Resonance assignment and structural studies of larger
proteins by nuclear magnetic resonance (NMR) can be challenging when exchange broadening, multiple stable conformations, and
The extension of conventional two-dimensional
Such homonuclear
These multi-dimensional experiments provided a tremendous degree of spectral
simplification, in particular after appropriate analysis software became
available. However, it also quickly became clear that extension to large,
slowly tumbling proteins was hampered by low signal to noise, caused by the relative inefficiency of the magnetization transfer steps when the
dimensionality of a spectrum is increased. This decrease in sensitivity was
remedied by generating the protein in a highly perdeuterated state while keeping the solvent-exchangeable backbone amide protons protonated
(Torchia et al., 1988; Lemaster and Richards, 1988). Combining the
perdeuteration approach with both the triple-resonance assignment strategy (Grzesiek et al., 1993) and the subsequently introduced
powerful TROSY line-narrowing method (Pervushin
et al., 1997) made it possible to assign and analyze the structure of quite
large proteins, as exemplified by the 723-residue protein malate synthase G
(Tugarinov et al., 2002, 2005a). The sensitivity gained
by perdeuteration, enabling the recording of 4D
In the present report, we merge the above-mentioned prior advances, 3D
NOE-NOE and 3D
The gene encoding a C145A variant of M
Spectra were acquired on a sample containing 1.8 mM (0.9 mM
dimer)
Pulse schemes for four-dimensional
Considering that the measurements are carried out for perdeuterated protein,
the spectral windows in the indirect
Nonstandard processing was needed for the TROSY-NOESY-TROSY experiment
because the spectrum was recorded with sensitivity-enhanced gradient
selection in the
The full time-domain data matrix of the 4D NOESY-NOESY-TROSY experiment
(Fig. 1b) consists of 1536* (
Spectra were processed using NMRPipe software (Delaglio et al., 1995); peak picking and spectrum analysis was performed using SPARKY software (Goddard and Kneller, 2008; Lee et al., 2015) as well as NMRDraw (Delaglio et al., 1995). Programs for visualization and analysis were written using freely available python libraries (Hunter, 2007; Harris et al., 2020) as well as NMR-specific python libraries (Helmus and Jaroniec, 2013).
Two types of complementary 4D NOE experiments were recorded: (1) 4D
TROSY-NOESY-TROSY and (2) 4D NOESY-NOESY-TROSY (Fig. 1). While the former is
very similar to the HMQC-NOESY-TROSY experiment used recently for a single
The rotational correlation time of the C145A variant of M
The high quality and
Illustration of amide–amide NOEs in perdeuterated, amide-protonated SARS-CoV-2 Main Protease, observed by 4D TROSY-NOESY-TROSY.
The cross sections exemplify the power of 4D analysis for three types of
secondary structure:
The amides of L67 and Q69 in strand
It is interesting to compare the diagonal peak intensities in these various
cross sections of the TROSY-NOESY-TROSY spectrum. Diagonal intensity is a
function of the amount of amide
As highlighted by the work of Kaptein and co-workers, 3D NOE-NOE experiments
provided an effective method for studying the
The pulse scheme of this 4D NOESY-NOESY-TROSY is shown in Fig. 1b. It
represents a straightforward extension of the original NOE-NOE 3D experiment
(Boelens et al., 1989) but with the detection period substituted by the gradient-enhanced 2D
(
Compared to the 4D TROSY-NOESY-TROSY pulse scheme, the 4D NOESY-NOESY-TROSY
experiment avoids the lossy magnetization transfer step from
As expected,
The NOESY-NOESY-TROSY spectrum also shows multiple NOEs to sidechain amide
protons that are not visible in the TROSY-NOESY-TROSY spectrum because the
TROSY element does not select magnetization transfer for NH
The spectra shown in this study were recorded during the summer of 2020,
when access to campus facilities was strongly restricted due to COVID-19
pandemic mitigation efforts. These restrictions allowed for much lengthier
acquisition of spectra than commonly used, for a total of 8 d for the two 4D spectra. As a benefit of NUS reconstruction, it is possible to
generate spectra of the same resolution recorded in any fraction of that
time. Alternatively, we can discard the data recorded at the longest values
of
Use of the lengthy data acquisition times needed to collect the 4D spectra
requires a high stability sample, which in our case benefited from the C145A
active site mutation, protecting the sample from auto-proteolysis. As with
all NMR experiments,
We note that the TROSY-NOESY-TROSY experiment used a long NOE mixing time of
200 ms, such as to increase the number of observed connectivities by adding
indirect NOE effects, including spin diffusion through hydroxyl protons
(Koharudin et al., 2003), thereby aiding the assignment
process. The use of a 50 ms NOE mixing period in the subsequent 4D
NOESY-NOESY-TROSY experiment then provided a semi-quantitative measure of
distance between these protons and their neighbors. Indeed, as pointed out
by Kaptein and co-workers, recording of NOE-NOE spectra provides important
experimental data on the pathway of magnetization transfer during NOE
mixing. Such information could be used to convert these data into more quantitative distance information than the typical qualitative analysis of
NOE intensities, potentially leading to the generation of higher resolution
structures (Vogeli et al., 2009, 2012). Quantitative NOE
interpretation traditionally relied on the recording of a series of NOE
buildup data, which can become comparably time-consuming as the recording of
4D NMR spectra if resonance overlap is a limiting factor, as typically is
the case for NOE spectra. This problem is further exacerbated by the
spectral crowding of large proteins, particularly in the
While the high signal to noise and spectral simplicity of working with perdeuterated proteins has long been recognized (Torchia et al., 1988; Lemaster and Richards, 1988; Grzesiek et al., 1993; Tugarinov et al., 2004) the number of structural restraints accessible used to be small. Our present study demonstrates that a much larger number of NOE interactions becomes available by the recording of 4D NOE spectra. Moreover, it highlights the exquisite detail and value of NOE-NOE interaction analysis explored by the Kaptein group and it demonstrates that this approach is highly suitable for the larger biomolecules and biomolecular complexes being explored today, in particular when using extensive perdeuteration. Therefore, we believe that the recording of high quality 4D NMR spectra of the type presented in this study is entirely practical and invaluable for the structural and functional analysis of large proteins and their complexes, with possible extension to the study of nucleic acids. We note, however, that in the absence of extensive deuteration the dilution of nuclear magnetization over sidechain resonances will strongly lower the sensitivity of the experiment, which is further exacerbated by decreased effectiveness of TROSY-based line narrowing in such samples. On the other hand, adaptations of the NOESY-NOESY-TROSY experiment to methyl-protonated but otherwise perdeuterated proteins (Tugarinov et al., 2005b) are expected to be readily feasible.
The raw Bruker NMR data sets including the acquisition parameters and NUS
sampling lists, pulse programs, include file, and NMRPipe processing scripts
are available for download from Zenodo:
The supplement related to this article is available online at:
AJR expressed and purified protein samples, collected and analyzed the data, and edited the manuscript; JY optimized pulse sequence parameterization and processing and edited the manuscript; AB supervised the project and wrote the manuscript.
The authors declare that they have no conflict of interest.
This article is part of the special issue “Robert Kaptein Festschrift”. It is not associated with a conference.
We thank John M. Louis, Joseph Courtney, Yang Shen, James L. Baber and Dennis A. Torchia for helpful discussions. This work was supported by the Intramural Research Program of the NIDDK and by the Intramural Antiviral Target Program of the Office of the Director, NIH.
This study is dedicated to Robert Kaptein on the occasion of his 80th birthday.
This research has been supported by the National Institute of Diabetes and Digestive and Kidney Diseases (grant nos. DK075023 and DK029046).
This paper was edited by Isabella Felli and reviewed by four anonymous referees.