Related Experiment Video
Updated: Jun 15, 2026
14:44
Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR
Published on: December 16, 2013
BEST-HNN and 2D-(HN)NH experiments for rapid backbone assignment in proteins
Dinesh Kumar1, Subhradip Paul, Ramakrishna V Hosur
1Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, Mumbai 400005, India.
Journal of Magnetic Resonance (San Diego, Calif. : 1997)
|March 19, 2010
Summary
This study enhances protein backbone assignment speed using a modified HN(CO)NH experiment (BEST-HN(CO)NH) and a novel 2D-(HN)NH method. These techniques significantly accelerate data collection and analysis for (H(N), (15)N) resonance assignment in proteins.
Area of Science:
- Biochemistry
- Structural Biology
- Nuclear Magnetic Resonance (NMR) Spectroscopy
Background:
- The HN(CO)NH experiment is crucial for backbone (H(N), (15)N) assignment in labeled proteins.
- Low sensitivity and long acquisition times limit the application of traditional HN(CO)NH, particularly for proteins with short T(2) relaxation times.
Purpose of the Study:
- To improve the sensitivity and speed of backbone (H(N), (15)N) assignment in NMR.
- To introduce a rapid amino-acid type identification method complementary to backbone assignment.
Main Methods:
- Application of the Broadband Enhancement SpectroscopY (BEST) modification to the HN(CO)NH experiment.
- Development and implementation of 2D-(HN)NH experiments for amino acid type identification.
- Testing the methods on human ubiquitin and acetic-acid denatured HIV-1 protease.
Main Results:
- The BEST modification increased sensitivity per unit time by over 2.0-fold, enabling 3D data collection in 8-10 hours.
- 2D-(HN)NH experiments, recordable in under an hour, provide anchor points for Gly, Ala, Ser/Thr residues.
- The methods are effective for both folded and unfolded protein systems and applicable to deuterated samples.
Conclusions:
- BEST-HN(CO)NH and 2D-(HN)NH significantly accelerate protein backbone resonance assignment.
- These optimized NMR methods enhance efficiency for structural studies of diverse protein systems.
- The developed techniques offer practical advantages for rapid NMR data acquisition and analysis.
Related Concept Videos
01:182D NMR: Overview of Heteronuclear Correlation Techniques
Heteronuclear correlation spectroscopy is an analytical technique that investigates the coupling between different types of nuclei, often a proton and an X-nucleus, such as carbon-13 or nitrogen-15. This method is commonly used in nuclear magnetic resonance (NMR) spectroscopy to gain insights into complex chemical compounds' structural and compositional aspects. A typical heteronuclear correlation spectrum displays X-nucleus chemical shifts on one axis and a proton spectrum on the other axis.
01:162D NMR: Overview of Homonuclear Correlation Techniques
Homonuclear correlation spectroscopy (COSY) is a powerful technique used in Nuclear Magnetic Resonance (NMR) spectroscopy to study the correlations between nuclei of the same type within a molecule. It provides information about scalar couplings between adjacent nuclei, which helps determine connectivity and structural information. There are several COSY variants, each with its unique strengths and experimental parameters.
COSY90 is the standard two-dimensional (2D) COSY experiment that...
COSY90 is the standard two-dimensional (2D) COSY experiment that...
00:52¹H NMR of Conformationally Flexible Molecules: Temporal Resolution
At room temperature, the chair conformer of cyclohexane undergoes rapid ring flipping between two equivalent chair conformers at a rate of approximately 105 times per second. These two chair conformers are in equilibrium. The rapid ring flipping results in the interconversion of the axial proton to an equatorial proton and an equatorial to the axial proton. Such interconversions are too rapid and cannot be detected on the NMR timescale. Hence, the NMR spectrometer cannot distinguish between the...
01:192D NMR: Heteronuclear Single-Quantum Correlation Spectroscopy (HSQC)
Heteronuclear single-quantum correlation spectroscopy (HSQC) is a 2D NMR technique that reveals one-bond correlations between hydrogen and a heteronucleus. The HSQC experiment is similar to the heteronuclear correlation experiment (HETCOR) but is more sensitive. In the HSQC spectrum, the proton chemical shift is plotted on the horizontal F2 axis, while the 13C chemical shift is plotted on the vertical F1 axis. The corresponding proton and 13C spectra are also shown. The HSQC contour plot does...
02:06Newman Projections
Different notations are used to represent the three-dimensional structure of molecules on two-dimensional surfaces. One of the most commonly used representations is the dash-wedge formula. The dashed wedges, solid wedges, and the plane lines indicate the groups situated behind the plane, coming out of the plane, and in the plane, respectively.
The organic molecules rotate across the single bonds leading to numerous temporary three-dimensional structures of varying energy known as conformers.
The organic molecules rotate across the single bonds leading to numerous temporary three-dimensional structures of varying energy known as conformers.
01:12Two-Dimensional (2D) NMR: Overview
The 1D NMR spectrum of large and complex molecules like natural products has complicated splitting patterns and overlapping signals, which can be easily interpreted using 2-dimensional (2D) NMR. Unlike 1D NMR, 2D NMR has two frequency axes that provide the coupling information between the nucleus A and nucleus B in a molecule. The process from which 2D spectra are obtained has four steps.
The first step is the preparation period, during which nucleus A is excited with a radiofrequency pulse.
The first step is the preparation period, during which nucleus A is excited with a radiofrequency pulse.