Dynamics of the aromatic amino acid residues in the globular conformation of the basic pancreatic trypsin inhibitor (BPTI)

Summary The molecular conformation of the basic pancreatic trypsin inhibitor (BPTI) is known in considerable detail from both X-ray studies in single crystals and NMR studies in solution. The NMR experiments showed that the aromatic rings of the phenylalanyl and tyrosyl residues can undergo rapid rotational motions about the C-C bond. The present paper describes a model investigation of the mechanistic aspects of these intramolecular rotational motions. From calculations of the conformational energies for molecular species derived from the X-ray structure by rotations of individual aromatic rings, it was apparent that the rotational motions of the aromatics could only be understood in a flexible structure. Flexibility was simulated by allowing the protein to relax to an energetically favorable conformation for each of the different rotation states of the aromatic rings. It was then of particular interest to investigate how the perturbations caused by different rotation states of the aromatic rings were propagated in the protein structure. It was found that the rotation axes C-C were only slightly affected ( ¹20°). The most sizeable perturbations are caused by through space interactions with nearby atoms, which move away from the ring center and thus release the steric hindrance opposing the rotational motions. The values for the energy barriers obtained from the energy minimization are of the same order of magnitude as those measured by NMR.     

Dynamics of the aromatic amino acid residues in the globular conformation of the basic pancreatic trypsin inhibitor (BPTI). I. 1H NMR studies.

The basic pancreatic trypsin inhibitor (BPTI) was investigated by high resolution 1H NMR techniques at 360 MHz. Observation of the amide proton resonances of the polypeptide backbone showed that the globular conformation of BPTI determined by X-ray studies in single crystals is maintained in aqueous solution over the temperature range from 4 degrees to 87 degrees. NMR studies over this temperature range of the aromatic amino acid residues of BPTI. i.e. 4 tyrosines and 4 phenylalanines, led to complete assignments of all the aromatic spin systems in the protein. From this, information was obtained on the rotational motions about the C beta--Cv bond axis of the aromatic rings in the globular form of PBTI. At 25 degrees, two tyrosine rings and one phenylalanine ring are rotating rapidly on the NMR time scale. For the other rings the transitions from slow to rapid rotational motions were investigated at variable temperatures and energy barriers for these intramolecular rate processes determined. The studies of the tyrosine resonances had been described in detail in a previous publication. The present paper describes the identification of the phenylalanine resonances and comments on some technical aspects which might be of quite general interest for the analysis of highly resolved 1H NMR spectra of proteins. Data for the tyrosines and the phenylalanines are compiled in three tables, i.e. the pK alpha-values for the tyrosines, the NMR parameters for all eight aromatics, and the parameters delta G not equal to, and, where available, delta H not equal to and delta S not equal to for the rotational motions of the rings.     

Ring current effects in the conformation dependent NMR chemical shifts of aliphatic protons in the basic pancreatic trypsin inhibitor.

Previously, a highly refined crystal structure and energy refined atomic coordinates were obtained for the basic pancreatic trypsin inhibitor, as well as numerous individual resonance assignments in the 1H NMR spectrum. These data were now used to investigate the contributions from the local ring current fields of the aromatic rings to the overall conformation dependent chemical shifts in this globular protein. A program was written which allowed the consideration of certain aspects of internal mobility of the protein, and the different commonly used ring current equa tions were compared. These studies indicate that ring current shifts are the dominant contribution to the observed conformation dependent chemical shifts of the peripheral aliphatic side chain protons. On the other hand, it appears that ring current shifts do not make dominant contributions to the conformation dependent shifts of the backbone alpha- and amide protons or the aromatic protons in the inhibitor. On the basis of the empirical calibration with the peripheral aliphatic side chain protons, the Johnson-Bovey ring current equation was selected for an analysis of the ring geometries of two prolines in the inhibitor.     

Conformations of N-(2-fluorophenyl)-N-phenylcarbamoyl-alpha-chymotrypsin

N-(2-Fluorophenyl)-N-phenylcarbamoyl chloride is shown to react with alpha-chymotrypsin to give a catalytically inactive material. A crystal structure determination shows that the chloride exists in the solid state in two conformations. In both of these the aromatic rings are tilted substantially relative to the plane through the atoms of the carbamoyl chloride group; the structures differ by a 180 degrees rotation of the 2-fluorophenyl ring. Fluorine NMR studies of alpha-chymotrypsin modified with this carbamoyl chloride show that, when bound to the enzyme, one aromatic ring of the diphenylcarbamoyl group likely rotates slowly while the other rotates much more rapidly or else is frozen in one dominant conformation. In the denatured enzyme (8 M urea) at room temperature and above, both aromatic rings of the diphenylcarbamoyl group appear to be rapidly rotating although differential linewidth changes observed at lower sample temperatures suggest that rotation of one ring becomes slow under these conditions. Rotation about the carbamoyl carbon-nitrogen bond is detected in fluorine NMR spectra of both the native and the denatured modified enzymes as the sample temperature is increased. Rates of carbamoyl rotation in the chloride, in the native modified enzyme, and in the denatured enzyme at 25 degrees C are approximately 66, 10, and 200 s-1, respectively.     

Solution structure and fluctuation of the Mg(2+)-bound form of calmodulin C-terminal domain

Calmodulin (CaM) is a Ca(2+)-binding protein that functions as a ubiquitous Ca(2+)-signaling molecule, through conformational changes from the "closed" apo conformation to the "open" Ca(2+)-bound conformation. Mg(2+) also binds to CaM and stabilizes its folded structure, but the NMR signals are broadened by slow conformational fluctuations. Using the E104D/E140D mutant, designed to decrease the signal broadening in the presence of Mg(2+) with minimal perturbations of the overall structure, the solution structure of the Mg(2+)-bound form of the CaM C-terminal domain was determined by multidimensional NMR spectroscopy. The Mg(2+)-induced conformational change mainly occurred in EF hand IV, while EF-hand III retained the apo structure. The helix G and helix H sides of the binding sequence undergo conformational changes needed for the Mg(2+) coordination, and thus the helices tilt slightly. The aromatic rings on helix H move to form a new cluster of aromatic rings in the hydrophobic core. Although helix G tilts slightly to the open orientation, the closed conformation is maintained. The fact that the Mg(2+)-induced conformational changes in EF-hand IV and the hydrophobic core are also seen upon Ca(2+) binding suggests that the Ca(2+)-induced conformational changes can be divided into two categories, those specific to Ca(2+) and those common to Ca(2+) and Mg(2+).     

Envisioning the Loop Movements and Rotation of the Two Subdomains of Dihydrofolate Reductase by Elastic Normal Mode Analysis

Abstract

Normal mode analysis, using the elastic network model, has been employed to envision the low frequency normal mode motion trends in the structures of five intermediates and a transition state in the kinetic pathway of E. coli dihydrofolate reductase (DHFR). Five of the reaction pathway analog structures and a crystal structure resembling the transition state, using X-ray analyses determined by Kraut et al., have been adapted as structural models. The motions that poise pathways of the M20 loop transitions from closed to occluded conformations and sub domain rotation to close the substrate cleft, have been predicted and envisioned for the first time by this study. Pathway entries to the movement of the substrate binding cleft helices are also envisioned. These motions play roles in transition structure stabilization and in regulating the release of the product tetrahydrofolate (THF). The motions observed push the ground state conformation of each intermediate towards a higher energy sub state conformation. A set of conserved residues involved in the catalytic reactions and conformational changes, previously studied by kinetic, theoretical and NMR, have been analyzed. The importance of these motions in terms of protein dynamics are revealed and envisioned by the normal mode analysis. Additional residues are proposed as candidates for further study of their potential promotional function.     

Identification of the substrate interaction region of the chitin-binding domain of Streptomyces griseus chitinase C

Chitinase C from Streptomyces griseus HUT6037 was discovered as the first bacterial chitinase in family 19 other than chitinases found in higher plants. Chitinase C comprises two domains: a chitin-binding domain (ChBD(ChiC)) for attachment to chitin and a chitin-catalytic domain for digesting chitin. The structure of ChBD(ChiC) was determined by means of 13C-, 15N-, and 1H-resonance nuclear magnetic resonance (NMR) spectroscopy. The conformation of its backbone comprised two beta-sheets composed of two and three antiparallel beta-strands, respectively, this being very similar to the backbone conformations of the cellulose-binding domain of endoglucanase Z from Erwinia chrysanthemi (CBD(EGZ)) and the chitin-binding domain of chitinase A1 from Bacillus circulans WL-12 (ChBD(ChiA1)). The interaction between ChBD(ChiC) and hexa-N-acetyl-chitohexaose was monitored through chemical shift perturbations, which showed that ChBD(ChiC) interacted with the substrate through two aromatic rings exposed to the solvent as CBD(EGZ) interacts with cellulose through three characteristic aromatic rings. Comparison of the conformations of ChBD(ChiA1), ChBD(ChiC), and other typical chitin- and cellulose-binding domains, which have three solvent-exposed aromatic residues responsible for binding to polysaccharides, has suggested that they have adopted versatile binding site conformations depending on the substrates, with almost the same backbone conformations being retained.     

Studying excited states of proteins by NMR spectroscopy.

Protein structure is inherently dynamic, with function often predicated on excursions from low to higher energy conformations. For example, X-ray studies of a cavity mutant of T4 lysozyme, L99A, show that the cavity is sterically inaccessible to ligand, yet the protein is able to bind substituted benzenes rapidly. We have used novel relaxation dispersion NMR techniques to kinetically and thermodynamically characterize a transition between a highly populated (97%, 25 degrees C) ground state conformation and an excited state that is 2.0 kcal mol(-1) higher in free energy. A temperature-dependent study of the rates of interconversion between ground and excited states allows the separation of the free energy change into enthalpic (Delta H = 7.1 kcal mol(-1)) and entropic (T Delta S = 5.1 kcal mol(-1), 25 degrees C) components. The residues involved cluster about the cavity, providing evidence that the excited state facilitates ligand entry.     

The Free Energy Barrier for Arginine Gating Charge Translation Is Altered by Mutations in the Voltage Sensor Domain

The gating of voltage-gated ion channels is controlled by the arginine-rich S4 helix of the voltage-sensor domain moving in response to an external potential. Recent studies have suggested that S4 moves in three to four steps to open the conducting pore, thus visiting several intermediate conformations during gating. However, the exact conformational changes are not known in detail. For instance, it has been suggested that there is a local rotation in the helix corresponding to short segments of a 3-helix moving along S4 during opening and closing. Here, we have explored the energetics of the transition between the fully open state (based on the X-ray structure) and the first intermediate state towards channel closing (C), modeled from experimental constraints. We show that conformations within 3 Å of the X-ray structure are obtained in simulations starting from the C model, and directly observe the previously suggested sliding 3-helix region in S4. Through systematic free energy calculations, we show that the C state is a stable intermediate conformation and determine free energy profiles for moving between the states without constraints. Mutations indicate several residues in a narrow hydrophobic band in the voltage sensor contribute to the barrier between the open and C states, with F233 in the S2 helix having the largest influence. Substitution for smaller amino acids reduces the transition cost, while introduction of a larger ring increases it, largely confirming experimental activation shift results. There is a systematic correlation between the local aromatic ring rotation, the arginine barrier crossing, and the corresponding relative free energy. In particular, it appears to be more advantageous for the F233 side chain to rotate towards the extracellular side when arginines cross the hydrophobic region.     

NMR studies of multiple conformations in complexes of Lactobacillus casei dihydrofolate reductase with analogues of pyrimethamine

1H and 19F NMR signals from bound ligands have been assigned in one- and two-dimensional NMR spectra of complexes of Lactobacillus casei dihydrofolate reductase with various pyrimethamine analogues (including pyrimethamine [1, 2,4-diamino-5-(4'-chlorophenyl)-6-ethylpyrimidine], fluoropyrimethamine [2, 2,4-diamino-5-(4'-fluorophenyl)-6-ethylpyrimidine], fluoronitropyrimethamine [3, 2,4-diamino-5-(4'-fluoro-3'-nitrophenyl) -6-ethylpyrimidine], and methylbenzoprim [4, 2,4-diamino-5-[4'- (methylbenzylamino)-3'-nitrophenyl]-6-ethylpyrimidine]). The signals were identified mainly by correlating signals from bound and free ligands by using 2D exchange experiments. Analogues (such as 1 and 2) with symmetrically substituted phenyl rings give rise to 1H signals from four nonequivalent aromatic protons, clearly indicating the presence of hindered rotation about the pyrimidine-phenyl bond. Analogues containing asymmetrically substituted aromatic rings (such as 3 and 4) exist as mixtures of two rotational isomers (an enantiomeric pair) because of this hindered rotation and the NMR spectra revealed that both isomers (forms A and B) bind to the enzyme with comparable, though unequal, binding energies. In this case two complete sets of bound proton signals were observed. The phenyl ring protons in each of the two forms experience essentially the same protein environment (same shielding) as that experienced by the corresponding protons in bound pyrimethamine: this confirms that forms A and B correspond to two rotational isomers resulting from approximately 180 degrees rotation about the pyrimidine-phenyl bond, with the 2,4-diaminopyrimidine ring being bound similarly in both forms. The relative orientations of the two forms have been determined from NOE through-space connections between protons on the ligand and protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural and conformational analysis of pentostatin (2'-deoxycoformycin), a potent inhibitor of adenosine deaminase

X-ray, NMR and molecular mechanics studies on pentostatin (C11H16N4O4), a potent inhibitor of the enzyme adenosine deaminase, have been carried out to study the structure and conformation. The crystals belong to the monoclinic space group P21 with the cell dimensions of a = 4.960(1), b = 10.746(3), c = 11.279(4)A, beta = 101.18(2) degrees and Z = 2. The structure was solved by direct methods and difference Fourier methods and refined to an R value of 0.047 for 997 reflections. The trihydrodiazepine ring is nonplanar and adopts a distorted sofa conformation with C(7) deviated from the mean plane by 0.66A. The deoxyribose ring adopts a C3'-endo conformation, different from coformycin where the sugar has a C2'-endo conformation. The observed glycosidic torsion angle (chi = -119.5 degrees) is in the anti range. The conformation about the C(4')-C(5') bond is gauche+. The conformation of the molecule is compared with that of coformycin and 2-azacoformycin. 1 and 2D NMR studies have been carried out and the dihedral angles obtained from coupling constants have been compared with those obtained from the crystal structure. The conformation of deoxyribose in solution is approximately 70% S and 30% N. Molecular mechanics studies were performed to obtain the energy minimized conformation, which is compared with X-ray and NMR results.     

1H NMR studies at 360 MHz of the aromatic amino acid residues in ferrocytochrome c-552 from Euglena gracilis.

The resonances of the aromatic rings in the 1H NMR spectra at 360 MHz of ferrocytochrome c-552 of Euglena gracilis were investigated by double resonance techniques. The spin systems of the two tryptophan and four of the tyrosine residues could be identified. This analysis of the aromatic region of the 1H NMR spectrum provided evidence that His-14 is bound to the heme iron. It gave also some insight into the molecular dynamics of ferrocytochrome c-552 in that it showed that of the six aromatic rings, four tyrosines were rotating rapidly about the Cbeta-Cgamma bond, while one tyrosine and the single phenylalanine were restricted in their rotational mobilities by their environmnent in the protein.     

Nuclear magnetic resonance of the filamentous bacteriophage fd.

The filamentous bacteriophage fd and its major coat protein are being studied by nuclear magnetic resonance (NMR) spectroscopy. 31P NMR shows that the chemical shielding tensor of the DNA phosphates of fd in solution is only slightly reduced in magnitude by motional averaging, indicating that DNA-protein interactions substantially immobilize the DNA packaged in the virus. There is no evidence of chemical interactions between the DNA backbone and the coat protein, since experiments on solid virus show the 31P resonances to have the same principle elements of its chemical shielding tensor as DNA. 1H and 13C NMR spectra of fd virus in solution indicate that the coat proteins are held rigidly in the structure except for some aliphatic side chains that undergo relatively rapid rotations. The presence of limited mobility in the viral coat proteins is substantiated by finding large quadrupole splittings in 2H NMR of deuterium labeled virions. The structure of the coat protein in a lipid environment differs significantly from that found for the assembled virus. Data from 1H and 13C NMR chemical shifts, amide proton exchange rates, and 13C relaxation measurements show that the coat protein in sodium dodecyl sulfate micelles has a native folded structure that varies from that of a typical globular protein or the coat protein in the virus by having a partially flexible backbone and some rapidly rotating aromatic rings.     

Crystal Structures of a Group II Chaperonin Reveal the Open and Closed States Associated with the Protein Folding Cycle

Chaperonins are large protein complexes consisting of two stacked multisubunit rings, which open and close in an ATP-dependent manner to create a protected environment for protein folding. Here, we describe the first crystal structure of a group II chaperonin in an open conformation. We have obtained structures of the archaeal chaperonin from Methanococcus maripaludis in both a peptide acceptor (open) state and a protein folding (closed) state. In contrast with group I chaperonins, in which the equatorial domains share a similar conformation between the open and closed states and the largest motions occurs at the intermediate and apical domains, the three domains of the archaeal chaperonin subunit reorient as a single rigid body. The large rotation observed from the open state to the closed state results in a 65% decrease of the folding chamber volume and creates a highly hydrophilic surface inside the cage. These results suggest a completely distinct closing mechanism in the group II chaperonins as compared with the group I chaperonins.     

The role of slow and fast protein motions in allosteric interactions

Allostery is fundamentally thermodynamic in nature. Long-range communication in proteins may be mediated not only by changes in the mean conformation with enthalpic contribution but also by changes in dynamic fluctuations with entropic contribution. The important role of protein motions in mediating allosteric interactions has been established by NMR spectroscopy. By using CAP as a model system, we have shown how changes in protein structure and internal dynamics can allosterically regulate protein function and activity. The results indicate that changes in conformational entropy can give rise to binding enhancement, binding inhibition, or have no effect in the expected affinity, depending on the magnitude and sign of enthalpy–entropy compensation. Moreover, allosteric interactions can be regulated by the modulation a low-populated conformation states that serve as on-pathway intermediates for ligand binding. Taken together, the interplay between fast internal motions, which are intimately related to conformational entropy, and slow internal motions, which are related to poorly populated conformational states, can regulate protein activity in a way that cannot be predicted on the basis of the protein’s ground-state structure.     

The Relationship Between Structure and Activity Among Opioid Peptides

Summary Since the discovery and isolation of the endogenous opioid peptides Leu- and Met-enkephalin, structural studies have been focused on deducing the bioactive conformation of the peptide ligands. Theoretically, linear peptides can have many different backbone conformations, yet early, X-ray studies on enkephalin and its analogues showed only two different backbone conformations: extended and single β-bend. More recent reports include a third conformation for Leu-enkephalin and constrained opioid peptides from two ‘new’ classes (i.e. cyclic and ‘allaromatic’ peptides). In this report the relationship between solid-state X-ray structure and opioid peptide activity is examined. The N-terminal amine nitrogen and the two aromatic rings have previously been identified as structural features important to the biological activity of opioid peptides. From X-ray studies we find that the distances between the centroids of the aromatic rings, and between the N-terminal amine nitrogen and the centroid of the phenylalanine ring, vary over a large range. There is a discernible relationship, however, between the separation of the two rings and their orientation that correlates with activity.     

Crystal structure of N-(tri-O-acetyl-alpha-D-xylopyranosyl)pyridinium bromide.

The structure of N-(2,3,4-tri-O-acetyl-alpha-D-xylopyranosyl)pyridinium bromide was determined by X-ray crystallography and 1H NMR spectroscopy. Two xylopyranosyl moieties crystallize with three water molecules and there is a novel pattern of Br(-) and H(2)O contacts. Both xylopyranosyl rings in the asymmetric unit have the 1C(4) conformation, with all three axial O-Ac groups.     

Synthesis of the β-1,3-glucan, laminarahexaose: NMR and conformational studies

The synthesis of laminarahexaose is described. NMR studies of several of the intermediates leading to the β-1,3-glucan show anomalously small coupling constants for some of the C-1 hydrogens. An X-ray structure for the protected hexasaccharide shows that the small coupling constants are due to some of the glucopyranose rings adopting a twist-boat conformation. The X-ray studies also explain other unexpected NMR observations.     

Rotations of the 2B sub-domain of E. coli UvrD helicase/translocase coupled to nucleotide and DNA binding

Escherichia coli UvrD is a superfamily 1 DNA helicase and single-stranded DNA (ssDNA) translocase that functions in DNA repair and plasmid replication and as an anti-recombinase by removing RecA protein from ssDNA. UvrD couples ATP binding and hydrolysis to unwind double-stranded DNA and translocate along ssDNA with 3′-to-5′ directionality. Although a UvrD monomer is able to translocate along ssDNA rapidly and processively, DNA helicase activity in vitro requires a minimum of a UvrD dimer. Previous crystal structures of UvrD bound to a ssDNA/duplex DNA junction show that its 2B sub-domain exists in a “closed” state and interacts with the duplex DNA. Here, we report a crystal structure of an apo form of UvrD in which the 2B sub-domain is in an “open” state that differs by an ∼ 160° rotation of the 2B sub-domain. To study the rotational conformational states of the 2B sub-domain in various ligation states, we constructed a series of double-cysteine UvrD mutants and labeled them with fluorophores such that rotation of the 2B sub-domain results in changes in fluorescence resonance energy transfer. These studies show that the open and closed forms can interconvert in solution, with low salt favoring the closed conformation and high salt favoring the open conformation in the absence of DNA. Binding of UvrD to DNA and ATP binding and hydrolysis also affect the rotational conformational state of the 2B sub-domain, suggesting that 2B sub-domain rotation is coupled to the function of this nucleic acid motor enzyme.     

Molecular orbital calculations on the conformation of polypeptides and proteins. II. Conformational energy maps and stereochemical rotational states of aromatic residues

Our previous quantum-mechanical calculations, by an all-valence-electrons method (PCILO) taking into account simultaneously the σ and π electrons of the system, on the conformation energy maps of the glycyl and alanyl residues are extended to the evaluation of these maps and of the stereochemical rotational states of the aromatic residues, phenylalanyl, tyrosyl, histidyl, and tryptophanyl in dipeptides. Calculations on model compounds are used for the predetermination of the side-chain rotational angles χ₁ and χ₂ which are then used as selected parameters for the evaluation of the conformational energy maps as function of the backbone rotational angles Φ and ψ. The theory predicts that the most stable conformation for these aromatic residues should occur in the same region, around Φ = 200, ψ = 140°, in which it was predicted to occur for the glycyl and alanyl residues and which was completely overlooked by most of the previous “empirical” computations. Recent experimental work by a group of Russian authors using NMR and infrared techniques seems to confirm the theoretical result for the alanyl and phenylalanyl residues. The paper indicates also the secondary local minima which appear for the different residues. The theoretically allowed general conformational area for the four aromatic residues, within the reasonable value of 5 kcal/mole above the deepest minimum, is somewhat larger than the similar area allowed by the “hard sphere” empirical calculations. Practically all available representative experimental points from the study of small molecules and of the proteins lysozyme and myoglobin fall within the allowed area, the agreement being better with the results of the quantum mechanical calculations than with those of the “hard sphere” approximation. The values of the side-chain rotational angles χ₁ and χ₂ and of their allowed combinations agree less satisfactorily with experiment, the experimentally observed combinations being more varied than the theoretically allowed ones. These last ones having, however, been predetermined on studies with model compounds, this situation is not astonishing. It is proposed to refine these results by a minimization with respect to the four parameters Φ, ψ, χ₁, and χ₂ involved.