NMR Spectroscopy of Large Biological Macromolecules in Solution

2006 ◽  
pp. 89-128
Author(s):  
César Fernández ◽  
Gerhard Wider
Keyword(s):  
Nmr Spectroscopy ◽  
Biological Macromolecules

scholarly journals Fast and Robust 2D Inverse Laplace Transformation of Single-Molecule Fluorescence Lifetime Data

2021 ◽  
Author(s):  
Saurabh Talele ◽  
John T. King
Keyword(s):  
Nmr Spectroscopy ◽  
Fluorescence Spectroscopy ◽  
Single Molecule ◽  
Fluorescence Lifetime ◽  
Conformational Dynamics ◽  
Correlation Spectroscopy ◽  
Great Promise ◽  
Biological Macromolecules ◽  
Single Molecule Fluorescence ◽  
Laplace Transformations

AbstractFluorescence spectroscopy at the single-molecule scale has been indispensable for studying conformational dynamics and rare states of biological macromolecules. Single-molecule 2D-fluorescence lifetime correlation spectroscopy (sm-2D-FLCS) is an emerging technique that holds great promise for the study of protein and nucleic acid dynamics as it 1) resolves conformational dynamics using a single chromophore, 2) measures forward and reverse transitions independently, and 3) has a dynamic window ranging from microseconds to seconds. However, the calculation of a 2D fluorescence relaxation spectrum requires an inverse Laplace transition (ILT), which is an ill-conditioned inversion that must be estimated numerically through a regularized minimization. The current methods for performing ILTs of fluorescence relaxation can be computationally inefficient, sensitive to noise corruption, and difficult to implement. Here, we adopt an approach developed for NMR spectroscopy (T1-T2 relaxometry) to perform 1D and 2D-ILTs on single-molecule fluorescence spectroscopy data using singular-valued decomposition and Tikhonov regularization. This approach provides fast, robust, and easy to implement Laplace inversions of single-molecule fluorescence data.Significance StatementInverse Laplace transformations are a powerful approach for analyzing relaxation data. The inversion computes a relaxation rate spectrum from experimentally measured temporal relaxation, circumventing the need to choose appropriate fitting functions. They are routinely performed in NMR spectroscopy and are becoming increasing used in single-molecule fluorescence experiments. However, as Laplace inversions are ill-conditioned transformations, they must be estimated from regularization algorithms that are often computationally costly and difficult to implement. In this work, we adopt an algorithm first developed for NMR relaxometry to provide fast, robust, and easy to implement 1D and 2D inverse Laplace transformations on single-molecule fluorescence data.

scholarly journals Introduction to high-resolution cryo-electron microscopy

2016 ◽  
Vol 62 (3) ◽  
pp. 383-394
Author(s):  
Mariusz Czarnocki-Cieciura ◽  
Marcin Nowotny
Keyword(s):  
Electron Microscopy ◽  
Nmr Spectroscopy ◽  
Structural Biology ◽  
Atomic Resolution ◽  
Biological Macromolecules ◽  
X Ray ◽  
X Ray Crystallography ◽  
Cryo Electron Microscopy ◽  
Transmission Electron ◽  
Electron Microscopes

For many years two techniques have dominated structural biology – X-ray crystallography and NMR spectroscopy. Traditional cryo-electron microscopy of biological macromolecules produced macromolecular reconstructions at resolution limited to 6–10 Å. Recent development of transmission electron microscopes, in particular the development of direct electron detectors, and continuous improvements in the available software, have led to the “resolution revolution” in cryo-EM. It is now possible to routinely obtain near-atomic-resolution 3D maps of intact biological macromolecules as small as ~100 kDa. Thus, cryo-EM is now becoming the method of choice for structural analysis of many complex assemblies that are unsuitable for structure determination by other methods.

Choice of Problems in the Early Days of Biological NMR Spectroscopy

1997 ◽  
Author(s):  
M. Cohn
Keyword(s):  
Amino Acids ◽  
Nmr Spectroscopy ◽  
Structural Information ◽  
Chemical Shifts ◽  
Biological Macromolecules ◽  
Relaxation Rates ◽  
Complex Molecules ◽  
Weak Complexes ◽  
Unique Method ◽  
Rate Processes

It was a mere ten years after the discovery of NMR that Oleg Jardetzky under the mentorship of the physical chemist John Wertz (Wertz and Jardetzky, 1956) began using 23Na NMR with the aim of studying Na+ transport in biological systems as suggested by William Lipscomb. Jardetzky found that Na+ NMR provided a unique method for following the binding of Na+ in weak complexes. Advantage was taken of the sensitivity of quadrupolar nuclei to their chemical environment as reflected in their relaxation rates which could be readily observed at a field of 7,030 gauss available at the time (Jardetzky and Wertz, 1956). From the very first, Jardetzky limited his choice to those problems that could be investigated uniquely or most effectively by NMR Spectroscopy. One of Jardetzky’s principal goals was to elucidate, at least in part, the threedimensional structures of biological macromolecules in aqueous solution, a distant goal in the late 1950’s. He realized that before attempting to tackle the structure of these complex molecules, proteins and nucleic acids, by NMR it was essential to initially characterize the spectra of their components, amino acids andnucleosides. In 1957, he published a note in the Journal of Chemical Physics (Takedaand Jardetzky, 1957) on a few amino acids, not only reporting the chemical shifts of all the protons but also showing that in a dipeptide, for example, glycylglycine, the two CH2 groups are non-equivalent. In 1958, he published an NMR paper, a systematic study of the proton NMR spectra of amino acids, in the Journal of Biological Chemistry (Jardetzky and Jardetzky, 1958), thus introducing many facets of NMR Spectroscopy to the biochemical community. This seminal paper included: l) the chemical shifts of the protons of 22 amino acids and their dependence on pH, concentration and ionic strength and 2) the effect of rate processes on the NMR spectrum as exemplified by the exchange of the guanidino protons of arginine with water. Increased structural information from peptide NMR spectroscopy attracted many investigators to this area of research.

Applications of 2DFT NMR Spectroscopy to the Study of Biological Macromolecules

1979 ◽  
pp. 19-21
Author(s):  
O. Jardetzky ◽  
W. W. Conover ◽  
G. R. Sullivan ◽  
V. J. Basus
Keyword(s):  
Nmr Spectroscopy ◽  
Biological Macromolecules

Heteronuclear three-dimensional NMR spectroscopy of isotopically labelled biological macromolecules

1990 ◽  
Vol 23 (2) ◽  
pp. 97-131 ◽  
Author(s):  
Stephen W. Fesik ◽  
Erik R. P. Zuiderweg
Keyword(s):  
Nmr Spectroscopy ◽  
Fourier Transformation ◽  
Structural Information ◽  
Three Dimensional ◽  
2D Nmr ◽  
Biological Macromolecules ◽  
Transformation Techniques ◽  
Vast Number ◽  
Small Proteins ◽  
Nmr Techniques

Due to the development of two-dimensional Fourier transformation techniques (for reviews see Bax, 1982; Ernst et al. 1987), NMR spectroscopy has become a powerful tool for determining the 3D structures of small proteins (MW ≤ 10 kDa); for reviews see Wüthrich, 1986; Clore & Gronenborn, 1987. For larger molecules, however, the amount of detailed structural information that can be obtained using homonuclear 2D NMR techniques is limited because of the vast number of overlapping signals. In order to extend the capabilities of NMR to the study of larger systems, new approaches are required.

scholarly journals Protein in-cell NMR spectroscopy at 1.2 GHz

2021 ◽  
Author(s):  
Enrico Luchinat ◽  
Letizia Barbieri ◽  
Matteo Cremonini ◽  
Lucia Banci
Keyword(s):  
Nmr Spectroscopy ◽  
Nmr Spectra ◽  
Atomic Resolution ◽  
Biological Macromolecules ◽  
Functional Information ◽  
Relaxation Rates ◽  
Unfolded Proteins ◽  
Cellular Environment ◽  
Low Sensitivity ◽  
Ultrahigh Fields

AbstractIn-cell NMR spectroscopy provides precious structural and functional information on biological macromolecules in their native cellular environment at atomic resolution. However, the intrinsic low sensitivity of NMR imposes a big limitation in the applicability of the methodology. In this respect, the recently developed commercial 1.2 GHz NMR spectrometer is expected to introduce significant benefits. However, cell samples may suffer from detrimental effects at ultrahigh fields, that must be carefully evaluated. Here we show the first in-cell NMR spectra recorded at 1.2 GHz on human cells, and we compare resolution and sensitivity against those obtained at 900 and 950 MHz. To evaluate the effects of different spin relaxation rates, SOFAST-HMQC and BEST-TROSY spectra were recorded on intracellular α-synuclein and carbonic anhydrase. Major improvements are observed at 1.2 GHz when analyzing unfolded proteins, such as α-synuclein, while the TROSY scheme improves the resolution for both globular and unfolded proteins.

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