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1.
J Chem Phys ; 159(16)2023 Oct 28.
Article in English | MEDLINE | ID: mdl-37870142

ABSTRACT

Enzymes accelerate the rates of biomolecular reactions by many orders of magnitude compared to bulk solution, and it is widely understood that this catalytic effect arises from a combination of polar pre-organization and electrostatic transition state stabilization. A number of recent reports have also implicated ultrafast (femtosecond-picosecond) timescale motions in enzymatic activity. However, complications arising from spatially-distributed disorder, the occurrence of multiple substrate binding modes, and the influence of hydration dynamics on solvent-exposed active sites still confound many experimental studies. Here we use ultrafast two-dimensional infrared (2D IR) spectroscopy and covalently-tethered substrate analogs to examine dynamical properties of the promiscuous Pyrococcus horikoshii ene-reductase (PhENR) active site in two binding configurations mimicking proposed "inactive" and "reactive" Michaelis complexes. Spectral diffusion measurements of aryl-nitrile substrate analogs reveal an end-to-end tradeoff between fast (sub-ps) and slow (>5 ps) motions. Fermi resonant aryl-azide analogs that sense interactions of coupled oscillators are described. Lineshape and quantum beat analyses of these probes reveal characteristics that correlate with aryl-nitrile frequency fluctuation correlation functions parameters, demonstrating that this anisotropy is an intrinsic property of the water-exposed active site, where countervailing gradients of fast dynamics and disorder in the reactant ground state are maintained near the hydration interface. Our results suggest several plausible factors leading to state-selective rate enhancement and promiscuity in PhENR. This study also highlights a strategy to detect perturbations to vibrational modes outside the transparent window of the mid-IR spectrum, which may be extended to other macromolecular systems.


Subject(s)
Nitriles , Water , Spectrophotometry, Infrared/methods , Catalytic Domain , Anisotropy , Water/chemistry
2.
J Phys Chem A ; 127(42): 8911-8921, 2023 Oct 26.
Article in English | MEDLINE | ID: mdl-37819373

ABSTRACT

The high transition dipole strength of the azide asymmetric stretch makes aryl azides good candidates as vibrational probes (VPs). However, aryl azides have complex absorption profiles due to Fermi resonances (FRs). Understanding the origin and the vibrational modes involved in FRs of aryl azides is critically important toward developing them as VPs for studies of protein structures and structural changes in response to their surroundings. As such, we studied vibrational couplings in 4-azidotoluene and 4-azido-N-phenylmaleimide in two solvents, N,N-dimethylacetamide and tetrahydrofuran, to explore the origin and the effects of intramolecular group and solvent on the FRs of aryl azides using density functional theory (DFT) calculations with the B3LYP functional and seven basis sets, 6-31G(d,p), 6-31+G(d,p), 6-31++G(d,p), 6-311G(d,p), 6-311+G(d,p), 6-311++G(d,p), and 6-311++G(df,pd). Two combination bands consisting of the azide symmetric stretch and another mode form strong FRs with the azide asymmetric stretch for both molecules. The FR profile was altered by replacing the methyl group with maleimide. Solvents change the relative peak position and intensity more significantly for 4-azido-N-phenylmaleimide, which makes it a more sensitive VP. Furthermore, the DFT results indicate that a comparison among the results from different basis sets can be used as a means to predict more reliable vibrational spectra.

3.
J Biol Chem ; 299(5): 104700, 2023 05.
Article in English | MEDLINE | ID: mdl-37059184

ABSTRACT

Ribonucleoproteins (RNPs) comprise one or more RNA and protein molecules that interact to form a stable complex, which commonly involves conformational changes in the more flexible RNA components. Here, we propose that Cas12a RNP assembly with its cognate CRISPR RNA (crRNA) guide instead proceeds primarily through Cas12a conformational changes during binding to more stable, prefolded crRNA 5' pseudoknot handles. Phylogenetic reconstructions and sequence and structure alignments revealed that the Cas12a proteins are divergent in sequence and structure while the crRNA 5' repeat region, which folds into a pseudoknot and anchors binding to Cas12a, is highly conserved. Molecular dynamics simulations of three Cas12a proteins and their cognate guides revealed substantial flexibility for unbound apo-Cas12a. In contrast, crRNA 5' pseudoknots were predicted to be stable and independently folded. Limited trypsin hydrolysis, differential scanning fluorimetry, thermal denaturation, and CD analyses supported conformational changes of Cas12a during RNP assembly and an independently folded crRNA 5' pseudoknot. This RNP assembly mechanism may be rationalized by evolutionary pressure to conserve CRISPR loci repeat sequence, and therefore guide RNA structure, to maintain function across all phases of the CRISPR defense mechanism.


Subject(s)
CRISPR-Cas Systems , RNA, Guide, CRISPR-Cas Systems , RNA , Ribonucleoproteins , Gene Editing , Phylogeny , Ribonucleoproteins/genetics , RNA, Guide, CRISPR-Cas Systems/genetics , Protein Folding
4.
Spectrochim Acta A Mol Biomol Spectrosc ; 267(Pt 2): 120596, 2022 Feb 15.
Article in English | MEDLINE | ID: mdl-34801392

ABSTRACT

Guanine-rich nucleic acid sequences have a tendency to form four-stranded non-canonical motifs known as G-quadruplexes. These motifs may adopt a wide range of structures characterized by size, strand orientation, guanine base conformation, and fold topology. Using three K+-bound model systems, we show that vibrational coupling between guanine C6 = O and ring modes varies between parallel-stranded and antiparallel-stranded G-quadruplexes, and that such structures can be distinguished by comparison of the polarization dependences of cross-peaks in their two-dimensional infrared (2D IR) spectra. Combined with previously defined vibrational frequency trends, this analysis reveals key features of a 30-nucleotide unimolecular variant of the Bcl-2 proximal promoter that are consistent with its reported structure. This study shows that 2D IR spectroscopy is a convenient method for analyzing G-quadruplex structures that can be applied to complex sequences where traditional high-resolution methods are limited by solubility and disorder.


Subject(s)
G-Quadruplexes , Base Sequence , Circular Dichroism , DNA/genetics , Guanine , Nucleic Acid Conformation
5.
Protein Sci ; 30(5): 1072-1080, 2021 05.
Article in English | MEDLINE | ID: mdl-33641228

ABSTRACT

Mitochondrial outer membrane permeabilization, which is a critical step in apoptosis, is initiated upon transmembrane insertion of the C-terminal α-helix (α9) of the proapoptotic Bcl-2 family protein BAX. The isolated α9 fragment (residues 173-192) is also competent to disrupt model membranes, and the structures of its membrane-associated oligomers are of interest in understanding the potential roles of this sequence in apoptosis. Here, we used ultrafast two-dimensional infrared (2D IR) spectroscopy, thioflavin T binding, and transmission electron microscopy to show that the synthetic BAX α9 peptide (α9p) forms amyloid aggregates in aqueous environments and on the surfaces of anionic small unilamellar vesicles. Its inherent amyloidogenicity was predicted by sequence analysis, and 2D IR spectra reveal that vesicles modulate the ß-sheet structures of insoluble aggregates, motivating further examination of the formation or suppression of BAX amyloids in apoptosis.


Subject(s)
Amyloid/chemistry , Protein Multimerization , bcl-2-Associated X Protein/chemistry , Humans , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand
6.
Chembiochem ; 21(19): 2792-2804, 2020 10 01.
Article in English | MEDLINE | ID: mdl-32372560

ABSTRACT

Infrared spectroscopy detects the formation of G-quadruplexes in guanine-rich nucleic acid sequences through shifts in the guanine C=O stretch mode. Here, we use ultrafast 2D infrared (IR) spectroscopy and isotope substitution to show that these shifts arise from vibrational delocalization among stacked G-quartets. This provides a direct measure of the sizes of locally ordered motifs in heterogeneous samples with substantial disordered regions. We find that parallel-stranded, potassium-bound DNA G-quadruplexes are limited to five consecutive G-quartets and 3-4 consecutive layers are preferred for longer polyguanine tracts. The resulting potassium-dependent G-quadruplex assembly landscape reflects the polyguanine tract lengths found in genomes, the ionic conditions prevalent in healthy mammalian cells, and the onset of structural disorder in disease states. Our study describes spectral markers that can be used to probe other G-quadruplex structures and provides insight into the fundamental limits of their formation in biological and artificial systems.


Subject(s)
DNA/chemistry , DNA/chemical synthesis , G-Quadruplexes , Humans , Nucleic Acid Conformation , Particle Size , Spectrophotometry, Infrared
7.
Science ; 353(6303): 1040-1044, 2016 09 02.
Article in English | MEDLINE | ID: mdl-27701114

ABSTRACT

Potassium channels are responsible for the selective permeation of K+ ions across cell membranes. K+ ions permeate in single file through the selectivity filter, a narrow pore lined by backbone carbonyls that compose four K+ binding sites. Here, we report on the two-dimensional infrared (2D IR) spectra of a semisynthetic KcsA channel with site-specific heavy (13C18O) isotope labels in the selectivity filter. The ultrafast time resolution of 2D IR spectroscopy provides an instantaneous snapshot of the multi-ion configurations and structural distributions that occur spontaneously in the filter. Two elongated features are resolved, revealing the statistical weighting of two structural conformations. The spectra are reproduced by molecular dynamics simulations of structures with water separating two K+ ions in the binding sites, ruling out configurations with ions occupying adjacent sites.


Subject(s)
Bacterial Proteins/chemistry , Models, Chemical , Potassium Channels/chemistry , Bacterial Proteins/chemical synthesis , Binding Sites , Carbon Isotopes/chemistry , Crystallography, X-Ray , Isotope Labeling , Molecular Dynamics Simulation , Oxygen Isotopes/chemistry , Potassium Channels/chemical synthesis , Protein Conformation , Sodium/chemistry , Spectrophotometry, Infrared , Water/chemistry
8.
Methods Mol Biol ; 1345: 21-41, 2016.
Article in English | MEDLINE | ID: mdl-26453203

ABSTRACT

This chapter provides protocols for isotope-labeling the human islet amyloid polypeptide (hIAPP or amylin) involved in type II diabetes and γD-crystallin involved in cataract formation. Because isotope labeling improves the structural resolution, these protocols are useful for experiments using Fourier transform infrared (FTIR), two-dimensional infrared (2D IR), and NMR spectroscopies. Our research group specializes in using 2D IR spectroscopy and isotope labeling. 2D IR spectroscopy provides structural information by measuring solvation from 2D diagonal lineshapes and vibrational couplings from cross peaks. Infrared spectroscopy can be used to study kinetics, membrane proteins, and aggregated proteins. Isotope labeling provides greater certainty in the spectral assignment, which enables new structural insights that are difficult to obtain with other methods. For amylin, we provide a protocol for (13)C/(18)O labeling backbone carbonyls at one or more desired amino acids in order to obtain residue-specific structural resolution. We also provide a protocol for expressing and purifying amylin from E. coli, which enables uniform (13)C or (13)C/(15)N labeling. Uniform labeling is useful for measuring the monomer infrared spectrum in an amyloid oligomer or fiber as well as amyloid protein bound to another polypeptide or protein, such as a chaperone or an inhibitor. In addition, our expression protocol results in 2-2.5 mg of amylin peptide per 1 L cell culture, which is a high enough yield to straightforwardly obtain the 2-10 mg needed for high resolution and solid-state NMR experiments. Finally, we provide a protocol to isotope-label either of the two domains of γD-crystallin using expressed protein ligation. Domain labeling makes it possible to resolve the structures of the two halves of the protein in FTIR and 2D IR spectra. With modifications, these strategies and protocols for isotope labeling can be applied to other amyloid polypeptides and proteins.


Subject(s)
Amyloid/genetics , Isotope Labeling/methods , Magnetic Resonance Spectroscopy/methods , Spectroscopy, Fourier Transform Infrared/methods , Amino Acids/genetics , Amyloid/metabolism , Humans , Islet Amyloid Polypeptide/genetics , Islet Amyloid Polypeptide/metabolism , Isotopes/chemistry
9.
J Phys Chem Lett ; 5(11): 1984-1993, 2014 Jun 05.
Article in English | MEDLINE | ID: mdl-24932380

ABSTRACT

There is an enormous amount of interest in the structures and formation mechanisms of amyloid fibers. In this Perspective, we review the most common structural motifs of amyloid fibers and discuss how infrared spectroscopy and isotope labeling can be used to identify their structures and aggregation kinetics. We present three specific strategies, site-specific labeling to obtain residue-by-residue structural information, isotope dilution of uniformly labeled proteins for identifying structural folds and protein mixtures, and expressed protein ligation for studying the domain structures of large proteins. For each of these methods, vibrational couplings are the source of the identifying features in the infrared spectrum. Examples are provided using the proteins hIAPP, Aß, polyglutamine, and γD-crystallin. We focus on FTIR spectroscopy but also describe new observables made possible by 2D IR spectroscopy.

10.
Proc Natl Acad Sci U S A ; 111(16): 5796-801, 2014 Apr 22.
Article in English | MEDLINE | ID: mdl-24550484

ABSTRACT

Polyglutamine (polyQ) sequences are found in a variety of proteins, and mutational expansion of the polyQ tract is associated with many neurodegenerative diseases. We study the amyloid fibril structure and aggregation kinetics of K2Q24K2W, a model polyQ sequence. Two structures have been proposed for amyloid fibrils formed by polyQ peptides. By forming fibrils composed of both (12)C and (13)C monomers, made possible by protein expression in Escherichia coli, we can restrict vibrational delocalization to measure 2D IR spectra of individual monomers within the fibrils. The spectra are consistent with a ß-turn structure in which each monomer forms an antiparallel hairpin and donates two strands to a single ß-sheet. Calculated spectra from atomistic molecular-dynamics simulations of the two proposed structures confirm the assignment. No spectroscopically distinct intermediates are observed in rapid-scan 2D IR kinetics measurements, suggesting that aggregation is highly cooperative. Although 2D IR spectroscopy has advantages over linear techniques, the isotope-mixing strategy will also be useful with standard Fourier transform IR spectroscopy.


Subject(s)
Amyloid/metabolism , Peptides/chemistry , Peptides/metabolism , Amino Acid Motifs , Animals , Carbon Isotopes , Models, Molecular , Optical Phenomena , Porphyrins/metabolism , Protein Binding , Spectrophotometry, Infrared , Sperm Whale , Spin Labels , Thermodynamics
11.
Protein Sci ; 23(3): 321-31, 2014 Mar.
Article in English | MEDLINE | ID: mdl-24415662

ABSTRACT

The eye lens protein γD-crystallin contributes to cataract formation in the lens. In vitro experiments show that γD-crystallin has a high propensity to form amyloid fibers when denatured, and that denaturation by acid or UV-B photodamage results in its C-terminal domain forming the ß-sheet core of amyloid fibers. Here, we show that thermal denaturation results in sheet-like aggregates that contain cross-linked oligomers of the protein, according to transmission electron microscopy and SDS-PAGE. We use two-dimensional infrared spectroscopy to show that these aggregates have an amyloid-like secondary structure with extended ß-sheets, and use isotope dilution experiments to show that each protein contributes approximately one ß-strand to each ß-sheet in the aggregates. Using segmental (13) C labeling, we show that the organization of the protein's two domains in thermally induced aggregates results in a previously unobserved structure in which both the N-terminal and C-terminal domains contribute to ß-sheets. We propose a model for the structural organization of the aggregates and attribute the recruitment of the N-terminal domain into the fiber structure to intermolecular cross linking.


Subject(s)
Amyloid/chemistry , gamma-Crystallins/chemistry , gamma-Crystallins/radiation effects , Amyloid/radiation effects , Humans , Microscopy, Electron, Transmission , Models, Molecular , Protein Denaturation , Protein Isoforms/chemistry , Protein Isoforms/radiation effects , Protein Structure, Secondary , Spectroscopy, Fourier Transform Infrared , Temperature
12.
Biochemistry ; 52(36): 6169-81, 2013 Sep 10.
Article in English | MEDLINE | ID: mdl-23957864

ABSTRACT

γD-Crystallin is an abundant structural protein of the lens that is found in native and modified forms in cataractous aggregates. We establish that UV-B irradiation of γD-Crystallin leads to structurally specific modifications and precipitation via two mechanisms: amorphous aggregates and amyloid fibers. UV-B radiation causes cleavage of the backbone, in large measure near the interdomain interface, where side chain oxidations are also concentrated. 2D IR spectroscopy and expressed protein ligation localize fiber formation exclusively to the C-terminal domain of γD-Crystallin. The native ß-sandwich domains are not retained upon precipitation by either mechanism. The similarities between the amyloid forming pathways when induced by either UV-B radiation or low pH suggest that the propensity for the C-terminal ß-sandwich domain to form amyloid ß-sheets determines the misfolding pathway independent of the mechanism of denaturation.


Subject(s)
Amyloid/chemistry , Lens, Crystalline/radiation effects , Ultraviolet Rays , gamma-Crystallins/radiation effects , Amino Acid Sequence , Amyloid beta-Peptides/chemistry , Cataract/etiology , Humans , Microscopy, Electron, Transmission , Protein Denaturation , Protein Structure, Quaternary , Protein Structure, Secondary , Spectrophotometry, Infrared , gamma-Crystallins/chemistry
13.
J Phys Chem B ; 117(17): 5009-18, 2013 May 02.
Article in English | MEDLINE | ID: mdl-23537223

ABSTRACT

A series of non-natural infrared probes is reported that consist of a metal-tricarbonyl modified with a -(CH2)n- linker and cysteine-specific leaving group. They can be site-specifically attached to proteins using mutagenesis and similar protocols for EPR spin labels, which have the same leaving group. We characterize the label's frequencies and lifetimes using 2D IR spectroscopy in solvents of varying dielectric. The frequency range spans 10 cm(-1), and the variation in lifetimes ranges from 6 to 19 ps, indicating that these probes are very sensitive to their environments. Also, we attached probes with -(CH2)-, -(CH2)3-, and -(CH2)4- linkers to ubiquitin at positions 6 and 63 and collected spectra in aqueous buffer. The frequencies and lifetimes were correlated for 3C and 4C linkers, as they were in the solvents, but did not correlate for the 1C linker. We conclude that lifetime measures solvation, whereas frequency reflects the electrostatics of the environment, which in the case of the 1C linker is a measure of the protein electrostatic field. We also labeled V71C α-synuclein in buffer and membrane-bound. Unlike most other infrared labels, this label has extremely strong cross sections and thus can be measured with 2D IR spectroscopy at sub-millimolar concentrations. We expect that these labels will find use in studying the structure and dynamics of membrane-bound, aggregated, and kinetically evolving proteins for which high signal-to-noise at low protein concentrations is imperative.


Subject(s)
Solvents/chemistry , Spectroscopy, Fourier Transform Infrared , alpha-Synuclein/chemistry , Electron Spin Resonance Spectroscopy , Mesylates/chemistry , Spin Labels , Static Electricity , Ubiquitin/chemistry , Ubiquitin/metabolism , alpha-Synuclein/metabolism
14.
J Am Chem Soc ; 134(44): 18410-6, 2012 Nov 07.
Article in English | MEDLINE | ID: mdl-23082813

ABSTRACT

Identifying the sequence and structural content of residues that compose the core of amyloid fibrils is important because core regions likely control the process of fibril extension and provide potential drug targets. Human γD-crystallin is an eye lens protein that aggregates into amyloid fibrils under acidic conditions. In this manuscript, we use a pepsin enzymatic digest to isolate the core of the amyloid fibrils. The sequence of the core is identified with MALDI MS/MS and its structure is probed with 2D IR spectroscopy and (13)C isotope labeling. Mass spectrometry of the digest identifies residues 80-163 as the amyloid core, which spans most of the C-terminal domain, the linker, and a small portion of the N-terminal domain. From 2D IR spectroscopy of the digested fibrils, we learn that only the C-terminal domain contributes to the amyloid ß-sheets while the N-terminal and linker residues are disordered. A comparison to the native crystal structure reveals that loops and α-helices in the native state must undergo conformational transitions to ß-strands upon aggregation. These locations may be good drug binding targets. Besides providing new information about γD-crystallin, this study demonstrates the complementarity of mass spectrometry and 2D IR spectroscopy to obtain both sequence and structure information that neither technique provides individually, which will be especially useful for samples only available in microgram quantities.


Subject(s)
Amyloid/chemistry , gamma-Crystallins/chemistry , Amino Acid Sequence , Carbon Isotopes/analysis , Humans , Isotope Labeling , Models, Molecular , Molecular Sequence Data , Protein Structure, Secondary , Protein Structure, Tertiary , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Spectrophotometry, Infrared , Tandem Mass Spectrometry
15.
Proc Natl Acad Sci U S A ; 109(9): 3329-34, 2012 Feb 28.
Article in English | MEDLINE | ID: mdl-22328156

ABSTRACT

The structural eye lens protein γD-crystallin is a major component of cataracts, but its conformation when aggregated is unknown. Using expressed protein ligation, we uniformly (13)C labeled one of the two Greek key domains so that they are individually resolved in two-dimensional (2D) IR spectra for structural and kinetic analysis. Upon acid-induced amyloid fibril formation, the 2D IR spectra reveal that the C-terminal domain forms amyloid ß-sheets, whereas the N-terminal domain becomes extremely disordered but lies in close proximity to the ß-sheets. Two-dimensional IR kinetics experiments show that fibril nucleation and extension occur exclusively in the C-terminal domain. These results are unexpected because the N-terminal domain is less stable in the monomer form. Isotope dilution experiments reveal that each C-terminal domain contributes two or fewer adjacent ß-strands to each ß-sheet. From these observations, we propose an initial structural model for γD-crystallin amyloid fibrils. Because only 1 µg of protein is required for a 2D IR spectrum, even poorly expressing proteins can be studied under many conditions using this approach. Thus, we believe that 2D IR and protein ligation will be useful for structural and kinetic studies of many protein systems for which IR spectroscopy can be straightforwardly applied, such as membrane and amyloidogenic proteins.


Subject(s)
Amyloid/chemistry , gamma-Crystallins/chemistry , Humans , Protein Structure, Secondary , Protein Structure, Tertiary , Spectrophotometry, Infrared
16.
Protein Eng Des Sel ; 21(11): 645-52, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18755707

ABSTRACT

The construction and characteristics of the stable and well-structured alpha(4)W protein are described. The 117-residue, single-chain protein has a molecular weight of 13.1 kDa and is designed to fold into a four-helix bundle. Experimental characterization of the expressed and purified protein shows a 69.8 +/- 0.8% helical content over a 5.5-10.0 pH range. The protein is thermostable with a T(M) > 355 K and has a free energy of unfolding as measured by chemical denaturation of -4.7 kcal mol(-1) at 25 degrees C and neutral pH. One-dimensional (1D) proton and 2D (15)N-HSQC spectra show narrow, well-dispersed spectral lines consistent with a uniquely structured alpha-helical protein. Analytical ultracentrifugation and NMR data show that the protein is monomeric over a broad protein concentration range. The 324 nm emission maximum of the unique Trp-106 is consistent with a sequestered position of the aromatic residue. Additionally, differential pulse voltammetry characterization indicates an elevated peak potential for Trp-106 when the protein is folded (pH range 7.0-8.5) relative to partly unfolded (pH range 11.4-13.2). The oxidation of Trp-106 is coupled to proton release as shown by a 53 +/- 3 mV/pH unit dependence of the peak potential over the 7.0-8.5 pH range.


Subject(s)
Oxidation-Reduction , Protein Structure, Secondary , Protein Structure, Tertiary , Proteins/chemistry , Amino Acid Sequence , Animals , Base Sequence , Cells, Cultured , Circular Dichroism , Gene Expression , Magnetic Resonance Spectroscopy , Protein Binding , Protein Conformation , Protein Folding , Protein Renaturation , Temperature , Thioredoxins/chemistry , Tryptophan/analysis , Ultracentrifugation
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