Complete tyrosine assignments in the high field proton nuclear magnetic resonance spectrum of the bovine pancreatic trypsin inhibitor

Snyder, Grayson H.; Rowan, Robert; Karplus, Susan; Sykes, Brian D.

doi:10.1021/bi00688a008

Cited by 159 publications

(99 citation statements)

References 41 publications

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“…3). This indicates that the tyrosine corresponding to Y 5 was near the 3-nitrotyrosine-1 15, which has a pK of 6.3 -7.1 [33]. Consequently, Y 5 might be assigned to tyrosine-73, which in the crystal structure of RNase is very close to tyrosine-1 15 (Fig.…”

Section: Comparison With Nitrated Rnases: Assignment Of Y L Y2 Y4 mentioning

confidence: 91%

The Aromatic Residues of Bovine Pancreatic Ribonuclease Studied by ¹H Nuclear Magnetic Resonance

Lenstra

Bolscher

Stob

et al. 1979

European Journal of Biochemistry

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1. The aromatic proton resonances in the 360-MHz 'H nuclear magnetic resonance (NMR) spectrum of bovine pancreatic ribonuclease were divided into histidine, tyrosine and phenylalanine resonances by means of pH titrations and double resonance experiments.2. Photochemically induced dynamic nuclear polarization spectra showed that one histidine (His-1 19) and two tyrosines are accessible to photo-excited flavin. This permitted the identification of the C-4 proton resonance of His-119.3. The resonances of the ring protons of Tyr-25, Tyr-76 and Tyr-1 15 and the C-4 proton of His-12 were identified by comparison with subtilisin-modified and nitrated ribonucleases. Other resonances were assigned tentatively to 4. On addition of active-site inhibitors, all phenylalanine resonances broadened or disappeared. The resonance that was most affected was assigned tentatively to Phe-120.5. Four of the six tyrosines of bovine RNase, identified as Tyr-76, Tyr-115 and, tentatively, Tyr-73 and Tyr-92, are titratable above pH 9. The rings of Tyr-73 and Tyr-115 are rapidly rotating or flipping by 180" about their CP-Cc" bond and are accessible to flavin in photochemically induced dynamic nuclear polarization experiments. Tyr-25 is involved in a pH-dependent conformational transition, together with Asp-14 and His-48. A scheme for this transition is proposed.6. Binding of active-site inhibitors to bovine RNase only influences the active site and its immediate surroundings. These conformational changes are probably not connected with the pH-dependent transition in the region of 7. In NMR spectra of RNase A at elevated temperatures, no local unfolding below the temperature of the thermal denaturation was observed. NMR spectra of thermally unfolded RNase A indicated that the deviations from a random coil are small and might be caused by interactions between neighbouring residues.

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Section: Comparison With Nitrated Rnases: Assignment Of Y L Y2 Y4 mentioning

confidence: 91%

The Aromatic Residues of Bovine Pancreatic Ribonuclease Studied by ¹H Nuclear Magnetic Resonance

Lenstra

Bolscher

Stob

et al. 1979

European Journal of Biochemistry

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“…In the 13C spectrum of lysozyme, resonances of most non-protonated aromatic carbon atoms have been assigned [3]. Similar progress has been made in assigning the spectra of the pancreatic trypsin inhibitor protein [9,10] and of cytochromes c [ll], but the lack of detailed assignments of the resAbbreviafions. NMR, nuclear magnetic resonance; ppm, parts per million; Nbf-C1, 4-chloro-7-nitro-benzofurazan; GlcNAc, Nacetyl-glucosamine.…”

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confidence: 99%

Study of the Tryptophan Residues of Lysozyme Using ¹H Nuclear Magnetic Resonance

Cassels

Dobson

Poulsen

et al. 1978

European Journal of Biochemistry

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The identification and complete assignment of the C-2 and N-1 proton nuclear magnetic resonances (NMR) of the six tryptophan residues of hen lysozyme are reported. Identification of the resonances required a detailed examination of the spectra of the protein in H20 and in 'HzO, and involved the application of spin-echo and Carr-Purcell-Meiboom-Gill pulse sequences. Assignment was achieved by observing the effects on the NMR spectra of performing specific chemical modifications, of binding paramagnetic species (lanthanide ions and spin labels), of binding inhibitors and protons and of carrying out solvent exchange experiments. The problems involved in completion of assignment are fully discussed. In the course of performing experiments to make assignments, several interesting aspects of the behaviour of the tryptophan residues in the protein structure were observed and are discussed.The application of nuclear magnetic resonance (NMR) spectroscopy to the study of proteins has made substantial progress over the last five years, and it is now possible to achieve well-resolved 'H and I3C spectra of small proteins [l -31. In our work on lysozyme, we are using NMR to investigate fundamental features of the structural and kinetic behavior of a protein in solution [4,5]. It is an essential feature of this work that we are able to study specific individual atoms or groups in the protein. It is therefore necessary that resonances of these individual groups are resolved and assigned to specific atoms in the protein. Indeed, the assignment process is an essential requirement in any detailed study of a molecule by NMR. In early studies of lysozyme by 'H NMR, assignments for a number of resolved resonances were proposed [6 -81. More recently we have been able to increase greatly the number of resolved resonances in the spectrum [l] and to apply rigorous techniques to assign resonances of a number of methyl groups and of several aromatic residues [5]. In the 13C spectrum of lysozyme, resonances of most non-protonated aromatic carbon atoms have been assigned [3]. Similar progress has been made in assigning the spectra of the pancreatic trypsin inhibitor protein [9,10] and of cytochromes c [ll], but the lack of detailed assignments of the resAbbreviafions. NMR, nuclear magnetic resonance; ppm, parts per million; Nbf-C1, 4-chloro-7-nitro-benzofurazan; GlcNAc, Nacetyl-glucosamine.Etzzyme. Lysozyme (EC 3.2.1.17).onances of other proteins reflects the difficuky of the required procedures. In this paper we describe the full assignment of the N-1 and (2-2 proton resonances of all the six tryptophdn residues of lysozyme. The strategy by which this assignment was achieved has been outlined in previous papers [1,4]. In essence, the process falls into two parts. The first stage involves assignment of a particular resonance to a nucleus of a type of amino acid residue. This stage makes use primarily of physical methods, such as determination of multiplet structure and coupling information. A number of different NMR techniques were used her...

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“…Such studies have demonstrated the essential role of protein structural relaxation in the rotation of the rings. Only by minimizing the protein energy for each ring orientation were the calculated rotation barriers in good agreement with the NMR measurements (3,4); the barriers obtained for a rigid protein matrix were much too large. From these static treatments it is clear that to determine the rate of an activated process inside a protein it is necessary to include the correlated atomic displacements that can occur.…”

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confidence: 72%

Dynamics of activated processes in globular proteins.

McCammon¹,

Karplus²

1979

Proc. Natl. Acad. Sci. U.S.A.

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A procedure for the dynamical simulation of activated processes, such as ligand binding and enzymatic reactions, in a globular protein is outlined. Preliminary calculations of the transition state geometry and barrier crossing trajectories are presented for a model reaction, the rotation of an aromatic ring in the bovine pancreatic trypsin inhibitor. The results show that repulsive nonbonded interactions between the ring atoms and the atoms in the surrounding protein matrix determine the dynamical character of the reorientation process; the nonbonded interactions are the source of the rotational barrier and of the impulses that speed up or slow down the ring motion during the barrier crossings. Activated processes play an important role in enzymatic reactions and more generally in the dynamics of protein molecules (1). In an activated process, the observed rate is limited by an energy barrier between the initial and the final state. Most processes in native proteins that take place on a time scale of microseconds or longer are likely to involve such an activation step. Measurements have yielded information concerning the free energy barriers associated with a variety of activated processes in proteins; these include the rotations of aromatic side chains (2-4), the motion of diatomic ligands through the globin of heme proteins (5, 6), and the interconversion of substrate and product in enzymes such as triosephosphate isomerase (7). For the cases listed the number and magnitudes of the free energy barriers involved have been determined (4-7). Further, measurements of the temperature dependence of the rates have demonstrated that there are large entropies of activation that point to the participation of the protein degrees of freedom in the activated process (3-6).The availability of these and other data suggests that an investigation of the detailed dynamics of activated processes in proteins is in order. Of primary interest are the protein contributions to the barriers and the dynamics of the motion across the barriers in the interior of a protein. In some cases (e.g., enzymatic reactions) the protein-substrate interactions may act to lower the activation barrier, whereas in others (e.g., ligand penetration) the interactions are themselves the source of the barrier. Because of the mobility of the interior of a protein (8), the measured barriers correspond to an average over a range of instantaneous interactions. Transient relaxation effects due to concerted displacements of the protein atoms may significantly lower the effective barriers. Because the density of proteins is high (9), frictional damping can occur during the transition and lead to diffusional rather than inertial barrier crossings (10-12).To provide a microscopic description of activated processes in proteins, it will be necessary to carry out a detailed analysis of the dynamics of the atoms involved. In one approach to the problem, which is essentially static in character, empirical energy functions have been used to estimate the activation...

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Complete tyrosine assignments in the high field proton nuclear magnetic resonance spectrum of the bovine pancreatic trypsin inhibitor

Cited by 159 publications

References 41 publications

The Aromatic Residues of Bovine Pancreatic Ribonuclease Studied by ¹H Nuclear Magnetic Resonance

The Aromatic Residues of Bovine Pancreatic Ribonuclease Studied by ¹H Nuclear Magnetic Resonance

Study of the Tryptophan Residues of Lysozyme Using ¹H Nuclear Magnetic Resonance

Dynamics of activated processes in globular proteins.

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Complete tyrosine assignments in the high field proton nuclear magnetic resonance spectrum of the bovine pancreatic trypsin inhibitor

Cited by 159 publications

References 41 publications

The Aromatic Residues of Bovine Pancreatic Ribonuclease Studied by 1H Nuclear Magnetic Resonance

The Aromatic Residues of Bovine Pancreatic Ribonuclease Studied by 1H Nuclear Magnetic Resonance

Study of the Tryptophan Residues of Lysozyme Using 1H Nuclear Magnetic Resonance

Dynamics of activated processes in globular proteins.

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Product

Resources

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The Aromatic Residues of Bovine Pancreatic Ribonuclease Studied by ¹H Nuclear Magnetic Resonance

The Aromatic Residues of Bovine Pancreatic Ribonuclease Studied by ¹H Nuclear Magnetic Resonance

Study of the Tryptophan Residues of Lysozyme Using ¹H Nuclear Magnetic Resonance