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Methods
journal homepage: www.elsevier.com/locate/ymeth
NMR-based investigations into target DNA search processes of proteins
Junji Iwaharaa,⁎
, Levani Zandarashvilib
, Catherine A. Kemmea
, Alexandre Esadzec
a
Department of Biochemistry and Molecular Biology, Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, United States
b
Department of Biochemistry and Biophysics, University of Pennsylvania, United States
c
Department of Pharmacology and Molecular Sciences, Johns Hopkins University, United States
A R T I C L E I N F O
Keywords:
DNA scanning
Dynamics
Kinetics
NMR
Protein-DNA interactions
Target search
A B S T R A C T
To perform their function, transcription factors and DNA-repair/modifying enzymes must first locate their
targets in the vast presence of nonspecific, but structurally similar sites on genomic DNA. Before reaching their
targets, these proteins stochastically scan DNA and dynamically move from one site to another on DNA. Solution
NMR spectroscopy provides unique atomic-level insights into the dynamic DNA-scanning processes, which are
difficult to gain by any other experimental means. In this review, we provide an introductory overview on the
NMR methods for the structural, dynamic, and kinetic investigations of target DNA search by proteins. We also
discuss advantages and disadvantages of these NMR methods over other methods such as single-molecule
techniques and biochemical approaches.
1. Introduction: DNA scanning by proteins
Protein-DNA interactions are vital for life. Living organisms regulate
expression of genes and maintain integrity of the genome through
protein-DNA interactions involving transcription factors or DNA-repair/modifying
enzymes. These DNA-binding proteins must first locate
their specific target sites in the vast presence of nonspecific but structurally
similar sites on genomic DNA. The genome of higher eukaryotes
contains billions of base pairs [1] and can potentially provide ∼109
nonspecific sites on DNA, whereas functional target sites of each transcription
factor or DNA-repair/modifying enzyme are far fewer (typically,
∼102
–103
sites) [2]. Functionality of these proteins should depend
on their efficiency in locating targets on DNA through stochastic
search processes. Therefore, it is important to understand at molecular
and atomic levels how proteins scan DNA, recognize sequences, and
locate the targets.
In the target search processes, the proteins nonspecifically interact
with DNA. Although affinity for nonspecific DNA is weaker than that for
targets, the vast quantity of nonspecific sites compensates for the weak
affinity, making profound overall impacts [3–5]. It should be noted that
DNA density in cell nuclei is as high as ∼100 mg/ml [6]. This high
density is understandable, considering that a total length of ∼2 m of
human genomic DNA (=2 × [3.2 × 109
bp] × [0.34 × 10−9
m/bp]) is
confined in the nucleus for which a typical diameter is ∼6 μm [1].
Histones make this condensation possible, occupying ∼80% of genomic
DNA as nucleosomes. The average length of linker DNA segments
between nucleosomes is ∼56 bp [7]. The overall concentration of
linker DNA segments is estimated to be as high as ∼500 μM in the
nuclei. Because this concentration alone is far higher than typical apparent
dissociation constants of sequence-specific or structure-specific
DNA-binding proteins for nonspecific DNA, the majority of these proteins
must be nonspecifically bound to genomic DNA before reaching
their targets.
When nonspecifically interacting with DNA, the DNA-binding proteins
rapidly move from one site to another, scanning DNA to locate
their targets (Fig. 1). Berg and von Hippel conceptually defined three
major mechanisms for protein translocation on DNA: sliding, dissociation
& re-association, and intersegment transfer [8]. Sliding corresponds
to one-dimensional (1D) diffusion along DNA and involves
random walks of protein being nonspecifically bound to DNA. Binding
to nonspecific sites near targets can accelerate target association because
sliding from nonspecific sites allows proteins to efficiently reach a
target through 1D diffusion [2,9–11]. The dissociation & re-association
mechanism is often called three-dimensional (3D) search. When DNA
concentration is high (e.g., in the nuclei), dissociation from DNA is the
rate-limiting step in this translocation mechanism. When translocation
through this mechanism occurs between two sites in close proximity
(e.g. within ∼1–7 nm [12]), it is called hopping. Intersegment transfer
(also known as direct transfer) is a unique mechanism that allows
protein to directly transfer from one DNA duplex to another without
going through the intermediary of free protein. This involves an intermediate
where a protein molecule transiently bridges two DNA
https://doi.org/10.1016/j.ymeth.2018.05.004
Received 28 February 2018; Accepted 4 May 2018
⁎
Corresponding author.
E-mail address: j.iwahara@utmb.edu (J. Iwahara).
Abbreviations: CSP, chemical shift perturbation; PRE, paramagnetic relaxation enhancement; RDC, residual dipolar coupling; ZF, zinc finger
Methods 148 (2018) 57–66
Available online 10 May 2018
1046-2023/ © 2018 Elsevier Inc. All rights reserved.
T
duplexes. Intersegment transfer mechanism may facilitate bypassing of
nucleosome because its two ends are close in the three-dimensional
structure [13]. These distinct translocation mechanisms coexist in solution,
and their relative contributions to the overall efficiency of target
DNA search should depend on various factors such as ionic strength,
protein structure, DNA density, DNA geometry, and the presence of
other proteins. However, details of the DNA scanning mechanisms are
not well understood.
Our goal in this review article is to provide general readers with an
introductory overview on nuclear magnetic resonance (NMR) methods
for investigating the target DNA search processes of proteins. These
NMR methods allow us to gain a great deal of information regarding
how proteins nonspecifically interact with DNA during the search
processes and how they move from one site to another on DNA. We first
compare these NMR methods with biochemical and single-molecule
methods for investigating target DNA search processes and explain
unique strengths of these methods. Then, we will elaborate on practical
details of the NMR methods that provide dynamic and kinetic information
on target DNA search processes.
2. Advantages of NMR over other methods for investigating target
DNA search of proteins
In the target DNA search processes, proteins dynamically move on
DNA, changing their locations. This movement creates a challenge in
studying these processes. Because proteins can change their locations
through distinct mechanisms that are simultaneously present in the
same system, characterizing a particular translocation mechanism is
difficult, particularly when molecular ensembles are measured in solution.
In the 21st century, there have been substantial advances in
methodology for research on the target DNA search processes. Progress
in single-molecule biophysics techniques is particularly remarkable,
allowing direct observations of protein translocation on DNA in vitro
and even in living cells. These single-molecule methods are recently
overviewed in excellent reviews [14–21]. Elegant biochemical methods
were also developed for kinetic investigations of target search and
protein translocation on DNA [12,22–29]. As discussed below, NMR
spectroscopy has provided unique insight into how proteins scan DNA
at an atomic level. Capability and suitability of these methods for target
search research are summarized in Table 1.
The greatest advantage of NMR methods over other methods is that
NMR spectroscopy can provide atomic-level information on the highly
dynamic processes whereby proteins scan DNA and locate their targets.
NMR spectroscopy is particularly suited to study structural dynamics of
biological macromolecules and can provide spatiotemporal information
on dynamics [30]. As described in Section 4, there are several NMR
methods that can provide different types of information on the DNAscanning
process. Some NMR data give structural information on
proteins scanning DNA. Other NMR data give information on conformational
mobility of particular domains or moieties within the
proteins bound to DNA. Dynamic behavior of each basic side chain at
protein-DNA interfaces can also be analyzed using NMR relaxation data
[31–34]. These NMR methods can provide structural and dynamic details
on the proteins moving on DNA, which are very difficult to analyze
at an atomic level by any other methods.
Through NMR experiments, one can also obtain quantitative information
on kinetics of protein translocation on DNA at equilibrium.
The range of kinetic rate constants that can be determined by NMR is as
wide as 0.1–10,000 s−1
, though suitable methods should be adopted
depending on the timescale of the analyzed processes. As described in
Section 5, several NMR methods are available for the kinetic investigations
of protein translocation on DNA and differ in the applicable
range in terms of analyzable timescale. These NMR experiments for
investigating the kinetics of protein-DNA interactions can be conducted
under various buffer conditions, offering flexibility in experimental
design. A qualitative in-cell NMR study of protein-DNA interactions in
E. coli cells has also been demonstrated [35]. NMR spectroscopy can
thus serve as a versatile tool for investigating target DNA search processes
of proteins.
3. NMR sample preparation for studying protein-DNA interactions
NMR studies on kinetics and dynamics of target DNA search by
proteins require solution samples containing protein and DNA in which
protein can move from one site to another. Because NMR spectroscopy
is not as sensitive as many other spectroscopic techniques, high concentrations
of proteins and DNA are required and high solubility of
these materials is crucial for NMR experiments. Although high-field
magnets and cryogenic probes have increased detection sensitivity for
modern NMR spectrometers, many NMR experiments still require
0.1–1 mM proteins and DNA in a 0.25–0.5 ml solution. Milligrams of
protein and DNA are necessary to prepare such a sample. Molecular size
is also a limiting factor for NMR application. For typical heteronuclear
multi-dimensional NMR experiments with backbone NH detection, the
total molecular weight should be < 100 kDa. For methyl-TROSY-type
experiments with side-chain CH3 detection, the total molecular weight
can be bigger. In fact, 200-kDa nucleosome core particles of histone
octamer and 167-bp DNA were studied by methyl TROSY methods [36].
Typical buffers for NMR experiments are at pH 5–8 and an ionic
strength less than 150 mM. We typically use 20–100 mM KCl (or NaCl)
for NMR measurements of protein-DNA complexes. In general, sensitivity
in NMR detection (particularly with cryogenic probes) is higher at
a lower conductivity of solution [37,38]. When a high concentration of
Fig. 1. Protein translocation on DNA through nonspecific interactions.
Table 1
Comparison of the experimental approaches for investigating target DNA search
of proteins.
Capability Methods
NMR Single-
molecule
Biochemical
Atomic details of DNA scanning +++a
– –
Direct visualization of protein
translocation on DNA
– +++ –
Kinetic analysis of protein translocation
on DNA
++b
++ ++
Kinetic analysis of target DNA
association
+ ++ +++
Analysis of target DNA search process
in cells
+ +++ +
Symbols are as follows: +++, well suited; ++, feasible; +, possible, but not
well explored; –, not possible.
a
See Section 4.
b
See Section 5.
J. Iwahara et al. Methods 148 (2018) 57–66
58
salt is required, use of sample tubes with a thinner sample diameter can
mitigate the adverse effect of salt [39,40].
Due to large amounts of materials required, protein-DNA complexes
might aggregate or precipitate during preparation of NMR samples,
particularly when high concentrations of proteins and DNA are mixed
at a low ionic strength. Fig. 2 shows our typical procedures of sample
preparation for NMR studies of protein-DNA interactions. To avoid the
aggregation or precipitation, we initially mix relatively low concentrations
(∼10–50 μM) of proteins and DNA at a relatively high ionic
strength (≥300 mM KCl). In our experience, nonspecific DNA complexes
of proteins exhibit a stronger tendency to precipitate at low ionic
strength. After mixing the protein and DNA components at high ionic
strength, we slowly reduce ionic strength. When ionic strength decreases
too rapidly, some proteins precipitates (in some cases, the
precipitated materials can be recovered by re-dissolving in a high ionicstrength
buffer). So, we often reduce ionic strength through dialysis. A
gradual decrease in salt concentration seems to generally help prevent
aggregation of protein-DNA complexes, as known for nucleosome reconstitutions
[41]. After reaching a desired ionic strength (typically,
20–100 mM KCl in our experiments), protein-DNA solutions are concentrated
and buffer-exchanged using centrifugal concentrators such as
Amicon Ultra-4 (Millipore) or Vivaspin-6 (Sartorius). A deuterated
buffer, such as Tris-d11 or succinate-d4, is used at this point if 1
H signals
from buffer molecules could interfere with planned NMR experiments.
For NMR studies of nonspecific DNA-protein complexes, concentration
of DNA should be sufficiently higher than that of protein. We
typically use molar ratios of protein to nonspecific DNA of 1:2–4. A
large excess of DNA helps prevent binding of more than one protein
molecule to the same DNA molecule. This is important because the
multiple bindings would complicate data analysis and also enhance
aggregation and precipitation. Use of mM concentrations of nonspecific
DNA mimics physiological conditions because it is comparable to the
concentration of linker DNA segments in the nuclei, as mentioned in
Section 1. DNA length is also an important parameter that affects solubility
and stability of protein-DNA complexes in NMR samples. When
nonspecific DNA complexes are analyzed, DNA must not contain any
high-affinity sequences that are similar to the target sequence. To
confirm the absence of any high-affinity site, we use fluorescence-based
competitive binding assays [42], which directly provide relative affinity
of competitor with respect to that of fluorescent-probe DNA containing
a high-affinity site.
4. NMR-based analysis of DNA scanning by proteins
DNA-scanning processes are highly dynamic, during which proteins
rapidly and stochastically change their locations on nonspecific DNA
before reaching their targets. Solution NMR spectroscopy is well suited
to study such dynamic systems. Although the DNA-scanning processes
represent intermediates before the proteins reach their targets, these
processes can be investigated most conveniently by using solutions of
nonspecific DNA complexes that do not contain any targets. NMR was
used to study nonspecific protein-DNA complexes in solution for generegulatory
proteins (e.g., the lac repressor [43], Egr-1 [13,44], Ets-1
[45], ETV6 [46], HMGB1 [47], HoxD9 [48], LmrR [49], Oct1 [50],
Sox2 [51], and ZNF217[52]) and as well as for DNA-repair/modifying
enzymes (e.g., EcoRI [53], M.HhaI [54], and UNG [55,56]). Because
proteins can move on nonspecific DNA, a solution of a nonspecific
complex contains many states with the protein being located at different
sites. However, NMR samples of nonspecific DNA-protein complexes
typically show only a single set of signals because transitions
between these states are so rapid that resonances of individual states
are averaged (i.e., the “fast exchange” regime in the terminology of
NMR spectroscopy). An example is shown in Fig. 3. Such samples of
nonspecific complexes can be used to study how proteins scan DNA
before reaching their targets. The following NMR methods are particularly
useful to study the DNA-scanning dynamics.
4.1. NMR relaxation
NMR relaxation data for backbone and side-chain moieties are
useful to study dynamics of proteins on timescales ranging from ps – ns
to μs – ms [57–60]. There are several different types of NMR relaxation
parameters (e.g., longitudinal and transverse relaxation rates, cross
relaxation rates, cross correlation rates, etc.), which reflect internal
motions of various moieties within proteins. Relaxation parameters of
15
N, 13
C, and 2
H nuclei are particularly useful for investigating protein
dynamics, whereas 1
H relaxation reflects more about structure due to
strong 1
H–1
H dipole-dipole interactions. Using NMR relaxation data,
one can obtain information on 1) conformational flexibility of protein
backbone and side chains, 2) conformational equilibrium between different
states, 3) inter-domain dynamics, and 4) kinetics of molecular
interactions. Analysis of NMR relaxation for nonspecific DNA complexes
of proteins allows us to learn how these proteins scan DNA.
Fig. 4A shows an example of such an analysis. This figure displays
some backbone 15
N relaxation data for the nonspecific and specific DNA
complexes of the Egr-1 zinc-finger protein. Egr-1 recognizes DNA via
three zinc fingers, ZF1, ZF2, and ZF3, each of which contacts with DNA
in the crystal structure of the complex with the target DNA [61–63].
Backbone 15
N relaxation data were compared with the nonspecific and
specific complexes with 28-bp DNA. The specific DNA contains the EgrFig.
2. Typical procedures for preparation of protein–DNA solutions for NMR
investigations of target DNA search processes.
J. Iwahara et al. Methods 148 (2018) 57–66
59
1 recognition sequence, GCGTGGGCG, whereas the nonspecific DNA
does not contain any high-affinity sequences. The backbone relaxation
data for all three zinc fingers ZF1, ZF2, and ZF3 were similar for the
specific DNA complex. However, the backbone 15
N relaxation data for
ZF1 in the nonspecific complex significantly differed from those for ZF2
and ZF3 in the nonspecific complex. These and other data suggested
that ZF1 is mainly dissociated from DNA when the Egr-1 zinc-finger
protein scans DNA. When ZF1 is locally dissociated from DNA and ZF2
and ZF3 are bound to DNA, ZF1 undergoes almost independent domain
motion, making 15
N longitudinal (R1) and transverse (R2) relaxation
rates of ZF1 differ from those of ZF2 and ZF3. The linker connecting ZF1
to ZF2 shows smaller heteronuclear 15
N NOE values in the nonspecific
complex than in the specific complex, suggesting that the linker is more
flexible in the nonspecific complex. This is also consistent with the
independent domain motion of ZF1 in the nonspecific complex.
4.2. Residual dipolar coupling (RDC)
Residual dipolar coupling (RDC) of nuclei in proteins weakly
aligned to the magnetic field provide information about orientations of
particular covalent bonds and are useful to study protein structure and
dynamics [64–67]. Analysis of RDC data for proteins in the nonspecific
and specific DNA complexes with DNA can allow us to assess structural
dynamics of the DNA-scanning processes [13,48,50,51]. For example,
RDC data for the HoxD9 homeodomain for the nonspecific and specific
DNA complexes suggested that this protein interacts with nonspecific
DNA in the same manner as in the specific complex [48]. In contrast,
RDC data for the Egr-1 zinc-finger protein clearly showed that the three
zinc fingers behave differently in the specific complex with target DNA
and in the nonspecific DNA complex [13]. As mentioned above, ZF1 in
the nonspecific DNA complex is locally dissociated from DNA while ZF2
and ZF3 are bound to DNA. Since ZF1 undergoes independent domain
motion with respect to the other part of the complex, the overall
magnitude of RDCs for ZF1 in the nonspecific complex was substantially
smaller than those for ZF2 and ZF3 in the nonspecific DNA
complex (Fig. 4B). Prediction of RDC for nonspecific DNA complexes is
difficult because the observed samples contain many states with the
protein being located at different sites. However, if structural models of
individual states can be built, the overall profile of RDC for nonspecific
complexes can be predicted using structure-based de novo RDC prediction
methods [68,69]. Comparing the experimental RDC data with
those predicted for the ensemble of various states in the nonspecific
complex allows us to examine models on how proteins scan DNA
[13,48].
4.3. Paramagnetic relaxation enhancement (PRE)
NMR paramagnetic relaxation enhancement (PRE) arising from dipole-dipole
interactions between 1
H nuclei of a protein and unpaired
electrons of an extrinsic paramagnetic group is a powerful tool for
structural and dynamic studies of protein-DNA complexes [70–72].
There are several methods for site-specific, covalent attachment of a
paramagnetic group to proteins or nucleic acids. For example, a paramagnetic
Mn2+
ion can be site-specifically incorporated into the EDTA
conjugated to DNA thymidine (dT-EDTA-Mn2+
) [73]. A paramagnetic
group can also be incorporated into DNA through conjugation of a
nitroxide spin label to a nucleotide base [74–76]. Intermolecular PRE
rates can be readily measured using DNA containing a paramagnetic
group and 15
N- and/or 13
C-labeled proteins [77,78]. In solution, proteins
moving on DNA can be located near the paramagnetic group attached
to a DNA base. Such a state causes strong PRE for the nuclei that
are in close proximity from the Mn2+
ion [47,48,79]. Due to their r−6
dependence, PRE rates are sensitive to the presence of states in which
the distances (r) between observed nuclei and the unpaired electrons of
the paramagnetic group are relatively short. Even if such states are as
minor as 1%, they can make a predominant contribution to observed
PRE rates, allowing us to detect the low-population states [79]. When
protein translocation on DNA occurs in the fast exchange regime, the
PRE data can show which parts of the protein can become proximal to
the paramagnetic group. This method thus provides structural information
on proteins in the target DNA search processes.
4.4. Solvent PRE
Random collisions with paramagnetic co-solute molecules (e.g.,
gadolinium-diethylenetriamine pentaacetic acid bismethylamide [GdDTPA-BME]
and 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl
[TEMPOL]) at relatively high concentrations cause sizable PRE for 1
H
nuclei of proteins [80–84]. This type of PRE, referred to as solvent PRE,
is stronger for 1
H nuclei near the molecular surface accessible to solvent
and can be used to identify molecular interfaces. This method is applicable
to highly dynamic complexes such as nonspecific DNA-protein
complexes. In the free state, the interfaces are more exposed to the
solvent and therefore exhibit larger solvent PRE than in the complex
with DNA even when the protein is changing its location on DNA. Although
chemical shift perturbation (CSP) upon complex formation may
provide similar information on the molecular interface, non-interfacial
regions often exhibit significant CSP as well [85]. In principle, solvent
PRE is more straightforward than CSP in identifying molecular interfaces.
Relative magnitudes of solvent PRE are predictable from structure,
assuming that spatial distribution of the paramagnetic co-solute
Fig. 3. 1
H–15
N heteronuclear correlation spectra recorded for the specific and
nonspecific DNA complexes of the Egr-1 zinc-finger protein [13]. Although the
protein can be located at various sites on nonspecific DNA with a mean residence
time being ∼1–10 μs at each site [23], the spectrum of the nonspecific
complex shows only a single set of signals due to rapid translocation that occurs
in the fast exchange regime.
J. Iwahara et al. Methods 148 (2018) 57–66
60
molecules is uniform [82,84]. Largely because this assumption is not
necessarily valid for commonly used paramagnetic co-solute molecules,
prediction of solvent PRE is only qualitatively accurate [81,84].
Nonetheless, by comparing solvent PRE data for the free protein and for
the nonspecific complex, the molecular interfaces during the DNA
scanning process can be mapped on the protein surface. This technique
was used to analyze how the HoxD9 homeodomain and the uracil DNA
glycosylate interact with nonspecific DNA [48,56].
5. NMR-based kinetic analysis of protein translocation on DNA
NMR spectroscopy can also be used to study kinetics of protein
translocation on DNA. Among various spectroscopic techniques for
analyzing molecular ensembles in solution, NMR is unique in that it
allows investigations of fast kinetics at equilibrium. There are a number
of NMR methods for kinetic investigations. Which one is the most appropriate
depends on the timescale of the processes of interest [30].
Some are suitable for kinetic analysis of processes on a timescale of
10−2
–1 s (e.g., z-exchange spectroscopy). Others are suitable for faster
processes on a timescale of 10−5
–10−3
s (e.g., line-shape analysis) or
10−4
–10−2
s (e.g., CPMG relaxation dispersion methods). Typically,
sliding of protein from one nonspecific site to an adjacent site on DNA
occurs on a μs timescale. Dissociation from a nonspecific site occurs on
a ms timescale, whereas dissociation from a specific, high-affinity site
may even require minutes to hours. Furthermore, association and intersegment
transfer are second-order processes and therefore their rates
depend on the concentration of free DNA.
5.1. Mixture approach
In the NMR sample, protein translocation on DNA occurs within the
same DNA molecule (i.e., intramolecular translocation) or between sites
on different DNA molecules (i.e., intermolecular translocation).
Distinguishing these two is not trivial. A general NMR approach called
the “mixture approach” has been developed to facilitate kinetic analysis
of intermolecular protein translocation on DNA [13,48,86–90]. This
approach makes use of two different DNA duplexes a and b of comparable
binding affinity and a 15
N-labeled protein, from which two
individual complexes of protein-DNA a and protein-DNA b and a mixture
of these two complexes are prepared (Fig. 5). DNA concentrations
in these samples should be high enough for the protein to completely
bind to either of these DNA. When the 1
H–15
N heteronuclear spectra are
recorded for these three samples, the two samples of the individual
complexes with DNA a and DNA b should show different 1
H/15
N chemical
shifts at least for some interfacial protein residues. For such residues,
the spectrum of the mixture should exhibit one of the following
features: 1) a single signal on the line between the two corresponding
signals of the individual complexes, 2) two separate signals corresponding
to the two complexes, or 3) very broad signal somewhere
between the two signals. In the terminology of NMR spectroscopy, these
three conditions are called fast, slow, and intermediate exchange regimes,
respectively. If the intermolecular translocation processes occur
in the fast exchange regime (this is usually the case for nonspecific DNA
duplexes), NMR line-shape analysis or relaxation dispersion methods
can be used to analyze the translocation kinetics (Section 5.2). If
translocation of the protein occurs in the slow exchange regime (this is
Fig. 4. DNA-scanning by the Egr-1 zinc-finger protein [13,44]. (A) Backbone heteronuclear 15
N NOE and 15
N R1 relaxation data for nonspecific and specific DNA
complexes of the Egr-1 zinc-finger protein. 28-bp DNA duplexes were used. Black and magenta data points are data obtained at the 1
H frequencies of 600 and
800 MHz, respectively. Blue dotted boxes show data points indicative of local dissociation of zinc finger 1 (ZF1). (B) RDC data for the nonspecific and specific DNA
complexes of the Egr-1 zinc-finger protein. RDC 1
DNH induced with 8 mg/ml Pf1 phage as a molecular alignment medium were analyzed. The main principal axis and
the magnitude Da of the alignment tensor for individual zinc fingers are also shown. (C) Dynamic equilibrium between the recognition and search modes. The search
mode facilitates translocation of the protein on DNA. The coarse-grained molecular dynamics simulations also showed the dynamic transitions between these states
[13,44]. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
J. Iwahara et al. Methods 148 (2018) 57–66
61
typically the case when DNA duplexes containing a high-affinity site are
used), the 15
N z-exchange method or the real-time approach can be
used to analyze kinetics of intermolecular translocations between two
DNA duplexes (Section 5.3).
5.2. Kinetics of protein translocation on nonspecific DNA
Protein translocation on nonspecific DNA occurs through sliding,
dissociation & re-association, and intersegment transfer, typically in the
fast exchange regime. A discrete-state kinetics model shown in Fig. 6A
can represent these translocation processes. This model assumes that a
nonspecific DNA duplex contains N distinct sites for binding. For example,
for 24-bp DNA and a protein that covers 9 bp at each site, N is
calculated to be 16 (=24 − 9 + 1). Due to the structural pseudo-C2
symmetry of each DNA site, two opposite orientations are possible for a
protein to bind to each site through short-range electrostatic interactions
with the DNA backbone. Each nonspecific site is assumed to exhibit
the same kinetic properties with the same rate constants for sliding
(ks), dissociation (koff), association (kon), and intersegment transfer (kit).
These kinetic rate constants are microscopic rate constants defined for
each site (not for the entire DNA molecule). The macroscopic association
rate constant for the DNA duplex is given by 2Nkon, and thus koff/
(2Nkon) corresponds to the macroscopic apparent dissociation constant
(Kd,app). The sliding rate constant ks is directly related to the macroscopic
one-dimensional diffusion coefficient for sliding (D1) by
D1 = l2
ks, where l represents the distance (3.4 Å) between adjacent sites
along the DNA axis [22]. D1 given in units of bp2
s−1
is equivalent to ks.
Because NMR experiments typically use DNA duplexes shorter than the
persistence length (i.e., ∼150 bp), intersegment transfers within the
same DNA duplex are neglected, and only those between two DNA
duplexes are considered. The kit constant is a second-order rate constant
for this type of intermolecular intersegment transfer [22].
In the mixture approach for kinetic analysis of these translocation
processes on nonspecific DNA [48,89], NMR line shapes of protein
backbone NH groups are analyzed for three samples: two containing
individual nonspecific complexes with DNA duplexes a and b and one
containing a mixture of the two nonspecific complexes. Some examples
of actual experimental data are shown in Fig. 6B. This NMR approach
provides accurate kinetic information on the intermolecular translocations
of proteins between two DNA molecules, although this approach
adopts simple two-state approximation. The validity of this approximation
has been confirmed in a recent study using more rigorous
McConnell equations that account for 4N + 1 microscopic states for a
system containing a protein and two nonspecific DNA duplexes [89].
The rate constants koff and kit can be determined through analysis of
apparent transverse relaxation rates from resonance line-shapes as a
function of DNA concentration. Interestingly, the same analysis also
provides semi-quantitative information on the rate constant ks and the
one-dimensional diffusion coefficient D1 for protein sliding on DNA
[89]. By the mixture approach, protein translocation on nonspecific
DNA was analyzed for Egr-1, HoxD9, and Sox2 proteins [13,48,51,89].
5.3. Kinetics of protein translocation between high-affinity sites on DNA
Since the residence time of protein at a high-affinity site is far longer
than that at nonspecific sites, protein translocation between two highaffinity
sites typically occurs in the slow exchange regime. If protein
translocation between two DNA duplexes a and b occurs with an exchange
rate constant kex (= kab + kba) being roughly ∼0.5–50 s−1
, the
15
N z-exchange method [88,91,92] can be used to determine the rate
constants for translocation from DNA a to b (kab) and that for translocation
from DNA b to a (kba). This method has been applied to intermolecular
translocation of proteins between high-affinity sites on two
different DNA duplexes [79,86–88,90,93,94] and also to intramolecular
translocation between two high-affinity sites on the same DNA duplex
[95]. In a 15
N z-exchange experiment, in addition to the signals from
the two complexes, signals are observed at the mixed positions with the
1
H resonance of the complex a and the 15
N resonance of the complex b
(and vice versa; see Fig. 7A), which are called ‘exchange cross peaks.’
These additional cross peaks arise due to protein translocation between
the two DNA duplexes a and b during the mixing time period in which
15
N nuclear magnetizations of interest remain along z, the direction of
the outer magnetic field. Because the mixing time is present between
the 15
N and 1
H evolution periods in the 15
N z-exchange experiment,
some proteins interact with DNA a during the 15
N evolution period and
interact with DNA b during the 1
H evolution period, causing the exchange
cross peak with the 15
N resonance of the complex a and 1
H
resonance of the complex b. By analyzing signal intensities of the exchange
cross peaks and the auto cross peaks as a function of the mixing
time, the rate constants kab and kba for protein translocation processes
between the two high-affinity sites can be determined. This translocation
can occur through the dissociation & re-association mechanism or
through the intersegment transfer mechanisms. By measuring the kab
and kba rate constants at some different concentrations of free DNA
duplexes, the kinetic contributions of these two mechanisms can be
determined individually [87].
If the residence time of a protein on a high-affinity site on DNA is on
the order of minutes or longer, the kinetics of translocation can be
analyzed using a real-time NMR approach [96]. In this approach, a
sample of protein-DNA complex is initially prepared. Then, another
DNA of comparable affinity is added to the solution and the series of 2D
NMR spectra are recorded to monitor translocation of the protein from
the original DNA to the added DNA. By analyzing intensities of signals
from the two complexes as a function of time, one can determine the
Fig. 5. The mixture approach for NMR-based investigations of kinetics of protein
translocation on DNA.
J. Iwahara et al. Methods 148 (2018) 57–66
62
kinetic rate constant for translocation between the two DNA duplexes.
Fast acquisition methods (e.g. SOFAST-HMQC) [97–99], which allow
recording of each 2D 1
H–15
N heteronuclear correlation spectrum within
a minute, are useful to shorten the time interval for the real-time kinetics
measurements. Translocation of the Egr-1 zinc-finger protein
between two target sites was analyzed using NMR in this manner [96].
It should be noted that slow translocation processes over minutes-hours
could readily be measured by biochemical assays requiring far less
amounts of proteins and DNA. Nonetheless, when the translocation
process turned out to be too slow to analyze with the 15
N z-exchange
method, NMR-based real-time kinetics approach could be convenient
because the same set of materials can be readily tested.
5.4. Self-decoupling of intermolecular hydrogen-bond scalar couplings
The above-mentioned NMR methods for kinetic analysis of protein
translocation on DNA require use of two DNA duplexes. Recently, a
unique NMR-based kinetic approach that does not require two DNA
duplexes has been proposed [94]. This NMR method utilizes intermolecular
hydrogen-bond scalar couplings (h3
JNP) between protein 15
N
and DNA 31
P nuclei. Ionic-strength dependence of h3
JNP could provide
information on the residence time of protein at a high-affinity site on
DNA [94]. This method does not require a mixture of two DNA duplexes.
The observation of intermolecular hydrogen-bond scalar couplings
is possible only if the residence time is sufficiently long
(> ∼10−2
s); otherwise, this coupling disappears through the process
called self-decoupling (Fig. 8A). Qualitatively, when an intermolecular
hydrogen-bond scalar coupling is observed with a magnitude comparable
to that of the intrinsic coupling constant h
J, the residence time of
the complex should be longer than (2π|h
J|)−1
. The intrinsic values of
hydrogen-bond scalar couplings can be calculated from structural information
by quantum chemical calculations or from the empirical relationship
between the coupling constants and the hydrogen-bond
geometry [100–102]. Detailed analysis of the self-decoupling of intermolecular
hydrogen-bond scalar couplings as a function of ionic
strength can provide more quantitative information about the residence
time of the complex. For the Antp homeodomain, the exchange rates
measured with the self-decoupling-based method were in good agreement
with those measured with the 15
N z-exchange methods (Fig. 8B)
[94]. This self-decoupling-based method is unique in that it does not
require different signatures for the states involved in the exchange,
although such conditions are typically crucial for other methods.
Fig. 6. NMR-based analysis of kinetics of protein translocation on nonspecific DNA. (A) A discrete-state kinetic model for protein translocation on nonspecific DNA
[89]. Note that the rate constants kon and kit are defined as microscopic rate constants for each site. The corresponding macroscopic rate constants are 2Nkon and
2Nkit. (B, C) Mixture approach data on HoxD9 translocation on nonspecific DNA. Panel B shows overlaid spectra recorded for the individual complexes and their
mixture. Panel C shows determination of the rate constants koff, kit, and ks from the apparent transverse relaxation rates of the three samples as a function of the
concentration of free DNA [89].
Fig. 7. 15
N z-exchange data on kinetics of translocation of the Antp homeodomain
between the two high-affinity sites on the DNA duplexes a and b [94].
(A) A 1
H–15
N spectrum recorded in 15
N z-exchange experiment. (B) Intensities
of the auto and exchange cross peaks as a function of the z-exchange mixing
time. The rate constants determined from this dataset are also shown.
J. Iwahara et al. Methods 148 (2018) 57–66
63
6. NMR instruments
The above-mentioned NMR methods require a high-field NMR
spectrometer (a magnetic field higher than 11 Tesla; 1
H frequency
≥500 MH) equipped with a multi-channel probe capable of heteronuclear
multidimensional experiments. Some quantitative NMR
methods require data collection at multiple magnetic fields (e.g., 1
H
frequencies at 600 and 800 MHz). We use Bruker Avance III NMR
spectrometers operated at 1
H frequencies of 800, 750, and 600 MHz.
Cryogenic 1
H, 13
C, 15
N triple-resonance TCI (800 and 750 MHz) or 1
H,
13
C, 15
N, 31
P quadruple-resonance QCI (600 MHz) probes are typically
used for these spectrometers. Cryogenic probes achieve ∼3-fold higher
signal-to-noise ratio in 1
H or 13
C NMR detection by cooling the detector
to a cryogenic temperature for reduction in thermal noise, while retaining
a physiological temperature (typically, 2–40 °C) for measured
samples. This high sensitivity is helpful for quantitative NMR methods
for investigating target DNA search processes of proteins. The 1
H, 13
C,
15
N, 31
P QCI cryogenic probe is particularly useful for studying proteinDNA
interactions because it allows for precise measurements of intermolecular
hydrogen-bond scalar couplings between DNA 31
P and protein
15
N nuclei [31,32,42,44,94,103,104]. Although this cryogenic
probe has an additional coil for 31
P nuclei, its sensitivity is almost as
good as that of a corresponding TCI cryogenic probe (lower only by
11%, which can readily be compensated by a slightly larger number of
scans).
7. Combining with other methods
Interpretation of NMR data on the target DNA search process can be
greatly facilitated by integrating with other methods. In our lab, we
routinely combine NMR and stopped-flow fluorescence data on the
target DNA search processes. Our stopped-flow fluorescence kinetic
methods allow us to obtain kinetic information on protein translocation
on DNA via sliding, dissociation & re-association, and intersegment
transfer mechanisms [22,23]. The kinetic results from the NMR and
fluorescence studies were consistent for the Egr-1 zinc-finger protein
[13,23]. The fluorescence data suggested that the Egr-1 zinc-finger
protein spends ∼1–10 μs at each nonspecific site and then slides to an
adjacent site. This was also consistent with NMR observation that
protein translocation on nonspecific DNA occurs in the fast exchange
regime. The NMR and fluorescence methods can be applicable under
the same buffer conditions and data obtained with these methods are
complementary. Computational studies are also complementary. The
coarse-grained molecular dynamics simulations showed that the search
mode of Egr-1 facilitates intersegment transfer between nonspecific
DNA duplexes (see Fig. 4C) [13]. Applying these experimental and
computational methods to the Egr-1 zinc-finger protein, we were able
to validate the theoretical model involving the dynamic conformational
equilibrium between the recognition and search modes during the
target DNA search process [13,44]. This model was originally proposed
by some theoretical researchers to explain how transcription factors can
simultaneously achieve two opposing factors: highly specific binding
and sufficiently rapid search [105–107]. Through mutagenesis, we
modulated the dynamic conformational equilibrium between the search
and recognition modes and directly assessed the conformational shifts
using NMR spectroscopy [44]. Using fluorescence and biochemical assays,
we analyzed how the shifts of the conformational equilibrium
influence binding affinity, target search kinetics, and efficiency in displacing
other proteins from the target sites. A shift toward the recognition
mode caused an increase in affinity for DNA and a decrease in
search efficiency. In contrast, a shift toward the search mode caused a
decrease in affinity and an increase in search efficiency. This demonstrated
that target search by these proteins can be accelerated via engineering
based on structural dynamic knowledge of the DNA-scanning
process. Thus, NMR spectroscopy can help us deepen our knowledge of
target DNA search by proteins and apply the knowledge to engineer
proteins that can find targets more efficiently for artificial gene regulation
or genome editing.
Acknowledgements
This work was supported by Grant R01-GM107590 from the
National Institutes of Health (to J.I.). We thank Ross Luu for proofreading.
We also appreciate various supports from the Sealy Center for
Structural Biology and Molecular Biophysics.
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