Regulation of Protein Structural Changes by Incorporation of a Small-Molecule Linker

Proteins have the potential to serve as nanomachines with well-controlled structural movements, and artificial control of their conformational changes is highly desirable for successful applications exploiting their dynamic structural characteristics. Here, we demonstrate an experimental approach for regulating the degree of conformational change in proteins by incorporating a small-molecule linker into a well-known photosensitive protein, photoactive yellow protein (PYP), which is sensitized by blue light and undergoes a photo-induced N-terminal protrusion coupled with chromophore-isomerization-triggered conformational changes. Specifically, we introduced thiol groups into specific sites of PYP through site-directed mutagenesis and then covalently conjugated a small-molecule linker into these sites, with the expectation that the linker is likely to constrain the structural changes associated with the attached positions. To investigate the structural dynamics of PYP incorporated with the small-molecule linker (SML-PYP), we employed the combination of small-angle X-ray scattering (SAXS), transient absorption (TA) spectroscopy and experiment-restrained rigid-body molecular dynamics (MD) simulation. Our results show that SML-PYP exhibits much reduced structural changes during photo-induced signaling as compared to wild-type PYP. This demonstrates that incorporating an external molecular linker can limit photo-induced structural dynamics of the protein and may be used as a strategy for fine control of protein structural dynamics in nanomachines.

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Unraveling the Mechanism of a LOV Domain Optogenetic Sensor: A Glutamine Lever Induces Unfolding of the Jα Helix

10.1101/2020.01.29.925040 ◽

2020 ◽

Author(s):

James N. Iuliano ◽

Jinnette Tolentino Collado ◽

Agnieszka A. Gil ◽

Pavithran T. Ravindran ◽

Andras Lukacs ◽

...

Keyword(s):

Structural Dynamics ◽

Conformational Changes ◽

Interdisciplinary Approach ◽

Site Directed Mutagenesis ◽

Side Chain ◽

Lov Domain ◽

Time Resolved ◽

Protein Structural Dynamics ◽

Dynamics Simulations ◽

Time Resolved Infrared Spectroscopy

AbstractLight-activated protein domains provide a convenient, modular, and genetically encodable sensor for optogenetics and optobiology. Although these domains have now been deployed in numerous systems, the precise mechanism of photoactivation and the accompanying structural dynamics that modulate output domain activity remain to be fully elucidated. In the C-terminal light, oxygen, voltage (LOV) domain of plant phototropins (LOV2), blue light activation leads to formation of an adduct between a conserved Cys residue and the embedded FMN chromophore, rotation of a conserved Gln (Q513), and unfolding of a helix (Jα-helix) which is coupled to the output partner. In the present work, we focus on the allosteric pathways leading to Jα helix unfolding in Avena sativa LOV2 (AsLOV2) using an interdisciplinary approach involving molecular dynamics simulations extending to 7 μs, time-resolved infrared spectroscopy, solution NMR spectroscopy, and in-cell optogenetic experiments. In the dark state, the side chain of N414 is hydrogen bonded to the backbone N-H of Q513. The simulations predict a lever-like motion of Q513 after Cys adduct formation resulting in loss of the interaction between the side chain of N414 and the backbone C=O of Q513, and formation of a transient hydrogen bond between the Q513 and N414 side chains. The central role of N414 in signal transduction was evaluated by site-directed mutagenesis supporting a direct link between Jα helix unfolding dynamics and the cellular function of the Zdk2-AsLOV2 optogenetic construct. Through this multifaceted approach, we show that Q513 and N414 are critical mediators of protein structural dynamics, linking the ultrafast (sub-ps) excitation of the FMN chromophore to the microsecond conformational changes that result in photoreceptor activation and biological function.

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Photoactivation mechanism of a carotenoid-based photoreceptor

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1700956114 ◽

2017 ◽

Vol 114 (24) ◽

pp. 6286-6291 ◽

Cited By ~ 38

Author(s):

Sepalika Bandara ◽

Zhong Ren ◽

Lu Lu ◽

Xiaoli Zeng ◽

Heewhan Shin ◽

...

Keyword(s):

Conformational Changes ◽

Structural Changes ◽

Direct Interaction ◽

Water Soluble ◽

Site Directed Mutagenesis ◽

Photochemical Property ◽

Direct Observations ◽

Absorbing State ◽

Molecular Events ◽

Orange Carotenoid Protein

Photoprotection is essential for efficient photosynthesis. Cyanobacteria have evolved a unique photoprotective mechanism mediated by a water-soluble carotenoid-based photoreceptor known as orange carotenoid protein (OCP). OCP undergoes large conformational changes in response to intense blue light, and the photoactivated OCP facilitates dissipation of excess energy via direct interaction with allophycocyanins at the phycobilisome core. However, the structural events leading up to the OCP photoactivation remain elusive at the molecular level. Here we present direct observations of light-induced structural changes in OCP captured by dynamic crystallography. Difference electron densities between the dark and illuminated states reveal widespread and concerted atomic motions that lead to altered protein–pigment interactions, displacement of secondary structures, and domain separation. Based on these crystallographic observations together with site-directed mutagenesis, we propose a molecular mechanism for OCP light perception, in which the photochemical property of a conjugated carbonyl group is exploited. We hypothesize that the OCP photoactivation starts with keto–enol tautomerization of the essential 4-keto group in the carotenoid, which disrupts the strong hydrogen bonds between the bent chromophore and the protein moiety. Subsequent structural changes trapped in the crystal lattice offer a high-resolution glimpse of the initial molecular events as OCP begins to transition from the orange-absorbing state to the active red-absorbing state.

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Protein Structural Dynamics of Photoactive Yellow Protein in Solution Revealed by Pump–Probe X-ray Solution Scattering

Journal of the American Chemical Society ◽

10.1021/ja210435n ◽

2012 ◽

Vol 134 (6) ◽

pp. 3145-3153 ◽

Cited By ~ 80

Author(s):

Tae Wu Kim ◽

Jae Hyuk Lee ◽

Jungkweon Choi ◽

Kyung Hwan Kim ◽

Luuk J. van Wilderen ◽

...

Keyword(s):

Structural Dynamics ◽

Photoactive Yellow Protein ◽

Pump Probe ◽

X Ray ◽

Protein Structural Dynamics

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Structural insights into SETD3-mediated histidine methylation on β-actin

10.1101/476705 ◽

2018 ◽

Author(s):

Qiong Guo ◽

Shanhui Liao ◽

Sebastian Kwiatkowski ◽

Weronika Tomaka ◽

Huijuan Yu ◽

...

Keyword(s):

Pulmonary Hypertension ◽

Enzyme Activity ◽

Small Molecule ◽

Conformational Changes ◽

Catalytic Mechanism ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Domain Protein ◽

First Time ◽

Structural Insights

SETD3 is a member of SET (Su(var)3-9, Enhancer of zeste, and Trithorax) domain protein superfamily and plays important roles in hypoxic pulmonary hypertension, muscle differentiation, and carcinogenesis. Recently, we have identified SETD3 as the actin-specific methyltransferase that methylates the N3 of His73 on β-actin. Here we present two structures of S-adenosyl-L-homocysteine-bound SETD3 in complex with either an unmodified β-actin peptide or its His-methylated variant. Structural analyses supported by the site-directed mutagenesis experiments and the enzyme activity assays indicated that the recognition and methylation of β-actin by SETD3 is highly sequence specific, and both SETD3 and β-actin adopt pronounce conformational changes upon binding to each other. In conclusion, the data show for the first time a catalytic mechanism of SETD3-mediated histidine methylation in β-actin, which not only throws light on protein histidine methylation phenomenon, but also facilitates the design of small molecule inhibitors of SETD3.

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Structure of HIV-1 RT/dsRNA initiation complex prior to nucleotide incorporation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1814170116 ◽

2019 ◽

Vol 116 (15) ◽

pp. 7308-7313 ◽

Cited By ~ 10

Author(s):

Kalyan Das ◽

Sergio E. Martinez ◽

Jeffrey J. DeStefano ◽

Eddy Arnold

Keyword(s):

Conformational Changes ◽

Reverse Transcription ◽

Transcription Initiation ◽

Structural Changes ◽

Structural Characteristics ◽

Primer Binding Site ◽

Energetic Cost ◽

Initiation Complex ◽

Nucleotide Incorporation ◽

Hiv 1

The initiation phase of HIV reverse transcription has features that are distinct from its elongation phase. The first structure of a reverse transcription initiation complex (RTIC) that trapped the complex after incorporation of one ddCMP nucleotide was published recently [Larsen KP, et al. (2018)Nature557:118–122]. Here we report a crystal structure of a catalytically active HIV-1 RT/dsRNA complex that mimics the state of the RTIC before the first nucleotide incorporation. The structure reveals that the dsRNA-bound conformation of RT is closer to that of RT bound to a nonnucleoside RT inhibitor (NNRTI) and dsDNA; a hyperextended thumb conformation helps to accommodate the relatively wide dsRNA duplex. The RNA primer 3′ end is positioned 5 Å away from the polymerase site; however, unlike in an NNRTI-bound state in which structural elements of RT restrict the movement of the primer, the primer terminus of dsRNA is not blocked from reaching the active site of RT. The observed structural changes and energetic cost of bringing the primer 3′ end to the priming site are hypothesized to explain the slower nucleotide incorporation rate of the RTIC. An unusual crystal lattice interaction of dsRNA with its symmetry mate is reminiscent of the RNA architecture within the extended vRNA–tRNALys3in the RTIC. This RT/dsRNA complex captures the key structural characteristics and components of the RTIC, including the RT conformational changes and interactions with the dsRNA primer-binding site region, and these features have implications for better understanding of RT initiation.

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ClpP protease activation results from the reorganization of the electrostatic interaction networks at the entrance pores

Communications Biology ◽

10.1038/s42003-019-0656-3 ◽

2019 ◽

Vol 2 (1) ◽

Cited By ~ 7

Author(s):

Mark F. Mabanglo ◽

Elisa Leung ◽

Siavash Vahidi ◽

Thiago V. Seraphim ◽

Bryan T. Eger ◽

...

Keyword(s):

Small Molecule ◽

Conformational Changes ◽

Electrostatic Interaction ◽

Structural Changes ◽

Structural Heterogeneity ◽

Interaction Networks ◽

Structural Basis ◽

X Ray ◽

X Ray Crystallography ◽

Bacterial Cell Death

Abstract Bacterial ClpP is a highly conserved, cylindrical, self-compartmentalizing serine protease required for maintaining cellular proteostasis. Small molecule acyldepsipeptides (ADEPs) and activators of self-compartmentalized proteases 1 (ACP1s) cause dysregulation and activation of ClpP, leading to bacterial cell death, highlighting their potential use as novel antibiotics. Structural changes in Neisseria meningitidis and Escherichia coli ClpP upon binding to novel ACP1 and ADEP analogs were probed by X-ray crystallography, methyl-TROSY NMR, and small angle X-ray scattering. ACP1 and ADEP induce distinct conformational changes in the ClpP structure. However, reorganization of electrostatic interaction networks at the ClpP entrance pores is necessary and sufficient for activation. Further activation is achieved by formation of ordered N-terminal axial loops and reduction in the structural heterogeneity of the ClpP cylinder. Activating mutations recapitulate the structural effects of small molecule activator binding. Our data, together with previous findings, provide a structural basis for a unified mechanism of compound-based ClpP activation.

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Photocage-initiated time-resolved solution X-ray scattering investigation of protein dimerization

IUCrJ ◽

10.1107/s2052252518012149 ◽

2018 ◽

Vol 5 (6) ◽

pp. 667-672 ◽

Cited By ~ 6

Author(s):

Inokentijs Josts ◽

Stephan Niebling ◽

Yunyun Gao ◽

Matteo Levantino ◽

Henning Tidow ◽

...

Keyword(s):

Small Molecule ◽

Conformational Changes ◽

Structural Changes ◽

Time Resolved ◽

Protein Dimerization ◽

X Ray ◽

Single Turnover ◽

X Ray Scattering ◽

Photoactivatable Proteins ◽

Ray Scattering

This work demonstrates a new method for investigating time-resolved structural changes in protein conformation and oligomerization via photocage-initiated time-resolved X-ray solution scattering by observing the ATP-driven dimerization of the MsbA nucleotide-binding domain. Photocaged small molecules allow the observation of single-turnover reactions of non-naturally photoactivatable proteins. The kinetics of the reaction can be derived from changes in X-ray scattering associated with ATP-binding and subsequent dimerization. This method can be expanded to any small-molecule-driven protein reaction with conformational changes traceable by X-ray scattering where the small molecule can be photocaged.

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Protein Structural Dynamics of Wild-Type and Mutant Homodimeric Hemoglobin Studied by Time-Resolved X-Ray Solution Scattering

International Journal of Molecular Sciences ◽

10.3390/ijms19113633 ◽

2018 ◽

Vol 19 (11) ◽

pp. 3633 ◽

Cited By ~ 2

Author(s):

Cheolhee Yang ◽

Minseo Choi ◽

Jong Kim ◽

Hanui Kim ◽

Srinivasan Muniyappan ◽

...

Keyword(s):

Structural Dynamics ◽

Conformational Changes ◽

Structural Transition ◽

Quaternary Structure ◽

Heme Binding ◽

Wild Type ◽

Time Resolved ◽

X Ray ◽

Protein Structural Dynamics ◽

Cooperative Transition

The quaternary transition between the relaxed (R) and tense (T) states of heme-binding proteins is a textbook example for the allosteric structural transition. Homodimeric hemoglobin (HbI) from Scapharca inaequivalvis is a useful model system for investigating the allosteric behavior because of the relatively simple quaternary structure. To understand the cooperative transition of HbI, wild-type and mutants of HbI have been studied by using time-resolved X-ray solution scattering (TRXSS), which is sensitive to the conformational changes. Herein, we review the structural dynamics of HbI investigated by TRXSS and compare the results of TRXSS with those of other techniques.

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BODIPY Dyes as Probes and Sensors to Study Amyloid-β-Related Processes

Biosensors ◽

10.3390/bios10120192 ◽

2020 ◽

Vol 10 (12) ◽

pp. 192

Author(s):

Sergei V. Dzyuba

Keyword(s):

Small Molecule ◽

Conformational Changes ◽

Self Assembly ◽

Structural Changes ◽

Photophysical Properties ◽

Amyloid Β ◽

Amyloid Formation ◽

Diverse Range ◽

Aβ Peptides ◽

Underlying Mechanisms

Amyloid formation plays a major role in a number of neurodegenerative diseases, including Alzheimer’s disease. Amyloid-β peptides (Aβ) are one of the primary markers associated with this pathology. Aβ aggregates exhibit a diverse range of morphologies with distinct pathological activities. Recognition of the Aβ aggregates by using small molecule-based probes and sensors should not only enhance understanding of the underlying mechanisms of amyloid formation, but also facilitate the development of therapeutic strategies to interfere with amyloid neurotoxicity. BODIPY (boron dipyrrin) dyes are among the most versatile small molecule fluorophores. BODIPY scaffolds could be functionalized to tune their photophysical properties to the desired ranges as well as to adapt these dyes to various types of conditions and environments. Thus, BODIPY dyes could be viewed as unique platforms for the design of probes and sensors that are capable of detecting and tracking structural changes of various Aβ aggregates. This review summarizes currently available examples of BODIPY dyes that have been used to investigate conformational changes of Aβ peptides, self-assembly processes of Aβ, as well as Aβ interactions with various molecules.

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Ultrafast base rotations mediate a solvent-assisted back-electron transfer in UV-excited DNA single strands

10.26434/chemrxiv-2021-5s5j8 ◽

2021 ◽

Author(s):

Benjamin Bauer ◽

Rahul Sharma ◽

Majed Chergui ◽

Malte Oppermann

Keyword(s):

Electron Transfer ◽

Structural Dynamics ◽

Conformational Changes ◽

Transient Absorption ◽

Relaxation Mechanism ◽

Polarization Sensitivity ◽

Back Electron Transfer ◽

Deep Uv ◽

Photochemical Systems ◽

Experimental Challenge

The photochemistry of DNA systems is characterized by the ultraviolet (UV) absorption of π-stacked nucleobases, resulting in exciton states delocalized over several bases. As their relaxation sensitively depends on local stacking conformations, disentangling the ensuing electronic and structural dynamics has remained an experimental challenge, despite their fundamental role in protecting the genome from potentially harmful UV radiation. Here we use transient absorption and transient absorption anisotropy spectroscopy with broadband femtosecond deep-UV pulses (250-360 nm) to resolve the exciton dynamics of UV-excited adenosine single strands under physiological conditions. Due to the exceptional deep-UV bandwidth and polarization sensitivity of our experimental approach, we simultaneously resolve the population dynamics, charge-transfer (CT) character and conformational changes encoded in the UV transition dipoles of the π-stacked nucleotides. Whilst UV excitation forms fully charge-separated CT excitons in less than 0.3 ps, we find that most decay back to the ground state via a solvent-assisted back-electron transfer. This deactivation mechanism is accompanied by a structural relaxation of the photoexcited base-stack, which we identify as an inter-base rotation of the nucleotides. Our results finally complete the exciton relaxation mechanism for adenosine single strands and offer a direct view into the coupling of electronic and structural dynamics in aggregated photochemical systems.

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