Site-specific Isotope Labeling Research Articles

Conformational transition of a hairpin structure to G-quadruplex within the WNT1 gene promoter.

The role of G-quadruplexes (G4s) in biological systems has been widely studied. It is found that they have an important function in gene transcription and regulation. In this work, we have identified two topologies of hairpin and G4 structures formed by a native G-rich sequence (WT22: 5'-GGGCCACCGGGCAGGGGGCGGG-3') from the WNT1 promoter region using nuclear magnetic resonance (NMR) spectroscopy. With the help of site-specific isotope labeling, the topologies of these two structures are unambiguously characterized. Circular dichroism and NMR results are analyzed to determine the kinetics associated with the potassium ion-induced hairpin-to-G4 transition, which is very slow-on the time scale of 4800 s-compared to the previously reported folding kinetics of G4 formation. In addition, the free energies of the unfolding of these two structures are obtained using differential scanning calorimetry. Combining the kinetic and thermodynamic data, we have established the free energy landscape of this two-state folding system. Considering that similar conformational change may exist in other native G-rich sequences, this work highlights an important hairpin to G4 conformational transition which can be used in manipulation of gene regulation or ligand modulation in vivo.

Chemical synthesis and biophysical applications of membrane proteins.

Chemical synthesis or semi-synthesis of membrane proteins can provide unique molecular tools, such as site-specific isotope labeling or post-translationally modified membrane proteins to gain insight into their biophysical and functional characteristics. However, during preparation, purification, and ligation of transmembrane peptides, tremendous challenges are encountered owing to their hydrophobic nature. This review focuses on the recent advances in chemical synthesis strategies of membrane proteins. These strategies help to solubilize the hydrophobic transmembrane peptide sequences under standard purification and chemical ligation conditions to improve their handling properties. Biophysical and functional studies of synthetic membrane proteins are reviewed as well.

ChemInform Abstract: Recent Advances in Solid‐State Nuclear Magnetic Resonance Techniques to Quantify Biomolecular Dynamics

AbstractReview: 52 refs.

Recent advances in solid-state nuclear magnetic resonance techniques to quantify biomolecular dynamics.

The emergence of solid-state NMR (SSNMR) techniques over the last decade has greatly expanded the range of accessible biomolecules amenable to structural study.1-2 These methods were developed and first applied to a variety of microcrystalline model proteins including BPTI,3 SH3,4-5 ubiquitin,6-7 kaliotoxin,8 and GB1.9-11 These studies demonstrated that individual 13C, 15N and 1H sites throughout the entire protein could be uniquely resolved and assigned for purposes of high-resolution structure determination. Whereas traditional solid-state NMR approaches required site-specific isotopic labeling, the new developments enable entire proteins to be examined by resolving signals in multiple dimensions. These capabilities also permit the measurement of dynamic parameters through correlation spectroscopy, which increases the throughput, sensitivity, reliability and reproducibility of such measurements. A major advantage of SSNMR is that a large range of sample conditions and types can be examined, including many that are not accessible to solution NMR methods, such as membrane proteins and fibrils in addition to microcrystals. Recent examples include fibrils of the HET-s prion,12 high molecular weight oligomers of αB crystallin,13 the tetrameric assembly of the M2 peptide with bound amantadine drug in physiologically relevant bilayers,14 the trimeric membrane protein YadA,15 the photoreceptor proteorhodopsin,16 fibrils of the core domain of transthyretin,17 and beta-amyloid plaques derived from the brains of Alzheimer's disease patients.18 Such studies have opened up new avenues for exploration of fundamental biophysics and biochemistry, and yielded insights into clinically relevant events in membranes and aggregated states not accessible to solution NMR or X-ray crystallography. Moreover, SSNMR experiments are applicable over a larger range of temperatures than can be accessed by solution NMR19 and thereby enable a continuum of experimental conditions from physiological temperatures to the cryogenic temperatures at which most crystal structures are solved. Another advantage of NMR methods in general is that they are non-perturbing and do not require covalent modification of the protein. Thus, the dynamic information procured from such experiments has the potential to accurately represent the native protein behavior. This review focuses on developments over the last five years, regarding methods and applications of dynamics studies by solid-state NMR over timescales ranging from nanoseconds to days.

Simulation of the Amide I Infrared Spectrum in Photoinduced Peptide Folding/Unfolding Transitions

The amide I' infrared spectrum of a α-helical photoswitchable peptide is calculated here by means of a mixed quantum mechanics/molecular dynamics theoretical-computational methodology based on the perturbed matrix method (PMM). The contribution of specific residues to the total spectrum is also analyzed and the results compared to previous experimental spectroscopic data, obtained by means of site-specific isotope labeling at different residues, resulting in good agreement. One of the residues (Ala7) shows atypical spectroscopic behavior in both the experimental and calculated spectra, i.e., the folded-state amide I' band is shifted to higher frequencies than the unfolded-state one, while the other residues show the opposite behavior. The calculations reveal the origin of this uncommon spectroscopic trend and point to a crucial role of the molecular switch, the presence of which perturbs the conformational sampling of the peptide. Indeed, infrared spectra of the same peptide calculated in the absence of the molecular switch show that the single-residue spectrum of Ala7 does not have any distinguishing feature, resembling the spectra of the other analyzed residues.

Effective Site-Specific Isotopic Labeling: Expression Optimization Using Specialized Media

Effective Site-Specific Isotopic Labeling: Expression Optimization Using Specialized Media

Dissection of Hydrogen Bond Interaction Network around an Iron–Sulfur Cluster by Site-Specific Isotope Labeling of Hyperthermophilic Archaeal Rieske-Type Ferredoxin

The electronic structure and geometry of redox-active metal cofactors in proteins are tuned by the pattern of hydrogen bonding with the backbone peptide matrix. In this study we developed a method for selective amino acid labeling of a hyperthermophilic archaeal metalloprotein with engineered Escherichia coli auxotroph strains, and we applied this to resolve the hydrogen bond interactions with the reduced Rieske-type [2Fe-2S] cluster by two-dimensional pulsed electron spin resonance technique. Because deep electron spin-echo envelope modulation of two histidine (14)N(δ) ligands of the cluster decreased non-coordinating (15)N signal intensities via the cross-suppression effect, an inverse labeling strategy was employed in which (14)N amino acid-labeled archaeal Rieske-type ferredoxin samples were examined in an (15)N-protein background. This has directly identified Lys45 N(α) as providing the major pathway for the transfer of unpaired electron spin density from the reduced cluster by a "through-bond" mechanism. All other backbone peptide nitrogens interact more weakly with the reduced cluster. The extension of this approach will allow visualizing the three-dimensional landscape of preferred pathways for the transfer of unpaired spin density from a paramagnetic metal center onto the protein frame, and will discriminate specific interactions by a "through-bond" mechanism from interactions which are "through-space" in various metalloproteins.

Deamidation Accelerates Amyloid Formation and Alters Amylin Fiber Structure

Deamidation of asparagine and glutamine is the most common nonenzymatic, post-translational modification. Deamidation can influence the structure, stability, folding, and aggregation of proteins and has been proposed to play a role in amyloid formation. However there are no structural studies of the consequences of deamidation on amyloid fibers, in large part because of the difficulty of studying these materials using conventional methods. Here we examine the effects of deamidation on the kinetics of amyloid formation by amylin, the causative agent of type 2 diabetes. We find that deamidation accelerates amyloid formation and the deamidated material is able to seed amyloid formation by unmodified amylin. Using site-specific isotope labeling and two-dimensional infrared (2D IR) spectroscopy, we show that fibers formed by samples that contain deamidated polypeptide contain reduced amounts of β-sheet. Deamidation leads to disruption of the N-terminal β-sheet between Ala-8 and Ala-13, but β-sheet is still retained near Leu-16. The C-terminal sheet is disrupted near Leu-27. Analysis of potential sites of deamidation together with structural models of amylin fibers reveals that deamidation in the N-terminal β-sheet region may be the cause for the disruption of the fiber structure at both the N- and C-terminal β-sheet. Thus, deamidation is a post-translational modification that creates fibers that have an altered structure but can still act as a template for amylin aggregation. Deamidation is very difficult to detect with standard methods used to follow amyloid formation, but isotope-labeled IR spectroscopy provides a means for monitoring sample degradation and investigating the structural consequences of deamidation.

Determination of noncovalent docking by infrared spectroscopy of cold gas-phase complexes.

Multidentate, noncovalent interactions between small molecules and biopolymer fragments are central to processes ranging from drug action to selective catalysis. We present a versatile and sensitive spectroscopic probe of functional groups engaged in hydrogen bonding in such contexts. This involves measurement of the frequency changes in specific covalent bonds upon complex formation, information drawn from otherwise transient complexes that have been extracted from solution and conformationally frozen near 10 kelvin in gas-phase clusters. Resonances closely associated with individual oscillators are easily identified through site-specific isotopic labeling, as demonstrated by application of the method to an archetypal system involving a synthetic tripeptide known to bind biaryl substrates through tailored hydrogen bonding to catalyze their asymmetric bromination. With such data, calculations readily converge on the plausible operative structures in otherwise computationally prohibitive, high-dimensionality landscapes.

Site-specific isotope labeling of long RNA for structural and mechanistic studies

A site-specific isotope labeling technique of long RNA molecules was established. This technique is comprised of two simple enzymatic reactions, namely a guanosine transfer reaction of group I self-splicing introns and a ligation with T4 DNA ligase. The trans-acting group I self-splicing intron with its external cofactor, ‘isotopically labeled guanosine 5′-monophosphate’ (5′-GMP), steadily gave a 5′-residue-labeled RNA fragment. This key reaction, in combination with a ligation of 5′-remainder non-labeled sequence, allowed us to prepare a site-specifically labeled RNA molecule in a high yield, and its production was confirmed with 15N NMR spectroscopy. Such a site-specifically labeled RNA molecule can be used to detect a molecular interaction and to probe chemical features of catalytically/structurally important residues with NMR spectroscopy and possibly Raman spectroscopy and mass spectrometry.

High-field EPR

Among the numerous spectroscopic techniques utilized in photosynthesis research, high-field/high-frequency EPR and its pulse extensions ESE, ENDOR, ESEEM, and PELDOR play an important role in the endeavor to understand, on the basis of structure and dynamics data, dominant factors that control specificity and efficiency of light-induced electron- and proton-transfer processes in primary photosynthesis. Short-lived transient intermediates of the photocycle can be characterized by high-field EPR techniques, and detailed structural information can be obtained even from disordered sample preparations. The chapter describes how multifrequency high-field EPR methodology, in conjunction with mutation strategies for site-specific isotope or spin labeling and with the support of modern quantum-chemical computation methods for data interpretation, is capable of providing new insights into the photosynthetic transfer processes. The information obtained is complementary to that of protein crystallography, solid-state NMR and laser spectroscopy.

Two-dimensional IR spectroscopy and isotope labeling defines the pathway of amyloid formation with residue-specific resolution

There is considerable interest in uncovering the pathway of amyloid formation because the toxic properties of amyloid likely stems from prefibril intermediates and not the fully formed fibrils. Using a recently invented method of collecting 2-dimensional infrared spectra and site-specific isotope labeling, we have measured the development of secondary structures for 6 residues during the aggregation process of the 37-residue polypeptide associated with type 2 diabetes, the human islet amyloid polypeptide (hIAPP). By monitoring the kinetics at 6 different labeled sites, we find that the peptides initially develop well-ordered structure in the region of the chain that is close to the ordered loop of the fibrils, followed by formation of the 2 parallel beta-sheets with the N-terminal beta-sheet likely forming before the C-terminal sheet. This experimental approach provides a detailed view of the aggregation pathway of hIAPP fibril formation as well as a general methodology for studying other amyloid forming proteins without the use of structure-perturbing labels.

Expanding the scope of chemoselective peptide ligations in chemical biology

Chemoselective ligation methods have increased the efficiency of bioconjugation, enabling complex macromolecules to be assembled. In particular, these methods have been utilized for the ligation and modification of peptides and proteins. The chemical synthesis of proteins from unprotected peptide fragments has enabled the introduction of unnatural amino acids, site-specific isotopic labeling, and the site-specific attachment of affinity tags or labels for imaging. A greater insight into current ligation methods has led to higher reaction rates, higher reaction yields, and greater biocompatibility, thereby increasing the impact of chemoselective ligation reactions in chemical biology.

Site-Specific Dichroism Analysis Utilizing Transmission FTIR

Infrared spectroscopy has long been used to examine the average secondary structure and orientation of membrane proteins. With the recent utilization of site-specific isotope labeling (e.g., peptidic 1–13C=18O) it is now possible to examine localized properties, rather than global averages. The technique of site-specific infrared dichroism (SSID) capitalized on this fact, and derives site-specific orientational restraints for the labeled amino acids. These restraints can then be used to solve the backbone structure of simple α-helical bundles, emphasizing the capabilities of this approach. So far SSID has been carried out in attenuated total internal reflection optical mode, with all of the respective caveats of attenuated total internal reflection. In this report we extend SSID through the use of transmission infrared spectroscopy tilt series. We develop the corresponding theory and demonstrate that accurate site-specific orientational restraints can be derived from a simple transmission experiment.

Cationic reduction of bastadin-4 to bastadin-5. Preparation of 5-[2h]-bastadin-5 by site-specific isotopic labeling.

A chemoselective conversion of bastadin-4 to the important Ca2+ channel modulator bastadin-5 (1a) has been achieved using cationic hydrogenation (Et3SiH, TFA, 60%). Specifically deuterated bastadin-5 (1b, >95 at. %) was prepared following this method and the simplified 1H NMR H-5/H2-6 spin system of 1b exploited to study temperature-dependent macrocyclic ring dynamics.

Investigation of Molecular Motions by Lee-Goldburg Cross-Polarization NMR Spectroscopy

We demonstrate the use of Lee-Goldburg cross-polarization (LG-CP) NMR under fast magic-angle spinning (MAS) to investigate the amplitude and geometry of segmental motions in biomolecular and polymeric solids. Motional geometry information was previously available only from 2 H NMR, which, however, has limited site resolution and requires site-specific isotopic labeling. Using a 2D LG-CP technique, we resolve the 1 3 C- 1 H or 1 5 N- 1 H dipolar couplings according to the 1 3 C or 1 5 N isotropic chemical shift. Applications to systems undergoing 180° phenylene ring flips show spectral line shapes reflecting the geometry of the motion. Using this LG-CP technique, we measured the 1 3 C- 1 H and 1 5 N- 1 H dipolar couplings in the water-soluble and membrane-bound states of the colicin la channel domain. The backbone motions of the membrane-bound colicin scale both the Ca-Ha and N-H couplings similarly, thus ruling out rotation of the α-helices around their axes as a specific mechanism of motion. We also show that the sensitivity of the LG-CP spectra can be enhanced by the addition of a phase-inverted 1 H- 1 3 C cross-polarization step, and the site resolution of the 1 5 N- 1 H LG-CP spectra can be enhanced by 1 3 C indirect detection.

Site-specific isotope labeling demonstrates a large mesomeric resonance effect of the methoxy groups on the carbonyl frequency in ubiquinones

The splitting of the carbonyl infrared bands of 2-methoxy-1,4-benzoquinone in solution can be related to a mesomeric resonance phenomenon leading to a conformation of the O-CH3 bond coplanar to the quinone ring. The delocalization of the electron density induces a frequency downshift of the C4=O carbonyl compared to 1,4-benzoquinone. This in turns decouples the two carbonyls leading to an upshift of the C1=O vibration. Using selective 13C-labeling of Q0 (2,3-dimethoxy-5-methyl-1,4-benzoquinone), we show that the effect of mesomeric resonance is an essential determinant of the carbonyl frequencies of ubiquinone in solution.

Microscopic pKa values of Escherichia coli thioredoxin.

Thiol:disulfide oxidoreductases have a CXXC motif within their active sites. To initiate the reduction of a substrate disulfide bond, the thiolate form of the N-terminal cysteine residue (CXXC) of this motif performs a nucleophilic attack. Escherichia coli thioredoxin [Trx (CGPC)] is the best characterized thiol:disulfide oxidoreductase. Previous determinations of the active-site pKa values of Trx have led to conflicting interpretations. Here, 13C-NMR spectroscopy, site-specific isotopic labeling, and site-directed mutagenesis were used to demonstrate that analysis of the titration behavior of wild-type Trx requires the invocation of microscopic pKa values for two interacting active-site residues: Asp26 (7.5 and 9.2) and Cys32 (CXXC; 7.5 and 9.2). By contrast, in two Trx variants, D26N Trx and D26L Trx, Cys32 exhibits a pKa near 7.5 and has a well-defined, single-pKa titration curve. Similarly, in oxidized wild-type Trx, Asp26 has a pKa near 7.5. In CVWC and CWGC Trx, Cys32 exhibits a single pKa near 6.2. In all five enzymes studied here, there is no evidence for a Cys35 (CXXC) pKa of < 11. This study demonstrates that a comprehensive approach must be used to unravel complex titration behavior of the functional groups in a protein.

Automated chemical synthesis of biologically active tRNA having a sequence corresponding to Ascaris suum mitochondrial tRNA(Met) toward NMR measurements.

RNA samples corresponding to Ascaris suum mitochondrial tRNA(Met) were chemically and automatically synthesized in amounts sufficient for NMR measurement. Conventional and rapid deprotection methods gave tRNA samples with the same amino acid-accepting activity as those prepared by other method; enzymatic synthesis, and enzymatic ligation of chemically synthesized fragments. The synthetic tRNA showed the same 1H-NMR spectrum in the iminoproton region as the ligated tRNA. This rapid and reliable preparation method thus provides biologically active tRNA for NMR measurement, and further, it is applicable for synthesis of other large synthetic RNAs, by combining the site-specific isotopic labeling method.

Site-specific isotopic labeling of proteins for NMR studies

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTSite-specific isotopic labeling of proteins for NMR studiesJonathan A. Ellman, Brian F. Volkman, David Mendel, Peter G. Schulz, and David E. WemmerCite this: J. Am. Chem. Soc. 1992, 114, 20, 7959–7961Publication Date (Print):September 1, 1992Publication History Published online1 May 2002Published inissue 1 September 1992https://doi.org/10.1021/ja00046a080RIGHTS & PERMISSIONSArticle Views407Altmetric-Citations58LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (403 KB) Get e-Alerts Get e-Alerts