Rotational dynamics of HIV‐1 nucleocapsid protein NCp7 as probed by a spin label attached by peptide synthesis

Zhang, Zhiwen; Xi, Xiangmei; Scholes, Charles P.; Karim, Christine B.

doi:10.1002/bip.21064

Cited by 14 publications

(16 citation statements)

References 62 publications

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“…In such cases, changes in the size of the system due to interactions with other macromolecules, such as a complimentary strand (Keyes and Bobst, 1998), a partner nucleic acid (Qin et al , 2001), or a protein (Keyes and Bobst, 1998; Xi et al , 2008; Zhang et al , 2008), will change the EPR spectrum. Such spectroscopic changes provide a reporter to monitor the interactions.…”

Section: Examples Of Applicationmentioning

confidence: 99%

Studying RNA Using Site-Directed Spin-Labeling and Continuous-Wave Electron Paramagnetic Resonance Spectroscopy

Zhang

Čekan

Sigurdsson

et al. 2009

Methods in Enzymology

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In site-directed spin-labeling (SDSL), a stable nitroxide radical is attached to a specific location within a macromolecule and electron paramagnetic resonance (EPR) spectroscopy is used to interrogate the local environment surrounding the nitroxide. The SDSL strategy enables probing sitespecific structural and dynamic features of RNA in solution without being limited by the size of the molecule, thus serving as a unique tool in biophysical studies of RNA. This chapter describes the use of continuous-wave (cw)-EPR to study dynamic features of RNAs as well as to monitor interactions between them. Various approaches for attaching nitroxide spin labels to nucleic acids are described, followed by detailed descriptions of cw-EPR spectral acquisition and processing procedures. Specific examples are subsequently used to illustrate analysis of EPR spectra, showing how information regarding the parent RNA can be extracted.Information about RNA structure and movement is critical for our understanding of how RNA is able to carry out its multifaceted functions. One spectroscopic technique that has shown great promise to study RNA, as well as other biopolymers, is electron paramagnetic resonance (EPR) spectroscopy, also named electron spin resonance (ESR) spectroscopy. EPR is a magnetic resonance technique that monitors the behaviors of unpaired electrons, and has long been used to study structure and dynamics of biomolecules (see recent reviews by Klug and Feix, 2008;Sowa and Qin, 2008). Structural information can be obtained by distance measurements, that is, by determination of distances between two spin-centers, and is the topic of another chapter in this volume (see Chapter 16 in this volume).This chapter focuses on continuous-wave (cw)-EPR, which has been commonly used for studying dynamic features and interactions between biomolecules. The technique is capable of covering motions ranging from picosecond (ps) to millisecond (ms). Other advantages of EPR are that small amount of material (typically 0.05-1.0 nmol) is required, samples are not limited by molecular size, and the measurements can be carried out under physiological conditions. However, RNA does not contain stable unpaired electrons and, therefore, spincenters must be introduced into the RNA in order to conduct EPR studies.This chapter first summarizes methods for attaching spin labels to nucleic acids, with a special focus on RNA (Section 1). Detailed protocols for these labeling methods have been described in a series of recent publications (Edwards and Sigurdsson, 2007;Qin et al., 2007;Schiemann et al., 2007), and will not be repeated here. Acquisition and processing of cw-EPR spectrum will then be described in detail (Section 2), followed by a brief discussion of spectral analysis (Section 3). Specific examples of using cw-EPR to study dynamics and interactions in RNA will subsequently be given (Section 4). NIH Public Access Author ManuscriptMethods Enzymol. Author manuscript; available in PMC 2010 October 19. Published in final edited form as:Met...

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Section: Examples Of Applicationmentioning

confidence: 99%

Studying RNA Using Site-Directed Spin-Labeling and Continuous-Wave Electron Paramagnetic Resonance Spectroscopy

Zhang

Čekan

Sigurdsson

et al. 2009

Methods in Enzymology

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“…SDSL has matured as a technique for probing protein structure and dynamics (Fanucci and Cafiso 2006), and has also been applied to investigate nucleic acids (Sowa and Qin 2008). However, SDSL studies of protein/RNA complexes are limited (Edwards et al 2005;Xi et al 2008;Zhang et al 2008) Here, two nitroxide derivatives, R1a and R1b (Fig. 1C), were attached at sites within the N-and C-fragment of two N-peptides without disrupting the native complex.…”

Section: Introductionmentioning

confidence: 99%

Conformational distributions at the N-peptide/boxB RNA interface studied using site-directed spin labeling

Zhang

Lee²,

Zhao³

et al. 2010

RNA

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In bacteriophage l, interactions between a 22-amino acid peptide (called the N-peptide) and a stem-loop RNA element (called boxB) play a critical role in transcription anti-termination. The N-peptide/boxB complex has been extensively studied, and serves as a paradigm for understanding mechanisms of protein/RNA recognition. Particularly, ultrafast spectroscopy techniques have been applied to monitor picosecond fluorescence decay behaviors of 2-aminopurines embedded at various positions of the boxB RNA. The studies have led to a model in which the bound N-peptide exists in dynamic equilibrium between two states, with peptide C-terminal fragment either stacking on (i.e., the stacked state) or peeling away from (i.e., the unstacked state) the RNA loop. The function of the N-peptide/boxB complex seems to correlate with the fraction of the stacked state. Here, the N-peptide/boxB system is studied using the site-directed spin labeling technique, in which X-band electron paramagnetic resonance spectroscopy is applied to monitor nanosecond rotational behaviors of stable nitroxide radicals covalently attached to different positions of the N-peptide. The data reveal that in the nanosecond regime the C-terminal fragment of bound N-peptide adopts multiple discrete conformations within the complex. The characteristics of these conformations are consistent with the proposed stacked and unstacked states, and their distributions vary upon mutations within the N-peptide. These results suggest that the dynamic two-state model remains valid in the nanosecond regime, and represents a unique mode of function in the N-peptide/boxB RNA complex. It also demonstrates a connection between picosecond and nanosecond dynamics in a biological complex.

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“…NCp7 was prepared by solid phase peptide synthesis and with analysis methods described previously (29)(30)(31)(32). The final NCp7 concentration was determined by using an extinction coefficient of…”

Section: Nucleocapsid Protein Ncp7mentioning

confidence: 99%

Pulse Dipolar ESR of Doubly Labeled Mini TAR DNA and Its Annealing to Mini TAR RNA

Sun

Borbat

Grigoryants

et al. 2015

Biophysical Journal

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Pulse dipolar electron-spin resonance in the form of double electron electron resonance was applied to strategically placed, site-specifically attached pairs of nitroxide spin labels to monitor changes in the mini TAR DNA stem-loop structure brought on by the HIV-1 nucleocapsid protein NCp7. The biophysical structural evidence was at Ångstrom-level resolution under solution conditions not amenable to crystallography or NMR. In the absence of complementary TAR RNA, double labels located in both the upper and the lower stem of mini TAR DNA showed in the presence of NCp7 a broadened distance distribution between the points of attachment, and there was evidence for several conformers. Next, when equimolar amounts of mini TAR DNA and complementary mini TAR RNA were present, NCp7 enhanced the annealing of their stem-loop structures to form duplex DNA-RNA. When duplex TAR DNA-TAR RNA formed, double labels initially located 27.5 Å apart at the 3'- and 5'-termini of the 27-base mini TAR DNA relocated to opposite ends of a 27 bp RNA-DNA duplex with 76.5 Å between labels, a distance which was consistent with the distance between the two labels in a thermally annealed 27-bp TAR DNA-TAR RNA duplex. Different sets of double labels initially located 26-27 Å apart in the mini TAR DNA upper stem, appropriately altered their interlabel distance to ~35 Å when a 27 bp TAR DNA-TAR RNA duplex formed, where the formation was caused either through NCp7-induced annealing or by thermal annealing. In summary, clear structural evidence was obtained for the fraying and destabilization brought on by NCp7 in its biochemical function as an annealing agent and for the detailed structural change from stem-loop to duplex RNA-DNA when complementary RNA was present.

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Rotational dynamics of HIV‐1 nucleocapsid protein NCp7 as probed by a spin label attached by peptide synthesis

Cited by 14 publications

References 62 publications

Studying RNA Using Site-Directed Spin-Labeling and Continuous-Wave Electron Paramagnetic Resonance Spectroscopy

Studying RNA Using Site-Directed Spin-Labeling and Continuous-Wave Electron Paramagnetic Resonance Spectroscopy

Conformational distributions at the N-peptide/boxB RNA interface studied using site-directed spin labeling

Pulse Dipolar ESR of Doubly Labeled Mini TAR DNA and Its Annealing to Mini TAR RNA

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