Applications of NMR in Protein Structure Prediction.pptx

APPLICATIONS OF NMR IN
PROTEIN STRUCTURE
PREDICTION
ANAGHA R ANIL
M.PHARM PHARMACOLOGY

CONTENTS
2
PROTEIN STRUCTURE PREDICTION
DIFFERENT METHODS OF PROTEIN PREDICTION
NMR SPECTROSCOPY
DETERMINING PROTEIN STRUCTURE USING NMR SPECTROSCOPY
WHY NMR SPECTROSCOPY?
RESEARCH ARTICLE
APPLICATIONS
OF
NMR
IN
PROTEIN
STRUCTURE
PREDICTION

PROTEIN STRUCTURE
PREDICTION
3
- Refers to finding the exact orientations and arrangements of different
amino acids present in protein.
- Biological system depend on structure & function of protein.
- To understand protein functions at molecular level ,determine the 3D
structure.
APPLICATIONS
OF
NMR
IN
PROTEIN
STRUCTURE
PREDICTION

4
DIFFERENT METHODS OF
PROTEIN PREDICTION
Computational
methods
Template based model
Homology modelling
Threading modelling
Template free
modelling
Ab- intio method
Experimental method
NMR spectroscopy
X ray spectroscopy
Electron spectroscopy
APPLICATIONS
OF
NMR
IN
PROTEIN
STRUCTURE
PREDICTION

NMR SPECTROSCOPY
• The NMR spectroscopy works on the principle of change in the energy state and
orientation of an atomic nuclei in a magnetic field. The change depends on the presence or
absence of electrons around the atomic nuclei of a molecule.
5
APPLICATIONS
OF
NMR
IN
PROTEIN
STRUCTURE
PREDICTION

DETERMINING PROTEIN STRUCTURE
USING NMR SPECTROSCOPY
6
Sample preparation
NMR Data
Collection
Resonance
assignments
Structure
determination from
the NMR data
Structure
Calculation and
Refinement
Structure Analysis
and Interpretation
APPLICATIONS
OF
NMR
IN
PROTEIN
STRUCTURE
PREDICTION

7
APPLICATIONS
OF
NMR
IN
PROTEIN
STRUCTURE
PREDICTION
• The target protein is expressed in a suitable system (e.g., E. coli) and purified
to homogeneity.
• Proteins are often labeled with isotopes like 15N and 13C to enhance sensitivity
and simplify the interpretation of NMR spectra. This involves growing the cells in
media containing these isotopes.
• The protein is dissolved in an appropriate buffer solution, typically at a
concentration of 0.5-1 mM. Conditions such as pH, temperature, and salt
concentration are optimized to ensure the protein is in a stable, soluble form.
SAMPLE PREPARATION

8
APPLICATIONS
OF
NMR
IN
PROTEIN
STRUCTURE
PREDICTION
• 1D NMR - assess the overall quality, purity, and stability of the sample.
• 2D NMR - HSQC (Heteronuclear Single Quantum Coherence): correlates the
resonance frequencies of hydrogen (1H) and nitrogen (15N) or carbon (13C) atoms and
makes it easier to assign the NMR signals to specific nuclei in the protein.
On theHSQC results, each cross peak indicates a pair of hydrogen and nitrogen or
carbon atoms that are close to each other in the protein.
NMR DATA COLLECTION

9
APPLICATIONS
OF
NMR
IN
PROTEIN
STRUCTURE
PREDICTION
• 3D NMR
HNCA (Heteronuclear Single Quantum Coherence - Alpha Carbon:
Correlates the amide proton (1H) with the directly bonded nitrogen (15N) and the alpha
carbon (13C). This helps in sequentially assigning the backbone atoms.
HNCO (Heteronuclear Single Quantum Coherence - Carbonyl:
Correlates the amide proton (1H) with the directly bonded nitrogen (15N) and the carbonyl
carbon (13C). It provides complementary information to HNCA and helps confirm
assignments.

1 0
APPLICATIONS
OF
NMR
IN
PROTEIN
STRUCTURE
PREDICTION 4D NMR
NOESY-HSQC: Combines NOESY (Nuclear Overhauser Effect Spectroscopy) with
HSQC to provide distance constraints between hydrogen atoms through space
This is crucial for determining the 3D structure of the protein.
NOESY measures the distances between hydrogen atoms (protons) in a protein by
observing the Nuclear Overhauser Effect (NOE).
Distance constraints obtained from NOESY-HSQC help in determining the
spatial arrangement of both the backbone and side chains of the protein.

1 1
APPLICATIONS
OF
NMR
IN
PROTEIN
STRUCTURE
PREDICTION
Resonance assignments refer to the process of identifying and matching the
observed NMR signals (resonances) to specific atoms within a protein.
Sequential Assignment
• The first step involves assigning the NMR signals to specific nuclei within the protein.
This is typically done using triple-resonance experiments (e.g., HNCA, HNCO) to
connect the backbone amide signals sequentially along the polypeptide chain.
Side-Chain Assignment
• Once the backbone assignments are complete, side-chain resonances are assigned
using additional experiments like HCCH-TOCSY.
RESONANCE ASSIGNMENTS

1 2
APPLICATIONS
OF
NMR
IN
PROTEIN
STRUCTURE
PREDICTION
• Distance Restraints: Derived from NOESY experiments, where cross peaks indicate
spatial proximity between nuclei. The intensity of these peaks is inversely
proportional to the sixth power of the distance. Stronger peaks correspond to shorter
distances, providing constraints that help in modeling the protein's 3D structure.
• Angle Restraints: Derived from torsion angles (Φ and Ψ) of the protein backbone, and
defines the protein's secondary structure elements, such as alpha-helices and beta-
sheets.
STRUCTURE DETERMINATION FROM THE NMR DATA

1 3
APPLICATIONS
OF
NMR
IN
PROTEIN
STRUCTURE
PREDICTION
• J-Coupling Constants: J-couplings (scalar couplings) are used to determine the
angles between atoms connected by bonds, contributing to the understanding
of the protein’s conformation.
• Residual Dipolar Couplings (RDCs): provide information on bond vector
orientations relative to a common reference frame and are useful for defining the
overall fold.
• Chemical Shifts: Chemical shifts can be used to predict secondary structure
elements (e.g., alpha-helices, beta-sheets).

1 4
APPLICATIONS
OF
NMR
IN
PROTEIN
STRUCTURE
PREDICTION
• Initial Structure Generation: Software programs (e.g., CYANA, ARIA) use the
experimental constraints to generate initial structural models.
• Energy Minimization: Energy minimization aims to find the lowest energy conformation
of the protein ensuring it is physically realistic and stable.
• Validation: The final structures are validated using various criteria, such as agreement
with experimental data, Ramachandran plot analysis, and comparison with known
structures.
STRUCTURE CALCULATION AND REFINEMENT

1 5
APPLICATIONS
OF
NMR
IN
PROTEIN
STRUCTURE
PREDICTION
• The quality of the NMR structure is assessed using parameters like RMSD (Root
Mean Square Deviation) between the calculated structures and experimental data.
• RMSD assess the quality of NMR structures by measuring the deviation between
the experimentally determined structures and the calculated models.
• The structural information is analyzed to gain insights into the protein's function,
dynamics, and interactions with other molecules.
STRUCTURE ANALYSIS AND INTERPRETATION

WHY NMR SPECTROSCOPY?
• It allows proteins to be studied in solution, mimicking their natural environment.
• It provides insights into protein dynamics, including conformational changes and folding
processes.
• It does not require protein crystallization, overcoming a major bottleneck in X-ray
crystallography.
• It offers detailed information on protein-ligand and protein-protein interactions, critical
for drug design.
• It utilizes isotopic labeling to simplify spectra and focus on specific molecule parts,
aiding the study of large proteins.
• It requires small amounts of protein sample, beneficial for proteins difficult to produce in
large quantities.
1 6
APPLICATIONS
OF
NMR
IN
PROTEIN
STRUCTURE
PREDICTION

1 7
APPLICATIONS
OF
NMR
IN
PROTEIN
STRUCTURE
PREDICTION
APPLICATIONS
Determination of Protein Structures
Study of Protein Dynamics
Mapping Protein-Protein Interactions
Probing Protein Folding and Misfolding
Characterization of Post-Translational
Modifications
Validation of Computational Models
Structural Studies of Membrane Proteins and
Complexes
Drug Discovery and Design

1 8
NMR data ambiguity and limitations with larger proteins (>50-70 kDa)
hinder accurate structure determination.
MELD(Modeling Employing Limited Data) integrates noisy NMR data into
molecular dynamics simulations (MELDxMD), using a Bayesian approach
(a probabilistic graphical model) to enhance accuracy of protein structure
predictions.
Advantages: Improves NMR data interpretation, enhances structural
prediction accuracy, and excels in complex scenarios like the CASP13
blind test.
APPLICATIONS
OF
NMR
IN
PROTEIN
STRUCTURE
PREDICTION

Applications of NMR in Protein Structure Prediction.pptx

Recommended

Recommended

More Related Content

Similar to Applications of NMR in Protein Structure Prediction.pptx

Similar to Applications of NMR in Protein Structure Prediction.pptx (20)

Recently uploaded

Recently uploaded (20)

Applications of NMR in Protein Structure Prediction.pptx