12.3 Protein structure determination by NMR

Nuclear Magnetic Resonance (NMR) spectroscopy is a powerful tool for determining protein structures in solution. It allows scientists to study proteins under near-physiological conditions, providing insights into their 3D structure and dynamics.

NMR experiments involve preparing isotopically labeled protein samples, exciting them with radiofrequency pulses, and analyzing the resulting signals. The data is then used to assign resonances, determine secondary structures, and calculate 3D structures using distance restraints from Nuclear Overhauser Effect measurements.

Sample Preparation and Data Acquisition

Protein Sample Preparation

• Protein NMR experiments require the preparation of a highly purified, isotopically labeled protein sample (15N and 13C) at a concentration of 0.1-1 mM in a suitable buffer
• The protein sample is placed in an NMR tube and subjected to a strong magnetic field, typically ranging from 500 to 1000 MHz, within an NMR spectrometer
• The quality of the NMR spectra depends on factors such as protein solubility, stability, and the presence of paramagnetic centers or aggregates

Data Acquisition and Processing

• The sample is excited with radiofrequency pulses, and the resulting NMR signals are detected and recorded as free induction decays (FIDs)
• The FIDs are processed using Fourier transformation to generate multidimensional NMR spectra, such as 2D 1H-15N HSQC, 3D HNCA, and 3D HNCACB
• These spectra provide information about the chemical environment of specific nuclei in the protein (amide protons, nitrogen atoms, alpha and beta carbons)
• Proper data acquisition and processing parameters (pulse sequences, spectral width, number of scans) are crucial for obtaining high-quality NMR spectra

Resonance Assignment and Secondary Structure

Backbone Resonance Assignment

• Resonance assignment involves identifying the specific amino acid residues corresponding to each peak in the NMR spectra
• The backbone resonance assignment is typically performed using a combination of 3D NMR experiments (HNCA, HN(CO)CA, HNCACB, CBCA(CO)NH)
• These experiments provide sequential connectivity information by correlating the chemical shifts of backbone nuclei (HN, N, Cα, Cβ) in adjacent residues
• The assigned chemical shift values are compared to reference values for random coil structures to identify deviations indicative of secondary structure elements

Secondary Structure Determination

• The presence of characteristic NOE patterns aids in the identification of secondary structure elements (strong HN-HN NOEs for α-helices, strong Hα-HN NOEs for β-sheets)
• The chemical shift index (CSI) method compares the observed chemical shifts to random coil values to predict the presence of α-helices and β-strands
• The TALOS program uses a database of known protein structures to predict backbone torsion angles (φ and ψ) based on the assigned chemical shifts
• Secondary structure elements are determined by integrating information from NOE patterns, CSI, and TALOS predictions
• The identified secondary structure elements (α-helices, β-strands) provide a foundation for the overall tertiary structure of the protein

Nuclear Overhauser Effect for Structure

Principles of Nuclear Overhauser Effect (NOE)

• The nuclear Overhauser effect (NOE) is a phenomenon in which the relaxation of one nuclear spin influences the relaxation of another nearby spin through dipolar coupling
• The strength of the NOE depends on the distance between the two spins, with the NOE intensity proportional to the inverse sixth power of the distance (1/r^6)
• NOE measurements provide distance restraints between pairs of protons that are spatially close (typically < 5 Å) in the protein structure, regardless of their positions in the primary sequence

Structure Calculation using NOE Restraints

• NOE restraints are typically obtained from 2D NOESY (Nuclear Overhauser Effect SpectroscopY) experiments, which show cross-peaks between protons that are close in space
• The intensities of the NOE cross-peaks are converted into distance restraints, which are used as input for structure calculation programs (CYANA, XPLOR-NIH)
• Structure calculation programs use the NOE-derived distance restraints, along with other restraints (dihedral angles, hydrogen bonds), to generate an ensemble of structures consistent with the experimental data
• The quality of the calculated structures is assessed using various validation tools (Ramachandran plots) to ensure that they satisfy the experimental restraints and have reasonable geometry
• The resulting NMR structure ensemble provides insights into the three-dimensional fold and conformation of the protein in solution

NMR Advantages vs Limitations

Advantages of NMR Spectroscopy

• NMR spectroscopy provides information about the structure and dynamics of proteins in solution, under near-physiological conditions
• NMR can be used to study proteins that are difficult to crystallize (intrinsically disordered proteins, membrane proteins in detergent micelles)
• NMR experiments can provide site-specific information about protein-ligand interactions, conformational changes, and protein folding
• NMR can be used to study protein dynamics on various timescales (picoseconds to seconds) using techniques such as relaxation measurements and hydrogen exchange

Limitations of NMR Spectroscopy

• NMR spectroscopy is typically limited to proteins with molecular weights less than 50-60 kDa due to faster relaxation and increased spectral complexity in larger proteins
• NMR experiments require relatively large amounts of purified, isotopically labeled protein samples (0.1-1 mM), which can be challenging to obtain for some proteins
• The quality of NMR structures depends on the number and distribution of NOE restraints; regions with few NOE restraints may have lower resolution or higher flexibility
• NMR structure determination can be time-consuming, as it requires extensive data collection, resonance assignment, and structure calculation steps
• The interpretation of NMR data can be challenging for proteins with multiple conformations or extensive dynamics, as the observed NMR parameters represent an ensemble average