Showing papers by "David A. Case published in 1994"

Use of chemical shifts and coupling constants in nuclear magnetic resonance structural studies on peptides and proteins.

Abstract: Publisher Summary This chapter describes advances in the use of coupling constants and chemical shifts in structural studies of peptides and proteins. Coupling constant information can be incorporated into the input data for structure calculations, giving direct information on dihedral angles and stereospecific assignments of prochiral groups such as β-methylene protons and the methyl groups of leucine and valine. Because of the complexity of the interactions that influence the chemical shift of each nucleus, this type of information is more useful at the structure refinement stage. However, the determination of protein and peptide structure using information from nuclear magnetic resonance (NMR) experiments in solution is now a well-established method, and it is frequently used as an adjunct to X-ray crystal structure determination, as well as in cases where crystals are unavailable. With increasing size of the proteins for which three-dimensional (3D) structure determination by NMR is attempted, there will be an increasing reliance on 3D and higher dimensional spectroscopy utilizing transfer pathways selective for particular coupling constants. The method of J doubling is suggested to increase accuracy of coupling constant measurements in large molecules.

Analysis of proton chemical shifts in regular secondary structure of proteins.

Abstract: The contribution of peptide groups to Hα and Hβ proton chemical shifts can be modeled with empirical equations that represent magnetic anisotropy and electrostatic interactions [Osapay, K. and Case, D.A. (1991) J. Am. Chem. Soc., 113, 9436–9444]. Using these, a model for the ‘random coil’ reference state can be generated by averaging a dipeptide over energetically allowed regions of torsion-angle space. Such calculations support the notion that the empirical constant used in earlier studies arises from neighboring peptide contributions in the reference state, and suggest that special values be used for glycine and proline residues, which differ significantly from other residues in their allowed ϕ,ψ-ranges. New constants for these residues are reported that provide significant improvements in predicted backbone shifts. To illustrate how secondary structure affects backbone chemical shifts we report calculations on oligopeptide models for helices, sheets and turns. In addition to suggesting a physical mechanism for the widely recognized average difference between α and β secondary structures, these models suggest several additional regularities that should be expected: (a) Hα protons at the edges of β-sheets will have a two-residue periodicity; (b) the Hα2 and Hα3 protons of glycine residues will exhibit different shifts, particularly in sheets; (c) Hβ protons will also be sensitive to local secondary structure, but in different directions and to a smaller extent than Hα protons; (d) Hα protons in turns will generally be shifted upfield, except those in position 3 of type I turns. Examples of observed shift patterns in several proteins illustrate the application of these ideas.

High-resolution X-ray study of deoxyhemoglobin Rothschild 37 beta Trp----Arg: a mutation that creates an intersubunit chloride-binding site.

Abstract: The mutation site in hemoglobin Rothschild (37 beta Trp----Arg) is located in the "hinge region" of the alpha 1 beta 2 interface, a region that is critical for normal hemoglobin function. The mutation results in greatly reduced cooperativity and an oxygen affinity similar to that of hemoglobin A [Gacon, G., Belkhodja, O., Wajcman, H., & Labie, D. (1977) FEBS Lett. 82, 243-246]. Crystal were grown under "low-salt" conditions [100 mM Cl- in 10 mM phosphate buffer at pH 7.0 with poly(ethylene glycol) as a precipitating agent]. The crystal structure of deoxyhemoglobin Rothschild and the isomorphous crystal structure of deoxyhemoglobin A were refined at resolutions of 2.0 and 1.9 A, respectively. The mutation-induced structural changes were partitioned into components of (1) tetramer rotation, (2) quaternary structure rearrangement, and (3) deformations of tertiary structure. The quaternary change involves a 1 degree rotation of the alpha subunit about the "switch region" of the alpha 1 beta 2 interface. The tertiary changes are confined to residues at the alpha 1 beta 2 interface, with the largest shifts (approximately 0.4 A) located across the interface from the mutation site at the alpha subunit FG corner-G helix boundary. Most surprising was the identification of a mutation-generated anion-binding site in the alpha 1 beta 2 interface. Chloride binds at this site as a counterion for Arg 37 beta. The requirement of a counterion implies that the solution properties of hemoglobin Rothschild, in particular the dimer-tetramer equilibrium, should be very dependent upon the concentration and type of anions present.

Collective NMR relaxation model applied to protein dynamics.

Abstract: A new dynamical model for the interpretation of nuclear magnetic resonance relaxation data is presented. It is based on a normal mode description, treating the low frequencies associated with collective motions as adjustable parameters to optimize agreement between calculated and experimental relaxation order parameters. This model provides a compact representation of many aspects of internal dynamics and characterizes motions affecting different spin pairs in a correlated way. Furthermore, it links together dynamical characteristics of different types of NMR observables and allows one to assess vibrational thermodynamic properties. Sample applications are given for a 25-residue zinc-finger peptide.