven more rapidly exchanging hydrogens, which are distributed throughout the molecule, primarily in a loop or turn. In both groups, most hydrogens exchange more slowly in crystals, but there is considerable variation in the degree to which the exchange is depressed in crystals. Many differences observed for the more rapidly exchanging hydrogens can be attributed to local surface effects arising from intermolecular contacts in the crystal lattice. Within the slower group, however, a very large effect on exchange of Ile 18 and Tyr 35 appears to be selectively transmitted through the matrix of the molecule.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure and dynamics of lipid-associated states of apocytochrome c.

Apocytochrome c (apocyt c), which in aqueous solution is largely unstructured, acquires an alpha-helical conformation upon association with lipid membranes. The extent of alpha-helix induced in apocyt c is lipid-dependent and this folding process is driven by both electrostatic and hydrophobic lipid-protein interactions. The structural and dynamic properties of apocyt c in lipid membranes were investigated by attenuated total reflection Fourier transform infrared spectroscopy combined with amide H-D exchange kinetics. Apocyt c acquires a higher content of alpha-helical structure with negatively charged membranes than with zwitterionic ones. For all membranes studied here, the helices of these partially folded states of apocyt c have a preferential orientation perpendicular to the plane of the lipid membrane. The H-D exchange revealed that a small fraction of amide protons of apocyt c, possibly associated with a stable folded domain protected by the lipid, remained protected from exchange over 20 min. However, a large fraction of amide protons exchanged in less than 20 min, indicating that the helical states of apocyt c in lipid membranes are very dynamic.     

Mechanism of surface peptide proton exchange in bovine pancreatic trypsin inhibitor. Salt effects and O-protonation

The acid-catalyzed hydrogen exchange rate constants kH, and the base-catalyzed rate constants kOH, have been determined (in the preceding paper) for the 25 most rapidly exchanging NH groups of bovine pancreatic trypsin inhibitor. Most of these NH groups are at the protein-solvent interface. The correlation of kH, but not kOH, with the static accessibility and hydrogen bonding of the peptide carbonyl O atom indicates that the mechanism of acid catalysis in proteins involves O-protonation. Agreement between the ionic strength dependence observed for kH and kOH and the ionic strength dependence calculated for an O-protonation mechanism supports this conclusion. N-protonation for acid catalysis, as well as N-deprotonation for base catalysis, have traditionally been assumed in the mechanism of the chemical step in peptide amide proton exchange. A preference for the alternative O-protonation mechanism has far-reaching implications in the interpretation of protein hydrogen exchange kinetics. With an O-protonation mechanism, acid-catalyzed rates of surface NH groups are primarily a function of the average solvent accessibility of the carbonyl O atoms in the dynamic solution structure, while base-catalyzed rates of surface NH groups measure solvent accessibility of the peptide N. The relative dynamic accessibilities of peptide O atoms, as measured by relative values of kH (corrected for electrostatic effects), correlate with O static accessibilities in the crystal structure. A lower correlation of static accessibility of N atoms with kOH is observed for surface NH groups in peptide groups in which the carbonyl O is not hydrogen bonded. For some surface NH groups, the observed pH of minimum rate, pHmin, deviates widely from the pHmin of model compounds. This is explained as the combined result of electrostatic effects and of the differences in accessibility of the carbonyl O and N atoms that result in a change in the relative values of kH and kOH as compared to those of model peptides. A mechanism whereby exchange of interior sites is catalyzed by interactions of catalysis ions with protein surface atoms via charge transfer is suggested.     

Individual amide proton exchange rates in thermally unfolded basic pancreatic trypsin inhibitor

A novel experiment is described for measurements of amide proton exchange rates in proteins with a time resolution of about 1 s. A flow apparatus was used to expose protein solutions in 2H2O first to high temperature for a predetermined time period, during which 1H-2H exchange proceeded, and then to ice-water. The technique was applied for exchange studies in thermally unfolded, selectively reduced basic pancreatic trypsin inhibitor. Measurements were made by 1H nuclear magnetic resonance after the exchange was quenched by rapid cooling. Thereby, the sequence-specific resonance assignments for the folded protein could be used, which had been previously obtained. The results of this study indicate that the exchange rates in the thermally unfolded protein are close to those expected for a random chain and that the NH exchange is catalyzed by 2H+ and O2H- up to high temperature, with no significant contributions from p2H-independent catalysis. We conclude that the parameters derived by Molday et al. [Molday, R. S., Englander, S. W., & Kallen, R. G. (1972) Biochemistry 11, 150-158] from measurements with small model peptides can be used to calculate intrinsic exchange rates in unfolded proteins and thus provide a reliable reference for the interpretation of exchange rates measured under native conditions.     

Chemically stabilized trypsin used in dipeptide synthesis

Bovine pancreatic trypsin was treated with ethylene glycol bis(succinic acid N-hydroxysuccinimide ester). Approximately 8 of 14 lysines per trypsin molecule were modified. This derivative (EG trypsin) was more stable than native between 30 degrees and 70 degrees C: T50 values were 59 degrees C and 46 degrees C, respective. EG trypsin's half-life of 25 min at 55 degrees C was fivefold greater than native's. EG trypsin had a decreased rate of autolysis and retained more activity in aqueous mixtures of 1,4-dioxan, dimethylformamide, dimethylsulfoxide, and acetonitrile. EG trypsin had lower Km values for both amide and ester substrates; its kcat values for two amides (benzoyl-L-arginine p-nitroanilide and benzyloxycarbonyl glycyl-glycyl-arginyl-7-amino-4-methyl coumarin) increased, whereas its kcat value for an ester (thiobenzoyl benzoyloxycarbonyl-L-lysinate) decreased slightly. The specific activity (kcat/Km) of EG trypsin was increased for both amide and ester substrates. EG trypsin gave higher yields and reaction rates than native in kinetically controlled synthesis of benzoyl argininyl-leucinamide in acetonitrile and in t-butanol. Highest peptide yields occurred with EG trypsin in 95% acetonitrile, where 90% of the substrate was converted to product. No peptide synthesis occurred in 95% DMF with either form of trypsin.     

Identification of a protein-binding surface by differential amide hydrogen-exchange measurements. Application to Bowman-Birk serine-protease inhibitor.

The binding surface of soybean trypsin/chymotrypsin Bowman-Birk inhibitor in contact with alpha-chymotrypsin has been identified by measurement of the change in amide hydrogen-exchange rates between free and chymotrypsin-bound inhibitor. Exchange measurements were made for the enzyme-bound form of the inhibitor at pH 7.3, 25 degrees C using fast-flow affinity chromatography and direct measurement of exchange rates in the protein complex from one-dimensional and two-dimensional nuclear magnetic resonance spectra. The interface is characterized by a broad surface of contact involving residues 39 through 48 of the anti-chymotryptic domain beta-hairpin as well as residues 32, 33 and 37 in the anti-chymotryptic domain loop of the inhibitor. A number of residues in the anti-tryptic domain of the protein also have an altered exchange rate, suggesting that there are changes in the protein conformation upon binding to chymotrypsin. These changes in amide exchange behavior are discussed in light of a model of the complex based on the X-ray crystallographic structure of turkey ovomucoid inhibitor third domain bound to a alpha-chymotrypsin, and the structure of free Bowman-Birk inhibitor determined in solution by two-dimensional nuclear magnetic resonance spectroscopy. The chymotrypsin-binding loop of Bowman-Birk inhibitor in the model is remarkably similar to the binding loop conformation in crystal structures of enzyme-bound polypeptide chymotrypsin inhibitor-I from potatoes, turkey ovomucoid inhibitor third domain, and chymotrypsin inhibitor-II from barley seeds.     

Perturbing the polar environment of Asp102 in trypsin: consequences of replacing conserved Ser214.

Much of the catalytic power of trypsin is derived from the unusual buried, charged side chain of Asp102. A polar cave provides the stabilization for maintaining the buried charge, and it features the conserved amino acid Ser214 adjacent to Asp102. Ser214 has been replaced with Ala, Glu, and Lys in rat anionic trypsin, and the consequences of these changes have been determined. Three-dimensional structures of the Glu and Lys variant trypsins reveal that the new 214 side chains are buried. The 2.2-A crystal structure (R = 0.150) of trypsin S214K shows that Lys214 occupies the position held by Ser214 and a buried water molecule in the buried polar cave. Lys214-N zeta is solvent inaccessible and is less than 5 A from the catalytic Asp102. The side chain of Glu214 (2.8 A, R = 0.168) in trypsin S214E shows two conformations. In the major one, the Glu carboxylate in S214E forms a hydrogen bond with Asp102. Analytical isoelectrofocusing results show that trypsin S214K has a significantly different isoelectric point than trypsin, corresponding to an additional positive charge. The kinetic parameter kcat demonstrates that, compared to trypsin, S214K has 1% of the catalytic activity on a tripeptide amide substrate and S214E is 44% as active. Electrostatic potential calculations provide corroboration of the charge on Lys214 and are consistent with the kinetic results, suggesting that the presence of Lys214 has disturbed the electrostatic potential of Asp102.     

Hydrogen kinetics of peptide amide protons at the bovine pancreatic trypsin inhibitor protein-solvent interface

Hydrogen exchange rate constants of the 25 most rapidly exchanging peptide amide protons in bovine pancreatic trypsin inhibitor have been determined over a range of pH that spans pH min, the pH of minimum rate. Most of these are on the protein surface, exposed to solvent and not hydrogen bonded in the crystal structure. Contrary to commonly held assumptions, the exchange kinetics of surface NH groups are not equivalent to the kinetics of NH groups in peptides in the extended configuration. All surface NH groups exchange more slowly than NH groups in model peptides, with rate constants distributed over a range of more than two orders of magnitude. In addition, their pH min values vary widely. For most of the surface NH groups, pH min is lower than in model compounds and, for several, pH min is less than 1. These results indicate that the local environment of the surface peptide groups when the exchange event occurs is very different from that of extended peptides. Analysis based on consideration of an O-protonation mechanism for acid catalysis and of electrostatic effects on exchange kinetics further indicates (see the accompanying paper) that, in general, exchange of surface NH groups occurs from a conformation of the protein approximated by the crystal structure. The 1H-2H exchange rate constants were measured from 300 MHz nuclear magnetic resonance spectra in which assigned surface N1H resonances are resolved by the use of partially deuterated protein samples. A marked pH dependence of the chemical shifts observed in the pH range 1 to 4.5 for several surface NH groups reflects the titration of nearby carboxyl groups.     

Rates of structural fluctuations of lysozyme in the range of thermal unfolding transition

The hydrogen–deuterium exchange reaction for the tryptophan residues in lysozyme have been followed in 4.5M LiBr at pH 7.2 in the temperature range of the unfolding transition by measuring the transmittance change at 293 nm. The exchange reaction proceeded in three phases at low temperature for native protein. The first and the second phases were ascribed to the H-D exchange reactions of three relatively exposed tryptophan residues on the molecular surface. The third phase corresponded to the H-D exchange reaction of the three tryptophan residues buried in the interior of the molecule. The H-D exchange reaction proceeded in two phases near the melting temperature and in a single phase at high temperature, where almost all molecules are unfolded. The H-D exchange of three tryptophan residues buried in folded molecules was caused by fluctuation between the folded and unfolded structure of the protein molecule. The rates of such a fluctuation were determined from the rates of the exchange reaction at various temperatures. These rates agreed very well with those determined from the temperature-jump method. This means that a protein molecule in solution fluctuates between the N- and D-states at every temperature within the transition region, where the N-form is the tightly folded native structure and the D-form the randomly coiled chain. From measurements of thermal unfolding of ester-108-lysozyme and the binding constant of (NAG)₃ to ester-108-lysozyme, it was found that almost all cross-linked molecules are in the folded state near 50°C and pH 7.2 in 4.5M LiBr, where intact molecules are unfolded. We also studied the H-D exchange reaction of ester-108-lysozyme. In the temperature region of 43–50°C, about 70% of the exchangeable tryptophan residues of ester-108-lysozyme were exchanged within 1 s immediately after the mixing of D₂O, in spite of the fact that almost all molecules are in the folded state. This was considered the premelting of the surface of a corss-linked molecule.