scholarly journals Structural analysis of proteins by isotope-edited FTIR spectroscopy

2010 ◽  
Vol 24 (1-2) ◽  
pp. 37-43 ◽  
Author(s):  
Suren A. Tatulian
Keyword(s):  
Structural Analysis ◽  
Ftir Spectroscopy ◽  
Structural Changes ◽  
Isotope Labeling ◽  
Structural Information ◽  
Attenuated Total Reflection ◽  
Site Specific ◽  
Segmental Isotope Labeling ◽  
Protein Membrane

Structure determination of multidomain proteins or protein–membrane complexes is one of the most challenging tasks in modern structural biology. High-resolution techniques, like NMR or X-ray crystallography, are limited to molecules of moderate size or those that can be crystallized easily. Both methods encounter serious technical obstacles in structural analysis of protein–membrane systems. This work describes an emerging biophysical technique that combines segmental isotope labeling of proteins with Fourier transform infrared (FTIR) spectroscopy, which provides site-specific structural information on proteins and allows structural characterization of protein–membrane complexes. Labeling of a segment of the protein with13C results in infrared spectral resolution of the labeled and unlabeled parts and thus allows identification of structural changes in specific domains/segments of the protein that accompany functional transitions. Segmental isotope labeling also allows determination of the precise configuration of protein–membrane complexes by polarized attenuated total reflection FTIR (ATR–FTIR) spectroscopy. These new developments offer solutions to functionally important site-specific structural changes in proteins and protein–membrane complexes that are hard to approach using conventional methods.

scholarly journals Investigation of protein–protein interactions by isotope‒edited Fourier transformed infrared spectroscopy

2004 ◽  
Vol 18 (3) ◽  
pp. 397-406 ◽  
Author(s):  
Tiansheng Li
Keyword(s):  
Ftir Spectroscopy ◽  
Conformational Changes ◽  
Protein Interactions ◽  
Isotope Labeling ◽  
Structural Information ◽  
Protein Complexes ◽  
Secondary Structures ◽  
Receptor Complex ◽  
Protein Protein Interactions

Recent advance in FTIR spectroscopy has shown the usefulness of13C uniform isotope labeling in proteins to study protein–protein interactions.13C uniform isotope labeling can significantly resolve the spectral overlap in the amide I/I′ region in the spectra of protein–protein complexes, and therefore allows more accurate determination of secondary structures of individual protein component in the complex than does the conventional FTIR spectroscopy. Only a limited number of biophysical techniques can be used effectively to obtain structural information of large protein–protein complex in solution. Though X‒ray crystallography and NMR have been used to provide structural information of proteins at atomic resolution, they are limited either by the ability of protein to crystallize or the large molecular weight of protein. Vibrational spectroscopy, including FTIR and Raman spectroscopies, has been extensively employed to investigate secondary structures and conformational dynamics of protein–protein complexes. However, significant spectral overlap in the amide I/Iʹ region in the spectra of protein–protein complexes often hinders the utilization of vibrational spectroscopy in the study of protein–protein complex. In this review, we shall discuss our recent work involving the application of isotope labeled FTIR to the investigation of protein–protein complexes such as cytokine–receptor complexes. One of the examples involves G‒CSF/receptor complex. To determine unambiguously the conformations of G‒CSF and the receptor in the complex, we have prepared uniformly13C/15N isotope labeled G‒CSF to resolve its amide Iʹ band from that of its receptor in the IR spectrum of the complex. Conformational changes and structural stability of individual protein subunit in G‒CSF/receptor complex have then been investigated by using FTIR spectroscopy (Li et al.,Biochemistry29 (1997), 8849–8859). Another example involves BDNF/trkB complex in which13C/15N uniformly labeled BDNF is complexed with its receptor trkB (Li et al.,Biopolymers67(1) (2002), 10–19). Interactions of13C/15N uniformly labeled brain‒derived neurotrophic factor (BDNF) with the extracellular domain of its receptor, trkB, have been investigated by employing FTIR spectroscopy. Conformational changes and structural stability and dynamics of BDNF/trkB complex have been determined unambiguously by FTIR spectroscopy, since amide I/Iʹ bands of13C/15N labeled BDNF are resolved from those of the receptor. Together, those studies have shown that isotope edited FTIR spectroscopy can be successfully applied to the determination of protein secondary structures of protein complexes containing either the same or different types of secondary structures. It was observed that13C/15N uniform labeling also affects significantly the frequency of amide IIʹ band, which may permit the determination of hydrogen–deuterium exchange in individual subunit of protein–protein complexes.

Differential Isotope Labeling of Glycopeptides for Accurate Determination of Differences in Site-Specific Glycosylation

2015 ◽  
Vol 15 (1) ◽  
pp. 326-331 ◽  
Author(s):  
Martin Pabst ◽  
Iva Benešová ◽  
Stephan R. Fagerer ◽  
Mathias Jacobsen ◽  
Klaus Eyer ◽  
...  
Keyword(s):  
Isotope Labeling ◽  
Accurate Determination ◽  
Site Specific

scholarly journals A multicrystal diffraction data-collection approach for studying structural dynamics with millisecond temporal resolution

IUCrJ ◽  
2016 ◽  
Vol 3 (6) ◽  
pp. 393-401 ◽  
Author(s):  
Robin Schubert ◽  
Svetlana Kapis ◽  
Yannig Gicquel ◽  
Gleb Bourenkov ◽  
Thomas R. Schneider ◽  
...  
Keyword(s):  
Data Collection ◽  
Radiation Damage ◽  
Temporal Resolution ◽  
Diffraction Data ◽  
Structural Changes ◽  
Structural Information ◽  
Data Sets ◽  
Enzymatic Reactions ◽  
Time Resolved ◽  
Site Specific

Many biochemical processes take place on timescales ranging from femtoseconds to seconds. Accordingly, any time-resolved experiment must be matched to the speed of the structural changes of interest. Therefore, the timescale of interest defines the requirements of the X-ray source, instrumentation and data-collection strategy. In this study, a minimalistic approach forin situcrystallization is presented that requires only a few microlitres of sample solution containing a few hundred crystals. It is demonstrated that complete diffraction data sets, merged from multiple crystals, can be recorded within only a few minutes of beamtime and allow high-resolution structural information of high quality to be obtained with a temporal resolution of 40 ms. Global and site-specific radiation damage can be avoided by limiting the maximal dose per crystal to 400 kGy. Moreover, analysis of the data collected at higher doses allows the time-resolved observation of site-specific radiation damage. Therefore, our approach is well suited to observe structural changes and possibly enzymatic reactions in the low-millisecond regime.

scholarly journals The application of ATR-FTIR Spectroscopy and the Reversible DNA Conformation as a Sensor to Test the Effectiveness of Platinum(II) Anticancer Drugs

2018 ◽  
Author(s):  
Khansa Al-Jorani ◽  
Anja Rüther ◽  
Miguela Martin ◽  
Rukshani Haputhanthri ◽  
Glen B. Deacon ◽  
...  
Keyword(s):  
Ftir Spectroscopy ◽  
Cancer Cells ◽  
Structural Changes ◽  
Isotonic Saline ◽  
Platinum Complexes ◽  
Attenuated Total Reflection ◽  
Dna Conformation ◽  
Acetone Water ◽  
Intensity Changes ◽  
And Shifts

Platinum(II) complexes have been found to be effective against cancer cells. Cisplatin curbs cell replication by interacting with the deoxyribonucleic acid (DNA), eventually leading to cell death and reducing cell proliferation. In order to investigate the ability of platinum complexes to affect cancer cells, two examples from the class of polyflurophenylorganoamidoplatinum(II) complexes were synthesised and tested on isolated DNA. The two compounds trans-[N,N’-bis(1,2,3,5,6-pentafluorophenyl)ethane-1,2-diaminato(1-)](2,3,4,5,6-pentafluorobenzoato)(pyridine)platinum(II) (PFB), and trans-[N,N’-bis(1,2,3,5,6-pentafluorophenyl)ethane-1,2-diaminato(1-)](2,4,6-trimethylbenzoato)(pyridine)platinum(II) (TMB) were compared with cisplatin through their reaction with DNA. Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) spectroscopy was applied to analyse the interaction of the Pt(II) complexes with DNA in the hydrated, dehydrated and rehydrated state. These were compared with control DNA in acetone/water (PFB, TMB) and isotonic saline (cisplatin) under the same conditions. Principle Component Analysis (PCA) was applied to compare the ATR-FTIR spectra of the untreated control DNA with spectra of PFB and TMB treated DNA samples. Disruptions in the conformation of DNA treated with the Pt(II) complexes upon rehydration were mainly observed by monitoring the position of the IR-band around 1711 cm-1 assigned to the DNA base-stacking vibration. Furthermore, other intensity changes in the phosphodiester bands of DNA at ~1234 cm-1 and 1225 cm-1 and shifts in the dianionic phosphodiester vibration at 966 cm-1 were observed. The isolated double stranded DNA (dsDNA) or single stranded DNA (ssDNA) showed different structural changes when incubated with the studied compounds. PCA confirmed PFB had the most dramatic effect by denaturing both dsDNA and ssDNA. Both compounds, along with cisplatin, induced changes in DNA bands at 1711, 1088, 1051 and 966 cm-1 indicative of DNA conformation changes. The ability to monitor conformational change with infrared spectroscopy paves the way for a sensor to screen for new anticancer therapeutic agents.

scholarly journals Structural changes of protein antigens due to adsorption onto and release from aluminium hydroxide using FTIR–ATR

2007 ◽  
Vol 21 (4) ◽  
pp. 211-226 ◽  
Author(s):  
Yiwu Zheng ◽  
Xuxin Lai ◽  
Henrik Ipsen ◽  
Jørgen Nedergaard Larsen ◽  
Henning Løwenstein ◽  
...  
Keyword(s):  
Immune Response ◽  
Aluminium Hydroxide ◽  
Structural Changes ◽  
Structural Integrity ◽  
Structural Information ◽  
Cd Spectroscopy ◽  
Attenuated Total Reflection ◽  
Protein Antigens ◽  
Recovery Curves ◽  
Atr Spectroscopy

Structural integrity of antigens upon adsorption and release is not only important for investigating vaccine immunogenicity, but also for the epitope specificity of the resulting immune response and hence therapeutic efficacy. Moreover, the structural information is also important for understanding the mechanism of how adjuvants can enhance the immune response. However, little is known about an antigen's structure when it is adsorbed on and subsequently released from aluminium adjuvants. In this study, the structures of two protein antigens, bovine serum albumin and β-lactoglobulin, were investigated using Fourier transform infrared–attenuated total reflection (FTIR–ATR) spectroscopy. The secondary structures of both model antigens change when adsorbed to aluminium hydroxide. The structural perturbation depends on the amount of adsorbed protein. Maximal adsorption gives a more native-like structure. This may indicate that protein is adsorbed in different manners depending on the concentration. The adsorbed antigens are released using phosphate buffer pH 7.4 (PB). The recovery is approximate 80% after 40 min in the presence of PB. The recovery curves of both proteins also indicate two different adsorption modes. FTIR–ATR and circular dichroism (CD) spectroscopy yield similar results suggesting that the adsorbed antigens refold to their native-like state after release.

scholarly journals The Application of ATR-FTIR Spectroscopy and the Reversible DNA Conformation as a Sensor to Test the Effectiveness of Platinum(II) Anticancer Drugs

Sensors ◽  
2018 ◽  
Vol 18 (12) ◽  
pp. 4297 ◽  
Author(s):  
Khansa Al-Jorani ◽  
Anja Rüther ◽  
Miguela Martin ◽  
Rukshani Haputhanthri ◽  
Glen Deacon ◽  
...  
Keyword(s):  
Ftir Spectroscopy ◽  
Cancer Cells ◽  
Structural Changes ◽  
Isotonic Saline ◽  
Platinum Complexes ◽  
Attenuated Total Reflection ◽  
Dna Conformation ◽  
Acetone Water ◽  
Intensity Changes ◽  
And Shifts

Platinum(II) complexes have been found to be effective against cancer cells. Cisplatin curbs cell replication by interacting with the deoxyribonucleic acid (DNA), reducing cell proliferation and eventually leading to cell death. In order to investigate the ability of platinum complexes to affect cancer cells, two examples from the class of polyfluorophenylorganoamidoplatinum(II) complexes were synthesised and tested on isolated DNA. The two compounds trans-[N,N′-bis(2,3,5,6-tetrafluorophenyl)ethane-1,2-diaminato(1-)](2,3,4,5,6-pentafluorobenzoato)(pyridine)platinum(II) (PFB) and trans-[N,N′-bis(2,3,5,6-tetrafluorophenyl)ethane-1,2-diaminato(1-)](2,4,6-trimethylbenzoato)(pyridine)platinum(II) (TMB) were compared with cisplatin through their reaction with DNA. Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) spectroscopy was applied to analyse the interaction of the Pt(II) complexes with DNA in the hydrated, dehydrated and rehydrated states. These were compared with control DNA in acetone/water (PFB, TMB) and isotonic saline (cisplatin) under the same conditions. Principle Component Analysis (PCA) was applied to compare the ATR-FTIR spectra of the untreated control DNA with spectra of PFB and TMB treated DNA samples. Disruptions in the conformation of DNA treated with the Pt(II) complexes upon rehydration were mainly observed by monitoring the position of the IR-band around 1711 cm−1 assigned to the DNA base-stacking vibration. Furthermore, other intensity changes in the phosphodiester bands of DNA at ~1234 cm−1 and 1225 cm−1 and shifts in the dianionic phosphodiester vibration at 966 cm−1 were observed. The isolated double stranded DNA (dsDNA) or single stranded DNA (ssDNA) showed different structural changes when incubated with the studied compounds. PCA confirmed PFB had the most dramatic effect by denaturing both dsDNA and ssDNA. Both compounds, along with cisplatin, induced changes in DNA bands at 1711, 1088, 1051 and 966 cm−1 indicative of DNA conformation changes. The ability to monitor conformational change with infrared spectroscopy paves the way for a sensor to screen for new anticancer therapeutic agents.

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