Structural proteomics
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This document discusses the application of nuclear magnetic resonance (NMR) in structural proteomics, highlighting its capability to study macromolecular structures at atomic resolution. It details the techniques involved in NMR structure determination, challenges in studying protein complexes, and alternative methods to derive distance restraints and orientational information. The conclusion emphasizes that NMR is unique in providing high-resolution structural data on protein-protein complexes in solution.
Structural proteomics
- 1. Structural Proteomics by NMR By Jacqueline De Vera Veenstra, T. (2006). Chapter 9. Structural Proteomicsby NMR. In Proteomics for biological discovery. Hoboken, N.J.: Wiley-Liss.
- 2. INTRODUCTION Nuclear magnetic resonance (NMR) powerful spectroscopic technique that permits the detailed study at atomic resolution of the three dimensional structure and dynamics of macromolecules and their complexes in solution. structure determination highly specialized, labor-intensive, and time- consuming technique isotopic labeling with 15N and 13C
- 3. Determining the structure of a single protein by NMR 1. sequential resonance assignment to identify through-bond connectivities along the backbone and side chains 2. assignment of cross-peaks in nuclear Overhauser enhancement spectra to obtain short (≤5–6 Å) interproton distance restraints 3. measurement of additional NMR observables (torsion angles) 4. calculation of the three-dimensional structure from the experimental NMR restraints using simulated annealing.
- 4. Contribution can NMR make to structural proteomics 2 major methods for deriving high-resolution structural information at atomic resolution 1. NMR spectroscopy in solution 2. single crystal X-ray diffraction Problems complexes are generally more difficult to crystallize than isolated proteins, In NMR, providing exchange is either fast (weak binding) or slow (tight binding) on the chemical shift time scale protein–protein complex by NMR is extremely time consuming.
- 5. Solution! shortened the amount of time required by making full use of prior knowledge in the form of existing high- resolution crystal structures of the free proteins
- 6. 9.2 INTERMOLECULAR DISTANCE RESTRAINTS Nuclear Overhauser effect (NOE) is the primary source of geometric information for NMR-based structure determination Proportional to the sixth root of the distance between two protons upper limit for interproton distances that can be detected using the NOE is 5–6 Å. To Detect NOEs combining various isotope (15N and 13C) labeling strategies with isotope fit ltering experiments
- 7. Other methods to derive intermolecular distance restraints 1. Combination of crosslinking 2. proteolytic digestion 3. mass spectrometry However, the data will not yield unique crosslinking partners but multiple possibilities. 4. NMR-based approach, which involves derivatizing a suitablesurface accessible cysteine (which may have to be introduced by site-directed mutagenesis) on one protein with either a spin label or a metal binding site (suchas EDTA) and measuring paramagnetic relaxation enhancement effects on theother protein to yield long-range (15– 35 Å) distance restraints can be applied if one already has a good idea of the interaction surfaces involved in complex formation.
- 8. Other methods to derive intermolecular distance restraints 15N/1HN chemical shift perturbation mapping cross-saturation experiments far more challenging experimentally since it necessitates that one of the proteins is not only 15N labeled but fully deuterated as well.
- 9. Typical isotope labeling schemes used in the study of protein– protein complexes and corresponding intermolecular NOEs observed.
- 10. 9.3 ORIENTATIONAL RESTRAINTS Long-range orientational restraints can be derived from the measurement of residual dipolar couplings and chemical shift anisotropy yield geometric information on the orientation of an interatomic vector(s) with respect to an external axis system θ, angle between the interatomic vector θ, angle that describes the position of the projection of the interatomic vector on the xy plane
- 11. Method for deriving orientational information Residual dipolar couplings- easiest Effectively measure dipolar couplings in solution Devise means of inducing only a small (ca. 10−3) degree oforder such that the N–H dipolar couplings lie in the ±20 Hz range. achieved by dissolving the protein or protein complex of interest in a dilute, water-soluble, liquid crystalline medium.
- 12. 9.4 CONJOINED RIGID BODY/TORSION ANGLE DYNAMICS protein complex formation involves no significant changes in backbone conformation. only the interfacial side chains are allowed to alter their conformation. The backbone and noninterfacial side chains of one protein are held fixed second protein are only allowed to rotate and translate as a rigid body. Conjoined rigid body/torsion angle dynamics can readily be extended to cases where significant changes in backbone conformation are localized to specific regions of the protein, such as the binding interface
- 13. 9.5 DOCKING BASED ON 15N/1HN CHEMICAL SHIFT PERTURBATION AND N–H DIPOLAR COUPLINGS Docking is a method which predicts the preferred orientation of one molecule to a second when bound to each other to form a stable complex. What makes the docking problem hard to solve? 1. scoring problem = calculating binding affinity given a protein-ligand complex - no general scoring function is available 2. large number of degrees of freedom - most important degrees of freedom: 1. relative orientation of the two molecules 2. conformation of the ligand 3. protein conformation 4. water molecules can be between ligand and protein 5. protonation state
- 14. 9.6 STRUCTURAL PROTEOMICS OF THE GLUCOSE ARM OF THE BACTERIAL PHOSPHOTRANSFERASE SYSTEM Phosphotransferase system (PTS) provides tight coupling of translocation and phosphorylation a signal transduction pathway involving phosphoryl transfer whereby a phosphoryl group originating on phosphoenolpyruvate is transferred to the translocated carbohydrate via a series of three bimolecular protein– protein complexes
- 15. 9.7 CONCLUSION NMR is the only solution technique capable of providing high-resolution structural information on protein–protein complexes at atomic resolution.
- 16. References Veenstra, T. (2006). Chapter 9. Structural Proteomicsby NMR. In Proteomics for biological discovery. Hoboken, N.J.: Wiley-Liss.
Editor's Notes
- #15: In bacteria, carbohydrate transport across the membrane is mediated by the phosphoenolpyruvate: sugar phosphotransferase system (PTS