Diverse Biomolecular and Biomedical Applications of NMR-Review

Natural Product Communications

NMR Methods to Characterize Protein-

May 2019: 1–17
© The Author(s) 2019
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DOI: 10.1177/1934578X19849296
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Sanhita Maity1, Ravi Kumar Gundampati1, and

Thallapuranam Krishnaswamy Suresh Kumar1

Abstract
Structural information pertaining to the interactions between biological macromolecules and ligands is of potential signifi-
cance for understanding of molecular mechanisms in key biological processes. Recently, nuclear magnetic resonance (NMR)
spectroscopic techniques has come of age and has widened its scope to characterize binding interactions of small molecules
with biological macromolecules especially, proteins. NMR spectroscopy-based techniques are versatile due to their ability to
examine weak binding interactions and for rapid screening the binding affinities of ligands with proteins at atomic resolution.
In this review, we provide a broad overview of some of the important NMR approaches to investigate interactions of small
organic molecules with proteins.

Keywords
protein-small molecule interactions, NMR methods, binding constant (KD)

Received: November 8th, 2018; Accepted: January 21st, 2019.

Specific molecular interactions of biological macromole-
cules with ligands of different sizes are central to the under-
standing of the regulation of key cellular processes.1 Proteins
are the most important class of biological macromolecules as
they govern the crucial structural and functional properties
of cellular systems. A good understanding of the molecular
mechanisms underlying important cellular processes will
obviously involve examination of structural interactions
forged by proteins with themselves or with other biological
macromolecules such as, nucleic acids, polysaccharides, and
lipids. Owing to the central role of proteins as drivers of cel-
lular functions, specific interactions of small molecular
organic ligands with proteins are critical for a rational drug
design. Several biophysical and biochemical techniques,
such as isothermal titration calorimetry, surface plasmon res-
onance, fluorescence resonance energy transfer, infrared
spectroscopy, circular dichroism, confocal microscopy,
enzyme-linked immunosorbent assay, have been success-
fully used to characterize the binding constant (KD), kinetics,
dynamics, and thermodynamics characterizing protein-li-
gand interactions. In addition, computational studies have
also proved to be useful in the virtual high throughput
screening of potential small molecule drug candidates
against protein targets.2 However, NMR spectroscopy has
proven to be the most valuable tool to obtain an atomic-level
view of the ligand-protein interactions.3-5 NMR spectros-
copy evolved as a powerful tool over the past few decades to
investigate protein-ligand binding affinities in the nanomolar
to millimolar regime.6-9 An additional advantage of NMR
has been its ability to not only provide information on bind-
ing affinity between ligands and their target protein(s) but
also its strength to shed valuable insights into the protein
conformational changes that plausibly occur upon binding to
their ligand(s). Therefore, the aim of this review is to provide
a comprehensive list of commonly used NMR-based tech-
niques that can be used to investigate different aspects of
protein interactions with small molecule organic ligands.
1D-1H Line Broadening
Ligand-observed 1D NMR methods are well established to
detect hits from chemical libraries because they prefer
ligands with KD values in the micromolar to millimolar range
and require lower data-acquisition times and reduced sample
requirements.10 The dissociation equilibrium constant for
protein-ligand complex can be measured by a change in
1
Department of Chemistry and Biochemistry, University of Arkansas,
Fayetteville, AR, USA
Corresponding Author:
Thallapuranam Krishnaswamy Suresh Kumar, Department of Chemistry
and Biochemistry, University of Arkansas, Fayetteville, AR 72701, USA.
Email: ​sthalla@​uark.​edu

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2 Natural Product Communications
Figure 1.  1D - 1H nuclear magnetic resonance (NMR)
spectra phenytoin (a) and naproxen (b) titrated with increasing
concentrations [(i) 0 µM, (ii) 0.4 µM, (iii) 1 µM, (iv) 2 µM, and (v) 4
µM] of HSA. The height of the NMR signal of the ligands decrease
because of the shorter relaxation times of the ligand-human
serum albumin complex.11 This figure has been reproduced from
ref.11 with permission from the publisher.
linewidth or chemical shift of the peaks corresponding to the
free ligand upon binding to a protein. Binding interactions
between a protein (MW >5000 Da) and a low molecular
weight ligand (MW <500 Da) can be measured by monitoring
the decrease in the NMR peak height of ligand upon addition
of ligands to a protein solution. Ligand that shows an observ-
able decrease in peak height is considered to be a binder.
Therefore, the KD values of a 1:1 protein (P)- ligand (L)
binding complex, can be represented by the equation,
‍ KD = [L]F [P]F /[PL]‍
wherein [L]F is the concentration of the free ligand, [P]F
is the concentration of the free protein at equilibrium, and
[PL] is the concentration of the protein-ligand complex.
Shortridge et al., assuming that [L]T > [P]T (wherein [P]T is
total protein concentration and [L]T is the total ligand con-
centration) transformed equation-1 using the Taylor series
expansion10 to calculate the NMR peak height ratio ‘R’ as,
HB c[P]T
R= 1− = 1 − 1/(1 + [L]T +KD )‍
‍ HF
wherein‍c = wBwF − 1‍
HF and HB are the sum of NMR peak heights of ligand in
the free state and the bound state, respectively. wF and wB are
the NMR-linewidth(s) of the ligand in their free and bound
states, respectively. NMR line width ratio constant (c) rep-
resents the change in the ligand linewidth upon binding of a
ligand to a protein. In the bound state, the resonance of the
free ligand linewidth (wF) gains the linewidth of the protein
(wB), and as a result, the increased linewidth causes a corre-
sponding decrease in the ligand peak height which is mea-
sured by the ratio of the NMR peak height (R). One can
obtain KD value from this equation (equation (2)) on the
basis of the relative changes in the ligand peak height.
Shortridge et al. rearranged equation-2 for measuring KD
(see equation (3)) values. The rearranged equation can also
be written in terms of fractional occupancy at a given protein
concentration (Rgiven) as,
[( [ ] ) ]
c P T [ ] [ ]
KD = Rgiven − c P − L T
(3)
‍ ‍
For proteins which possess multiple non-specific binding
sites, the nonspecific binding term n[P]T corresponds to a lin-
ear increase in fraction bound upon addition of protein,
accounts for the correction of the decrease in ligand signal at
high protein concentration. After the addition of the n[P]T
term, equation (2) can be represented as
HB 1[ ]
[ ]
R= 1− HF = 1− +n P T
c P
1+ [L] +KT (4)
‍ T D ‍
However, the 1D -1H NMR spectra of small molecules
have extremely sharp peaks. However, upon binding to pro-
tein(s), widening of the peak and subsequently decrease in
the ligand’s NMR signal height are observed due to shorter
relaxation time of the ligand-protein complex. . Human
serum albumin (HSA), which possesses multiple binding
sites, experiences 1D-1H line broadening effect upon binding
to small molecule drugs phenytoin and naproxen (Figure 1).11
The observed increase in the linewidth of the ligand
depends on the dissociation equilibrium constant for the pro-
tein-ligand interaction, KD. In general, the observed change
in the ligand’s linewidth (wobs) for the fast exchange limit
(1) will follow the equation shown below
‍ wobs = wF + fB (wB − wF ) where fB ≈ [P]T /([L]T + KD )‍(5)
wherein fB is the fraction of the bound protein-ligand
complex, wF is the free ligand NMR line-width, and wB is
the line-width of the bound state of the ligand.
There are some assumptions considered here. If it is
assumed that the linewidth of the protein-ligand complex is
higher than that for the free ligand, then the ratio of the
(2) ligand linewidth in the free state and in the bound state rep-
resent the remaining free ligand concentration which is
expressed by equation (2). This assumption is valid for
ligand screening against a single protein target wherein the
contribution of chemical and dynamic line-broadening of the
ligand(s) may remain almost constant. As a matter of fact,
the lower contribution of linewidth from the exchange pro-
cesses is not expected to change the ligand binding affinities.
This assumption is applicable for protein-ligand interaction
for single binding-site of a protein. The assumption is not
Maity et al.
applicable for multisite binding or other factors such as
allosteric interactions which would warrant a more complex
binding model.12,13 Upon binding to a protein target, the 1H
NMR resonances of the ligand would broaden significantly
or even disappear but background interference should be
expected to increase with concentration and the size of pro-
tein accounting for the enhancement in false-negative rates.
Worley et al. reported a statistical method uncomplicated sta-
tistical spectral remodeling (USSR) method to analyze
1D-1H NMR line-broadening spectra for removing interfer-
ing protein baseline signals.14 It was reported that a chemical
library of 456 compounds identified 43 binders using BSA
but the background signals of BSA completely concealing
the ligand spectra.14 Identification of the protein baseline
from screening data requires highly reproducible sample
preparation, data collection and processing. The later of
these requirements can be achieved by the use of phase-scat-
ter correction (PSC) method.15 When protein signals are the
major problem, PSC methods yield the good results com-
pared to the USSR method. Therefore, Worly et al. reported
that the combined use of PSC and USSR method would pro-
vide the convenient analysis for 1H line-broadening screen-
ing data in order to estimate the KD values.14
Relaxation Time to Identify Ligand Binding
The binding of ligand to the protein can be assessed by
line-broadening effect as described earlier. Line-broadening
effect also has limitation(s) for the detection of binding
activity of one or more components in a complex mixture.
Therefore, in the investigation of complex mixtures, the
Figure 2.  Relaxation experiments of a complex mixture of 2 p-hydroxybenzanilide and 4 nonbinding aromatic compounds without
FKBP (left), unlabeled FKBP (middle), and spin-labeled FKBP (right) with spin lock periods of 10 ms and 200 ms. Arrows indicate the
cancelation of signals generating from compounds with binding activity for FKBP.18 This figure has been reproduced from ref.18 with
permission from the publisher.
3
observation and quantification of such effects needs well-sep-
arated signals.16 Another experimental approach was pro-
posed that involves subtraction of the spectra responsible for
contributions stemming from binding ligands. However, this
approach requires the inclusion of critical steps that are often
time-consuming.17 As binding induces relatively minor
chemical shift perturbations (CSPs), as compared to the line-
width changes, the effects of relaxation rate are more effec-
tive and therefore preferred. A spin-labeled ligand is first
selected that has a propensity to bind to the primary binding
site of a protein. This ligand is used for screening for binding
of other ligands to second binding site of protein(s).18 As a
consequence, T2 relaxation is enhanced on other ligand by
the presence of the spin labeled first ligand and this effect
depends on the distance between the spin label and the sec-
ond binding site on the protein. As a matter of fact, due to
fast T2 relaxation, resonance signals of ligands that bind to a
second protein binding site is not detected in the T2-filtered
NMR spectrum. The binding of ligands with FKBP was
investigated by Jahnke et al. wherein spin labels (such as,
2,2,6,6-tetramethylpiperidine-1-oxyl; TEMPO) were
attached to the lysine side chains of FKBP, a FK506 binding
protein, to detect the binding ligands and these lysine resi-
dues were situated in the proximity of FK506 binding site
[Figure 2]. Therefore, any ligand that binds to this site would
experience increased T2 relaxation. This method was applied
to a complex mixture of two p-hydroxybenzanilide and 4
nonbinding aromatic compounds. Interestingly, the binding
affinity of p-hydroxybenzanilide to FKBP was detected by
using spin-labeled FKBP because p-hydroxybenzanilide
experienced significant quenching in the presence of
4 Natural Product Communications
spin-labeled FKBP. However, the method is limited when
covalent modification of the protein is required because it
affects the binding affinity.19
Chemical Shift Perturbations
Several parameters of protein and ligand change upon inter-
action of a protein with its ligand. (CSP) can occur due to the
spatial proximity of aromatic rings or due to the non-cova-
lent interactions with ligands and solvent molecules. In this
type of experiment, the chemical shift of the protein can be
assessed when ligand is titrated against protein into the solu-
tion.20,21 Saturation of the protein binding site is observed
when the ligand is titrated into the protein in excess. The
standard technique of 15N-heteronuclear single quantum cor-
relation (HSQC) titration requires 15N labeled protein pro-
duced in Escherichia coli with unlabeled ligand. The changes
in chemical shift during titration can be monitored by acquir-
ing 2D 15N-HSQC spectra within 30 minutes by using pro-
tein concentrations in the range 200 µM.22 When interactions
occur, specific 1H-15N cross peaks in the 15N-HSQC spectra
are displaced from their original position. CSPs allows cal-
culation of equilibrium dissociation constant (KD) and also
the ligand binding sites.23 It is essential to maintain some of
the physical parameters to remain constant during the course
of the experiment because small changes in sample pH, salt
concentration, temperature and buffer composition will sig-
nificantly affect the 1H-15N CSP.
When the ligand is added, some of the residues undergo
selective 1H-15N CSP.24 Generally, backbone amide protons of
residues in the protein located in close proximity to the ligand
Figure 3.  Overlap of 1H–15N HSQC spectra of newt FGF-
1(red) in the presence of sucrose octasulfate (SOS) (cyan). The
labeled residues are those in newt FGF-1 that undergo significant
chemical shift perturbations when the protein binds to SOS in
a 1:1 ratio.25 This figure has been reproduced from ref.25 with
permission from the publisher.
binding site exhibit significant 1H-15N CSP. Figure 3 represents
the titration of new acidic fibroblast growth factor (n FGF-1)
with sucrose octasulfate (SOS), and 2D 15N-HSQC spectra
were acquired at a nFGF-1 concentration of 1 mM. Sometimes
prominent 1H-15N CSP is also observed due to backbone con-
formational changes induced in the protein by the binding
ligand.25 In addition, 1H-15N CSP also furnishes information of
allosteric changes in the protein due to complexation to a
ligand. For the calculation of CSP two approaches are used.
One of them is the quantum chemical approach using Gaussian
98.26 In this method, CSP can be calculated from the shielding
of nuclei from the external magnetic field by their electrons.
The problem with this method is the accuracy and also it does
not provide significant information on the structural features
that generated the calculated shift. The second method is an
empirical one wherein the chemical shifts are the summation of
independent effects but any problem(s) in experimental data
leads to errors in calculation such as, differences between cal-
culated and the experimentally observed values.27 This is pri-
marily because the calculated values are based on crystal
structures which are in variance with the ensemble of solution
structures which are calculated from the solution weighted
average of the chemical shift changes. In general, protein (P)
binds to ligand (L) and forms a protein-ligand complex, P +
L↔PL, wherein the rate constant for forward and backward
reactions are represented as kon and koff. At equilibrium, KD is
equal to koff/ kon.28 In a HSQC spectrum representing weak
interactions/fast exchange, the koff value is higher than the
chemical shift difference between the free and bound protein.
As a consequence, the protein-ligand complex is only stable for
a short period of time. This aspect poses a challenge to acquire
reliable NMR data. Therefore, the observed chemical shift is
subjected to the weighted average of the chemical shifts of pro-
tein in free state and in the bound state. When koff is lower than
the chemical shift difference or for slow exchange (very strong
interactions), signals either for free protein or protein-ligand
binary complex are observed (Figure 4).
However, CSP of any protein signal measured at different
ligand concentrations can be used in a nonlinear least-square
fitting to predict the KD value using the following equation
reported by Becker et al.29 The equation can be expressed as
[ √
([P]T +[L]T +KD )
∆∂obs = ∆∂max 2[P]T − ([P]T + [L]T + KD )2 −
√ ] (6)
(4[P]T [L]T /2[P]T )
‍ ‍
wherein ‍∂ obs‍represents the change in the observed shift
relative to the free state, ‍∂ max ‍is the maximum shift change
when the protein is saturated with the ligand, [P]T is total
protein concentration and [L]T is total ligand concentration.
Paramagnetic Ligand Tagging
Ligand-transferred paramagnetic NMR is a commonly used
sensitive technique for the identification of protein binding
site of low affinity drugs. Chemical shift of a protein is
Maity et al.
Figure 4.  The effect of exchange rate on peak shape in a 2D 1H-15N HSQC spectrum. Figure 4: depicts peaks shifts representing a fast
exchange: the peak shifts from free (blue) to bound (red) and for slow exchange: the intensity decreases of a peak in the free (blue) state
but increases (red) when the protein is bound to the ligand.
dependent on metal binding site. Paramagnetic effects are
observed as a difference between NMR data that is acquired
in the presence of paramagnetic metal ion and a diamagnetic
metal ion. The data acquired in the presence of the diamag-
netic metal ion serves as reference. However, paramagnetic
shifts are observed through bond interactions as well as
through space interactions. The pseudocontact shifts are sub-
jected to through-space interactions when a paramagnetic
metal ion bound to a protein.30 This method is useful for the
detection of the paramagnetic properties of a metal ion bound
to the target protein adjacent to the ligand binding site. Metal
ions such as most of the lanthanide ions are paramagnetic
(except La3+ and Lu3+) and possess suitable ionic radii to
coordinate with the metal binding site of the target protein.
To determine the relative orientation of paramagnetic lantha-
nide ion in the area of binding site, pseudocontact shifts can
be expressed in terms of polar coordinates r, θ,‍ φ‍ of the
nuclear spin with respect to the paramagnetic susceptibility
tensor (ΔX) of the lanthanide ion. The pseudo-contact shift
(δPCS) measured chemical shift of nuclear spin of the protein
in paramagnetic and diamagnetic samples and can be repre-
sented as30
∂pcs = 1 [∆X (3 cos ∅ − 1) + 3/2∆X sin2 θ cos 2ϕ]
‍ 12πr3 ax rh
‍ calmodulin where the terminal fluorine of sevoflurane was
 (7)
wherein ΔXax and ΔXrh are the axial and rhombic compo-
nents of the magnetic susceptibility tensor (ΔX) of the lan-
thanide ion. The angles θ and φ denote its orientation with
respect to the protein coordination frame and r is the distance
between the lanthanoid and nuclear spin of protein. John et
5
al. reported the complex between a lanthanide (Ln3+)-labeled
protein, the N-terminal domain of the subunit ε bound to the
subunit ϴ of E. coli DNA polymerase III (ε186/ ϴ), and a
small ligand molecule, thymidine.31 Stoichiometric com-
plexes ε186/ ϴ /Ln3+ are easily obtained by the addition of
equimolar amounts of LnCl3 [where Ln3+ are Dy3+, Tb3+, and
Er3+, (except La3+ and Lu3+ because they are used as diamag-
netic references)]. Thymidine exchanges between the free
state and the ε186/ ϴ /Ln3+/thymidine complex state and the
PCS in the bound state are transferred to produce the aver-
aged NMR signal in 15N-HSQC spectrum. Therefore, the
transferred PCS and transferred paramagnetic line broaden-
ings are useful to measure binding affinities and the structure
of the protein-ligand complex.31 Paramagnetic tagging with
ligand also accounts for measuring binding affinities. The
lanthanide ion first recognizes the metal binding site of a
protein, and then a lanthanide ion binding tag is covalently
anchored to a protein side chain. The transient complex of
lanthanide ion-ligand then interacts with the protein.
Calmodulin-sevoflurane interaction was studied by para-
magnetic tagging where the ligand sevoflurane was attached
to DOTA.32 Brath et al. reported the interaction of complex
of DOTA-bound sevoflurane La3+, Eu3+, Yb3+, or Dy3+ with
substituted with oxygen because of similar electronic char-
acter of oxygen and fluorine.33 Intramolecular ligand δPCS
was measured for complexation Eu3+, Yb3+, or Dy3+ with
DOTA-bound sevoflurane and the binding of the paramag-
netically labeled drug to calmodulin binding site produced
chemical shift changes due to the pseudocontact shift effect
6 Natural Product Communications

Figure 5.  (a) Overlay of 2D NMR 1H−13C HSQC spectra of calmodulin C-lobe titrated with the lanthanoid La3+/Dy3+ complexed
with sevoflurane analogs (DOTA-bound sevoflurane). 1H−13C HSQC spectra of 0.1 mM calmodulin C-lobe (black) titrated with 0.05
mM (blue), 0.1 mM (purple), and 0.2 mM (red) Dy3+ complexed sevoflurane analog. (b) represents δPCS of Calmodulin lobe induced by
Dy3+ labeled sevoflurane and control ligands. Amide proton δPCS for calmodulin lobes in the presence of sevoflurane ligand and control
compound complexed to Dy3+ are indicated as black and gray filled circles, respectively.33 This figure has been reproduced from 33Pwith
permission from the publisher. [https://pubs.acs.org/doi/10.1021/jacs.5b06220].

(Figure  5 (a,b)). The tagged sevoflurane was observed to
possess KD of ∼4.0 mM for the N-lobe and ∼1.8 mM for the
C-lobe respectively.
This method has some limitations. Selective attachment
of the lanthanide ions to different side chain positions
enhances the accuracy of the location of the binding moiety
and therefore protein site-directed mutagenesis is required.
Moreover, detection of the low affinity binding ligands
sometimes may be challenging, because the observed para-
magnetic effect is small due to the low molar fraction of
bound ligand. Modification of the structure of the ligand may
influence its binding to protein, and therefore tag attachment
at several positions of the ligand may be necessary.
The Transferred-NOESY, ILOE, and NOE
Pumping Experiment
The 1H-1H NOESY experiment attributes to the correlation
signals caused by dipolar cross-relaxation between spa-
tially closed nuclei.34 Large molecules produce large nega-
tive NOE peaks. For small molecules (MW <500 Da) and
for medium-sized molecules (MW range of 500 to 1000 Da)
the corresponding NOE peaks are positive and very small
negative, respectively. Large molecules build-up NOE
quickly in contrast to smaller molecules and therefore for
large molecules the maximum NOE is shifted to shorter
mixing times. Transferred NOE (trNOEs) spectrum yields a
negative NOE because in the bound state (protein-ligand
complex), a small molecule (ligand) adopts subsequent
NOE behavior of protein. Transferred NOE measurements
provide information on the bound state of ligand that bind
weakly (KD in µM to mM range) to the target. Transferred
NOE measurements may increase the resolution and sig-
nal-to-noise ratio of intermolecular NOE cross peaks by
narrowing the linewidth in one of the two dimensions.
There are two important parameters for these measure-
ments: (1) The mixing time should be short enough for the
negligible contribution of NOE signals of free ligands, but
long enough when bound to protein, (2) The molar ratio of
ligand molecules to the target protein. Therefore, screening
of a library of compounds in the presence of a protein
receptor, the bound ligand molecules exhibit strong nega-
tive NOEs, whereas non-binders will show weak positive
NOEs.35 The trNOE cross peaks arise due to intramolecular
interactions between protons in the bound ligand or inter-
molecular interactions between protons in the protein and
protons of the ligand. The trNOE approach has been used
for studying the conformation of small ligand-large pro-
teins complexes that are characterized by fast off-rates but
if dissociation constant (KD) or dissociation rates (koff) are
not known, a titration as well as the recording of trNOE
spectra at each point is essential.36
trNOESY measurement were first employed to examine
the binding affinity carbohydrates to lectin, Aleuria aurantia
agglutinin (AAA).37 Lucas et al. demonstrated that
exchange-transferred NOE (trNOE) on diffusion time scale
can provide valuable information on the multiple binding
orientations of HSA with dansylglycine and caprylate
wherein both the ligands exhibited primary binding affinity
for HSA’s drug site II (Figure 6(b)).38
Maity et al. 7

Figure 6.  (a) 1H BPP-STE (bipolar gradient pulse pair-stimulated echo) spectra for dansylglycine (DG) and caprylate (CAP) at a
different ligand to HSA ratio. (i) 25:1 CAP:HSA, (ii) 25:1 DG:HSA, (iii) 25:1:1 DG:CAP:HSA, and (iv) 25:25:1 DG:CAP:HSA. The small
peaks observed in (ii) and (iii) between 0 and 2.5 ppm are spectral subtraction artifacts. (b) represents NOESY spectrum for the 25:25:1
DG:CAP:HSA solution. All cross-peaks are the same phase as the diagonal. Intra-ligand cross-peaks (circled) arise from trNOE and
indicate binding but ilNOE cross-peaks (boxed) between the two ligands suggest that they are their binding sites on the protein are in
close spatial proximity.38 This figure has been reproduced from ref.38 with permission from the publisher.

In general, NOESY experiments are recorded in the pres-
ence of two ligands that bind simultaneously to adjacent
binding pockets of the protein forming a ternary complex, to
yield intermolecular ligand-ligand NOEs (ILOEs).39 ILOEs
were successfully used to examine the complexation of
NAD+ and lycolate to lactate dehydrogenase.40 Intermolecular
trNOEs are important for the analysis of protein-ligand inter-
actions. There are two effects such as spin diffusion and
magnetic leakage to the protein can obscure the analysis of
the bound conformations of low-molecular weight ligands.
As spin diffusion and leakage of magnetism to the protein are
the results of intermolecular NOE between protein and
ligand. Therefore, in case of spin diffusion, false confirma-
tions of the complex may be obtained. Magnetic leakage to
the protein, which occurs due to use of longer mixing times,
an artifactual change in the conformation of the ligand-pro-
tein complex may be observed.19 Several techniques have
been developed to diminish these two effects.41
Diffusion-assisted NOE-pumping technique relies on
NOE to transfer the signal from the target protein to the
bound ligands wherein signal of the non-binding ligands can
be suppressed through diffusion filter.42 The non-binding
ligands are separated by the diffusion experiment at the
beginning of the NOE experiment and at the end of the NOE
experiment, only bound ligand signal is identified because
the magnetization is transferred from protein to the bound
ligand only. After ligand dissociation from the protein, the
delivered magnetization is preserved by the relatively long
T1 of the ligands in the free state. As a consequence, ligand
signals can still be observed at shorter T2. Figure  7 (a,b)
show (Bipolar gradient pulse pair-stimulated echo) BPP-
STE pulse sequence and diffusion-assisted NOE pumping
pulse sequence, respectively.42 Diffusion-assisted NOE-
pumping experiments were used for interactions of HSA
with a binding ligand (salicylic acid) and nonbinding ligands
(l-ascorbic acid and glucose).42,43 1D- 1H NMR spectrum of
salicylic acid, L-ascorbic acid, glucose, and HSA in D2O
with and without BPP-STE is shown in Figure 8a and stack
plot of 1D 1H spectra of HSA with three ligands is shown in
(Figure 8 (b)).42 A standard set-up of BPP-STE diffusion-fil-
tered experiment (Figure  8 (b)) is unable to distinguish a
mixture of binding ligand (Salicylic acid) and non-binding
ligands (L-ascorbic acid and glucose) with HSA because of
insignificant differences in diffusion co-efficient for 3 ligands
whereas in NOE-pumping experiment, sufficient gradient is
applied to discriminate the binding ligand signal from the
unbinding ligands signals, therefore binding interaction of
salicylic acid with HSA is easily detected. NOE-pumping
experiment can successfully identify the binding ligands
whereas the affinity NMR method fails to discriminate
between a binding and non-binding ligands.
A reverse of the previous experimental set-up was also
reported wherein for the first set-up involves intermolecular
trNOEs between ligand protons and protein protons are sup-
pressed. In the second reference set-up, the T2 filter is applied
after the mixing period so that during the mixing time inter-
molecular trNOEs build up and lead to a decrease of the
ligand signal intensities.44 As a result, the difference of those
experiments provide signals of the ligands which are specif-
ically bound to the protein.
8 Natural Product Communications

Figure 7.  (a and b) represent pulse sequence for the BPP-STE (bipolar gradient pulse pair-stimulated echo) diffusion-assisted NOE
pumping experiments.42 This figure is reproduced from ref.42 with permission from the publisher.

Interligand NOE for Pharmacophore
Mapping (INPHARMA)
INPHARMA peaks are observed when two ligands can bind
competitively to the same binding pocket of a target protein.
INPHARMA method is observed through spin diffusion
mediated (via protons of protein), transferred NOEs between
two ligands that bind competitively to the same target mole-
cule. This method was first successfully used for two ligands
such as epothilone A and baccatin III which can bind
competitively but not simultaneously to the same target pro-
tein, tubulin.45
In general, during the mixing time in NOESY experiment,
ligand LA binds to the receptor protein P and magnetization
is transferred from its proton HLA to the receptor proton HP
and ligand LA dissociates from the receptor protein P and
then ligand LB binds into the same binding site of the target
protein. Subsequently, magnetization transfers from the
receptor proton HP to a proton in the ligand LB (HLB)

Figure 8.  (a) (Top panel) 1D- 1H NMR spectrum and (Bottom panel) 1D- 1H NMR spectrum with BPP-STE of 10 mM salicylic acid
(S), 10 mM L-ascorbic acid (A), 10 mM glucose (G), and 100 µM HSA in D2O. (b) stack plot of 1D- 1H spectra of HSA (at 300 K) in the
presence of three ligands as the mixing time tm is increased from 5 ms to 1.2 s in intervals of 0.05 s.42 This figure is reproduced from
ref.42 with permission from the publisher.
Maity et al. 9

Figure 9.  (a), Schematic presentation of the INPHARMA method, two competitive binding ligands LA and LB can bind to the same
receptor (P) consecutively but not simultaneously. (b) represents the schematic representation of LOGSY-titration method, for reversibly
binding ligands magnetization received from water during the WaterLOGSY experiment is a combination of cross-relaxation obtained
from bulk water and cross-relaxation delivered via protein-bound water molecule as proposed in ref.46

resulting in the observation of an intermolecular transferred Therefore, difference in the spectrum (SSTD=S0-SSAT) only
NOE between HLA and HLB through spin diffusion-mediated measures the signal of the ligand that received saturation
effect via a proton of the protein. Figure  9a shows two transfer from the protein as compared to non-binding ligands.
ligands, LA and LB, bind to a common target consecutively However, in the presence of large excess of ligand, the satu-
and not simultaneously. This method is particularly useful to ration of free ligands in solution gets amplified because the
optimize the ligands from chemical library as it detects rela- relaxation of small molecules is slower than the saturation
tive orientation of different compounds in the same target transfer. Viegas et al. analyzed STD effect in terms of ampli-
binding site. fication factor (ASTD) which can be expressed in the follow-
ing form,49
Saturation Transfer Difference (STD) NMR S0 −SSAT
[ ]
L T SSTD
[ ]
L T
ASTD = × [ ] = × [ ] (8)
Spectroscopy ‍ S0 P S0 P ‍

The STD-NMR screening technique is useful to identify

ligand moieties that are important for binding to target pro-
teins. The STD-NMR experiment is observed on the ligand-
based resonance signals and the technique is based on the
principle of nuclear Overhauser effect (NOE). The target
protein is irradiated with a radiofrequency field which selec-
tively hits resonances of the protein generally known as
on-resonance spectrum. Therefore, in the fast exchange
between ligand in the free and in the bound state, the satura-
tion is delivered to the bound ligand via the protein and that
saturation is carried over to the free ligand. As a conse-
quence, the second spectrum has to be collected wherein sat-
uration takes place off-resonance. Therefore, the difference
measured in STD spectrum represents resonance which orig-
inates selectively from the saturated ligand in ligand-protein
complex.47 STD NMR was reported for the interaction of
Alisertib (MLN8237), an orally administered inhibitor of
Aurora A kinase, with HSA.48 A schematic representation of
STD method is shown in Figure 10a. STD measures the dif-
ference of saturated peak of protein (on-resonance spectrum)
with signal intensities SSAT, from without protein saturation
(off-resonance spectrum), with signal intensities S0.
wherein ASTD is the amplification factor which is defined
as the average number of ligand molecules saturated per
molecule of receptor. In equation (8), SSTD/S0 denotes the
relative STD effect of ligand concentration[L]T which is
present excess of the protein [P] at a molar ratio of [L]T/[P].
Viegas et al. rearranged equation (8) for measuring KD value
and which can be represented as
[ ]
aSTD L
ASTD = [ ]
KD + L ‍ (9)
‍
‍aSTD ‍is the maximum amplification factor and this expres-
sion is valid if [L] = [L]T. It was predicted that observed KD
value changes with saturation time and accurate KD values
could only be obtained for experiments set up at saturation
times that were close to zero. The deviation of KD was sub-
jected to the rebinding of partially saturated ligands to the
protein during the protein irradiation period. Moreover,
STD-NMR experiment does not distinguish the specific and
non-specific binding of ligand(s).
A novel method was introduced based on STD-NMR for
the structural determination of ligands complexed to target
10 Natural Product Communications

Figure 10.  (a) Schematic representation of the STD-NMR experiment. (b) STD-NMR spectra of 2-(3’-pyridyl)-benzimidazole in the
presence of unlabeled FKBP, perdeuterated, Ile-protonated FKBP, perdeuterated,Val-protonated FKBP, perdeuterated, Leu-protonated
FKBP, perdeuterated, Met-protonated FKBP, and perdeuterated FKBP. The resonances corresponding to the ligand are highlighted. (c)
STD-NMR spectra of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) in the presence of unlabeled MurA, perdeuterated, His-
protonated MurA, perdeuterated, Trp-protonated MurA, and perdeuterated, Phe protonated MurA. The resonances corresponding to
UDP-GlcNAc are highlighted.50 This figure has been reproduced from ref.50 with permission from the publisher.

protein independent of size and composition called SOS-
NMR.50 This method is focused on a ligand complexed to a
series of proteins (deuterated except for specific amino acids)
and the amino acid composition of the ligand-binding site
can be identified, thus leading to the definition of the 3-D
structure of the protein-ligand complex. This method is use-
ful when XRD and conventional NMR methods fail to pro-
duce the required information on the ligand binding site.
This method was successfully applied to 2 systems, FKBP-
2-(3’-pyridyl)-benzimidazole complex and MurA-uridine
diphosphate N-acetylglucosamine complex, respectively
(Figure 10 (b and c)).50 This method has some limitations.
For example, for the application of this method, moderately
soluble ligand is required in fast exchange NMR time scale
(typically KD >1 µM). Therefore in the case of “lead-like”
compounds, the potency will be small or moderate.51 Thus,
this method may be useful in preliminary stages for ligand
screening in the drug discovery process. Another novel
approach to STD-NMR was proposed by Jayalakshmi et al.52
This method relies on the optimization of intensity-restrained
procedure to predict the structures of ligand in the bound
protein. This is achieved by inclusion of a structure refine-
ment method involving CORCEMA (complete relaxation
and conformational exchange matrix) calculation. This
method was used for screening of carbohydrate and peptide
ligands.
Water-LOGSY Method
Water-LOGSY is one of the powerful techniques used to
screen ligand binding to target proteins. In this experiment,
1
H of bulk water is excited and magnetization is transferred
from 1H of bound water to 1H of bound ligand via indirect
spin diffusion.53 In this experiment, compounds that can
interact with the target protein usually yield positive reso-
nances (that have the same sign as protein resonances), and
compounds that do not bind to the target usually show-up as
negative resonances. Water-LOGSY experiments provide
information on the bound ligand population. This method
can be applied for the characterization of ligand-protein
interactions in relatively large molecular weight systems.54
The KD value was successfully measured for HSA-L-
tryptophan complex using Water-LOGSY method. The KD
values calculated are accurate and are consistent with those
calculated using other methods.17,55,56 Another reports
showed that KD values became higher for titration experi-
ment of varying concentrations of bovine serum albumin
(BSA) with L-tryptophan.57 The deviations of KD value was
also observed for binding of 3-fluorophenylboronic acid to
α-chymotrypsin at higher concentrations of α-chymotrypsin
and long mixing time. However, accurate KD value may be
obtained at lower concentrations of protein.58 The use of
Water-LOGSY method is restricted in fragment screening
where determination of KD is the needful step.58 Water-
LOGSY method also is limited due to its inability to differ-
entiate between buried and surface-accessible area of ligands.
A novel technique was emerged called LOGSY titration to
discriminate between buried and solvent-accessible area of
ligands and to map protein-bound water molecules in close
proximity to the ligand.59 The scheme of LOGSY titration is
shown in Figure 9 (b). The method was applied to determine
buried and solvent-accessible epitopes for binding of two
Maity et al. 11

Figure 11.  LOGSY titration of 6-(3,4-Dimethoxyphenyl)−3-methyl1,2,4 triazolo[4,3-b]-pyridazine (a) and 1-Methyl-5-phenoxy-

1H-pyrazole-4-carboxylic Acid Ethyl Ester (b)with perdeuterated bromodomain 1 present in protein 4 (Brd4-BD1) ligands. 100%
corresponds to the signal with the strongest slope. X-ray crystal structures of the complexes Brd4-BD1 with ligand are shown here. H2O
molecules resolved in the electron density map and within a distance of 6 Å from the ligand are shown as blue spheres.59 This figure has
been reproduced from ref.59 with permission from the publisher.[https://pubs.acs.org/doi/abs/10.1021/acs.jmedchem.7b00845

synthetic molecules 6-(3,4-Dimethoxyphenyl)-3-methyl1,2,4 It is observed that mapping of binding site of 15-nucleo-

triazolo[4,3-b]-pyridazine and 1-Methyl-5-phenoxy-1H-
pyrazole-4-carboxylic acid ethyl ester with bromodomain 1
present in protein 4 (Brd4-BD1), a prominent cancer target
(Figure 11 (a and b).59 The merit of this method as compared
to conventional ligand-based methods is its independence of
the molecular mass of the protein.
Cross Saturation and Transferred Cross
Saturation
The characterization of protein-large molecules interfaces is
a key factor to understanding of biological mechanisms at a
molecular level. Cross-saturation experiment provides accu-
rate information on the interface of larger protein com-
plexes.46 The cross-saturation mapping of protein–large
molecule interfaces is based on the steady-state NOE-
difference experiment wherein 1H resonance signals of a
non-deuterated target components (protein II) are saturated
and accounts for observation of magnetization of NH pro-
tons of a second deuterated and 15N-labeled protein (protein
I) (Figure 13(a)). However, in cross-saturation method resi-
due-specific information can be helpful to provide accurate
information about protein-large molecule interfaces but the
cost of sample preparation is high.
tide RNA on the RNA-binding protein, Nova1 KH3,
obtained from cross-saturation 1H/15N-HSQC experiments
was almost similar to the binding site predicted from the
CSP method and X-ray crystallography.60 Therefore, it can
be suggested that combined use of CSP and cross-saturation
data will be helpful to identify the interaction sites with high
accuracy. Ferrage et al. proposed a novel approach to the
direct determination of the likely pose of a peptide from the
proline-enriched tyrosine phosphatase, onto a protein Src
homology 3 (SH3) domain of the C-terminal Src kinase
(CSK) by utilizing frequency-selective cross-saturation
using a low stringency isotopic labeling method(s) and side-
chain protons as probes of the interface (Figure  12(a and
b).61 It is demonstrated that the advantages of the methods
related to the enhancement of overall efficiency of satura-
tion along with better coverage of interaction interface
could lead to the increase in accuracy of the overall results.
The biggest advantage of this approach involves significant
variations of the saturation transfer between experiments
could be associated to the local chemical shift distribution in
the protein to achieve site-specific restriction on both ele-
ments which will be helpful for molecular docking stud-
ies.62,63 This method will be very applicable for confirmation
of most occupied contacts in the particular cases wherein
12 Natural Product Communications

Figure 12.  Saturation of the signals from 13C bonded aromatic (a) and aliphatic (b) protons in side chains in SH3 by the sequence
employed for the selective saturation transfer. The ratio of intensities in HSQC with and without selective saturation is plotted along the
y axis.61 This Figure has been reproduced from ref.61 with permission from the publisher.

substantial motion is present in a complex which may lead
to design of potential ligands. However, cross-saturation
method is difficult to be applied to the protein complexes
with a molecular mass greater than 150 kDa. The molecular
weight limitation is overcome by introducing an extended
version of cross-saturation method called transferred
cross-saturation (TCS) method.64 In this method, saturation
in target protein (protein II) in the complex is transferred
significantly to the residues of the bound uniformly isotopi-
cally labeled (with 2H and 15N) ligand protein (protein I) at
the interaction interface, and the contact residues of protein
I in the complex are identified by observing the decrease of
the peak intensities on a 1H–15N shift correlation spectrum
(Figure  13(b)). The information on the contact residues is
transferred on the ligand proteins in the complex, from the
ligand proteins in the bound state to those in the free state,
under a fast exchange process (kex>0.1 s).65 The TCS method
has a higher sensitivity toward low-affinity transient interac-
tions. It is demonstrated that this method is effective for the
investigation of very large systems including

Figure 13.  Schematic representation of cross-saturation (a) and transferred cross-saturation (b) techniques.
Maity et al. 13

membrane-embedded protein interactions, liposomes, and
insoluble biomolecules.66
Diffusion-Ordered Spectroscopy (DOSY)
The translational diffusion coefficient of a protein is an use-
ful parameter to obtain information on a protein’s hydrody-
namic state in solution as well as to study conformational
changes upon addition of ligands.67 In the DOSY method, it
is possible to measure translational diffusion in solution by
utilizing pulse-field gradients (PFGs) with higher accuracy.68
This method is a valuable approach for the detection of bind-
ing of low-molecular-weight compounds to large molecular
weight protein receptors, ie specific for molecules with weak
binding affinity (KD>1 mM). Small molecules bound to pro-
tein diffuse at the slower rate of the protein than at the typical
rate for small molecules. Therefore, it is possible to distin-
guish compounds in the presence or absence of protein from
a mixture of compounds according to their diffusion proper-
ties.69 Ferrage et al. reported a novel pulsed field gradi-
ent(PFG) NMR method for measuring the translational
diffusion coefficient of an integral membrane protein/surfac-
tant complex.70 The novel pulsed field gradient NMR method
has advantage compared to the conventional NMR methods
with pulse field gradient because this method can be applied
to macromolecular assemblies (>25 kDa) in aqueous solu-
tion at room temperature. This new method was successfully
applied to a water-soluble complex of tOmpA, the hydropho-
bic transmembrane domain of bacterial outer membrane pro-
tein A, with the detergent octyltetraoxyethylene (C8E4; total
mass of complex ~ 45 kDa) (Figure 14(b)). Another novel
method was proposed for considering multiple diffusion
measurements associated with each molecule in a mixture.
In a mixture of ligands, multiple pairs of diffusion coeffi-
cients for each ligands arise from a pair of DOSY spectra in
the presence and absence of protein. Separation of diffusion
data characterizing ligands along two dimensions would
allow for more efficient clustering to distinguish between
ligands. Snyder et al. demonstrated the use of CoLD-CoP
(Clustering of Ligand Diffusion Coefficient Pairs) technique
to identify specific ligands that bind to target protein(s).71
Thus CoLD-CoP technique was useful to discriminate the
binders such as, N-acetylglucosamine (GlcNAc) and trypt-
amine with lysozyme and tartrate with wheat germ acid
phosphatase (WGAP) from non-binders. This method is not
applicable when the protein binding is weak and when pro-
tein concentration is limited. However, this method is partic-
ularly useful for fragment-based ligand screening.71 Didenko
et al. reported that 3D DOSY–heteronuclear multiple quan-
tum coherence (HMQC) pulse sequence that uses the TROSY
(transverse relaxation-optimized spectroscopy) effect of the
HMQC sequence on 13C methyl-labeled proteins and is
highly suitable for measuring the diffusion coefficient of
large proteins.72 3D DOSY–HMQC experiment is suitable
for a detailed analysis of the diffusion coefficients of com-
plex mixtures of small organic molecules.73
SAR (Surface Activity Relationship) by
NMR
SAR by NMR-based approach is introduced for discovering
high-affinity ligand binding to the individual binding sites of
protein.74 Binding of small molecules to proximal sites in a
protein are primarily identified and optimized by this method.
Small molecules are screened by 15N-1H- chemical shift

Figure 14.  (a) Pulse sequence for the measurement of diffusion coefficients using longitudinal magnetization of a heteronucleus such
as nitrogen-15 with stimulated echoes (X-STE). Narrow and wide pulses represent 90° and 180° pulses, respectively. (b), Nitrogen-15-
filtered proton spectra of [15N]-tOmpA in C8E4, pH 8) at 23°C. Arrows indicate the limits of the spectral region (7.6-8.9 ppm) over
which signals were integrated. The free induction decays were multiplied by exponentially decaying functions corresponding to 100 Hz
line broadening.70 This Figure has been reproduced from ref.70 with permission from the publisher.
14
Figure 15.  (a), Schematic representation of SAR by NMR. (b), list of compounds with different substituents tested in 1H/15N-HSQC
binding experiments to FKBP.74 The equilibrium binding constants(KD) are given in the table for different compounds. This Figure has been
reproduced from ref.74 with permission from the publisher.
changes monitored by 1H-15N HSQC using 15N labeled pro-
teins. The subsequent step requires the optimization of the
second weakly interacting ligand to an adjacent binding site
and the binding is identified via the observed chemical shift
changes. Final step involves, structural orientation of the
bound ligands in order to guide their linkage and maintain
this structural orientation and information to obtain high
binding specificity to the target compound (Figure 15(a)).
First a 15 N-1H HSQC spectrum of the labeled protein is
collected as a reference spectrum and if the resonance signal
is shifted in presence of one or many other molecules with
respect to reference spectrum it is marked as a sign of bind-
ing (Figure 16). The SAR by NMR method effectively leads
to the development of highly potent compounds and this
method has been emerging as one of most powerful tech-
niques for fragment-based lead discovery (FBLD) as it pro-
duced a large number of high-affinity ligands for target
macromolecules.75 The SAR by NMR approach has been
successfully used to screen ligands that bind to the FK506-
binding protein, FKBP.74 A number of compounds from a
designated chemical library were first screened for binding.
Natural Product Communications
Among all other compounds, trimethoxyphenyl pipecolinic
acid derivative (1) showed the highest binding affinity for
FKBP (KD = 2.0 µM). The binding site was predicted for (1)
was the same as for FK506. In the presence of saturating
amount of FKBP and compound 1 binary complex, screen-
ing of benzanilide derivative (compound 2) was examined
which bound to the adjacent binding site with a KD value of
about 100 µM. Self-linked fragment approach was applied to
produce a higher-affinity ligand by covalently conjugating
the two fragments 1 and 2 through a variety of linkers. By
using linkers, Compound 9 is the most optimal ligand as it
shows the highest binding affinity (KD = 19 µM for FKBP,
Figure 15, Panel-b).
Conclusion
Characterization of protein-ligand interface is key to ratio-
nal drug discovery. Detailed structural analysis of
ligand-protein interface will likely provide valuable clues
for the design of specific and potent small molecule antag-
onists against key cellular processes associated with
Maity et al.
Figure 16.  An overlay of 1H/15N-HSQC spectra for FKBP in the absence (magenta contours) and presence (black contours) of
benzanilide derivative (compound 2) with saturating amounts of trimethoxyphenyl pipecolinic acid derivative (Compound 1)(2 mM).
1 15
H/ N CSP observed for resonance signals of the residues are indicated.74 This figure is reproduced from ref.74 with permission from
the publisher.
disease(s). NMR spectroscopy has come of age. Recent
advancements in instrumentation technology in conjunc-
tion with the development of new methods has significantly
increased the versatility of NMR spectroscopy as the
method of choice in modern drug discovery. In addition,
advent of cryoprobes has significantly decreased the data
acquisition times and sample requirements. Further, with
availability of the microprobe technology, multidimen-
sional NMR data can now be acquired even under high salt
concentrations. Most importantly, NMR spectroscopy is
now amenable for high throughput ligand binding studies.
The applications of NMR spectroscopy are fast expanding
and in this context the recently emerging in-cell NMR
spectroscopy offers a new avenue(s) for investigating the
effects of small molecule drug candidates under ex-vivo
condition.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect
to the research, authorship, and/or publication of this article.
Funding
The author(s) disclosed receipt of the following financial support
for the research, authorship, and/or publication of this article: This
work was supported by the Department of Energy (grant number
DE-FG02-01ER15161), the National Institutes of Health/National
Cancer Institute (NIH/NCI) (1 RO1 CA 172631) and the NIH
through the COBRE program (P30 GM103450), and the Arkansas
Biosciences Institute (ABI-TKSK-2016-17).
15
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