New insights into the structural characteristics of irradiated crotamine
© Oliveira et al.; licensee BioMed Central. 2015
Received: 28 October 2014
Accepted: 6 May 2015
Published: 20 May 2015
Since ionizing radiation has the potential to alter the molecular structure and affect the biological properties of biomolecules, it has been successfully employed to attenuate animal toxins. The present study aimed to characterize the structural modifications on irradiated crotamine, a toxin from Crotalus durissus terrificus venom, using circular dichroism (CD), fluorescence, Fourier transformed infrared spectroscopy (FTIR), atomic force microscopy (AFM) and differential scanning calorimetry (DSC).
A combination of size exclusion and ion-exchange chromatography was used to purify the peptide using crude venom. The pure toxin was then submitted to 2 kGy gamma irradiation doses from a cobalt-60 source. Native and irradiated crotamine were analyzed using a fluorescence spectrophotometer. Wavelength was fixed at 295 nm and fluorescence emission scans were collected from 300 to 400 nm. CD and FTIR techniques were used to identify the secondary structure of both samples. DSC analyses were performed at a starting temperature of 20 °C up to a final temperature of 90 °C. AFM provided a 3D profile of the surfaces of both crotamine forms adsorbed on mica.
Fluorescence spectroscopy showed that the quantum yield of the irradiated form decreased. CD spectra of native and irradiated crotamine solutions showed differences between the samples in wavelength, indicating that irradiation induced a transition of a small portion of the random coil regions towards an α-helical conformation. FTIR and CD showed that the native and irradiated crotamine spectra were different with regard to secondary structure. The thermodynamic analysis showed that irradiation caused changes in the calorimetric profile and CD showed that temperature-induced changes also occur in the secondary structure. Finally, AFM showed the possible formation of insoluble aggregates.
Our results indicate that irradiation leads to progressive changes in the structure of the toxin, which could explain a decrease in myotoxic activity.
KeywordsGamma radiation Structural modifications Crotamine Snake venom Crotalus durissus terrificus
Serum therapy is the only specific and effective treatment against snakebites. In Brazil, it consists of the administration of antivenom produced in immunized horses. However, the high toxicity of snake venom reduces their lifespan and thus hinders the production of antivenom [1–5].
Several methods of snake venom detoxification involve chemical or physical modification of toxins [6–13]. Gamma irradiation is a potential tool for venom detoxification, since the structural changes caused by this radiation slow down or stop enzymatic activities of toxic proteins, while retain epitopes that induce efficient immune responses [14–20]. Gamma rays are forms of ionizing radiation that interact with biomolecules in solutions through two processes. The direct process, whereby radiation reaches the target biomolecules, and the indirect process, in which water radiolysis products interact with the target molecules. The indirect process is the main way of interaction and accounts for approximately 80 % of the total effects [21, 22]. Previous studies showed that the most appropriate radiation dose for reducing venom toxicity and maintaining immunogenicity in order to increase the production of antiserum is 2 kGy [23–27].
Envenomation resulting from Crotalus durissus terrificus bites accounts for 14 % of all snake accidents in Brazil and has a high mortality rate [19, 28]. The structural effects of gamma rays on venoms and toxins are not yet fully understood and this study therefore aimed to characterize the effects of radiation on crotamine from Crotalus durissus terrificus venom.
Crotamine is a small basic polypeptide myotoxin (pI – 10.3) with a number of isoforms and a molecular weight of 4882 Da. It is composed of 42 amino acid residues without free sulfhydryl groups and reticulated by three disulfide bonds. This toxin induces skeletal muscle spasms in mammals leading to spastic paralysis of peripheral origin [29, 30].
In 2013, Coronado et al.  isolated a single isoform and determined the 1.7-Å-resolution crystal structure of crotamine, revealing distinct cationic and hydrophobic surface areas located on opposite sides of the molecule. The topological analysis classified the crystal structure as α1β1α2β2. Residues Lys2-Lys7 form a single α-helical turn which flanks a two-stranded antiparallel β-sheet formed from residues Gly9-Pro13 (β1) and Trp34-Lys38 (β2) located in the core of the molecule. A short α-helical turn is formed from residues Pro20-Ser23 .
Determining protein structure is crucial to understand their mode of action, thus contributing to the development of their potential as molecular tools or new drugs. Although a number of studies have already shown the biological properties of irradiated proteins, doubts remain on what refers to the structural modifications suffered due to irradiation and the resulting effects on the mode of action of crotamine [25, 26, 32, 33]. Since the crystal structure of crotamine has already been described, this study aims to investigate the radiation-induced conformational changes suffered by the peptide.
Crude lyophilized venom extracted from Crotalus durissus terrificus was supplied by the Center for the Study of Venoms and Venomous Animals (CEVAP) of São Paulo State University (UNESP) in Botucatu, SP, Brazil.
Crude venom (70 mg) was dissolved in a 200 mM pH 3 ammonium formate buffer, centrifuged at 14,000 g for five minutes to remove insoluble material, and then fractionated using a Superdex 75 10/300 column (GE Healthcare, USA) equilibrated in the same buffer at a flow rate of 0.8 mL/minute. The absorbance of the eluate was monitored at 280 nm.
The crotamine fraction was pooled and refractionated using a 1 mL Resource S column (GE Healthcare, USA) equilibrated in 50 mM sodium phosphate buffer, pH 7.8 (buffer A). Buffer B was identical to buffer A, except that it was supplemented with 2.0 M NaCl. After an initial wash (5.0 mL) with 5 % buffer B, the protein was eluted with a linear gradient from 5 to 30 % buffer B. The fractions were pooled and then desalted by dialysis against water and lyophilized.
The purified toxin was dissolved in 0.15 M NaCl to a final concentration of 2 mg/mL and submitted to 2 kGy gamma irradiation doses from a cobalt-60 (60Co) source (Gammacell 220, Canada) in the presence of atmospheric oxygen, at room temperature and with a dose rate of 1.2 kGy/h.
After measuring absorbance at 280 nm of both samples to ensure that irradiation did not cause any alteration in peptide concentration, 100 μg/mL aliquots in PBS buffer (pH 7.2) of the native and irradiated crotamine were analyzed at 25 °C using a Varian Cary Eclipse fluorescence spectrophotometer (Varian Australia Pty Ltd, Australia). The excitation wavelength was fixed at 295 nm and fluorescence emission scans were collected from 300 to 400 nm.
Circular Dichroism Spectroscopy (CD)
Circular dichroism spectroscopy was used to quantify the secondary structure of the native and irradiated toxin using 500 μL of each sample at 100 μg/mL, placed in quartz cuvettes with a 1 mm path length using a Jasco J-810 L CD Spectropolarimeter (Japan). Spectra were measured between 190 nm and 260 nm.
Three runs were performed for each sample and the data were processed subtracting the blank spectrum. The temperature was increased in increments of 10 °C from 20 °C to 90 °C throughout the experiment. The alteration of structural elements was quantified using the K2d software (http://kal-el.ugr.es/k2d/spectra.html).
Fourier Transform Infrared Spectroscopy (FTIR)
The vibrational spectra of the molecules were analyzed using FTIR to determine the secondary structure of native and irradiated crotamine. The samples were scanned over the range 650 to 2,000 cm−1 at a resolution of 4 cm−1 and 120 scans. The spectra were baseline corrected and normalized to the amide band I to minimize homogeneity of variance. The area of each band was calculated based on the method used by Naumann [34, 35].
Differential Scanning Calorimetry (DSC)
DSC was performed using a Mettler Toledo Differential Scanning Calorimeter 822e after diluting the native and irradiated crotamine samples to 400 μg/mL in a 25 mM pH 7.2 phosphate buffer. The thermodynamic analysis was carried out by gradually heating the samples (1 °C/min) from a starting temperature of 20 °C to a final temperature of 90 °C. The signal was registered every 30 s and corrected against the buffer spectrum.
Atomic Force Microscopy (AFM)
AFM provides a 3D profile of the surface on a nanoscale, by measuring forces between a sharp probe (<10 nm) and a surface at a very short distance (0.2-10 nm probe-sample separation). The native and irradiated crotamine were diluted to 0.01, 0.1 and 1 μg/mL in phosphate buffer (10 mM KH2PO4, 150 mM KCl, pH 6). A drop of approximately 40 μL of crotamine solution was deposited on a pre-prepared mica support which was incubated for between 15 and 20 min. Mica surfaces are commonly used for obtaining topographic images of proteins using AFM because they are flat, hydrophilic, and because mica has a high affinity to protein molecules . Each sample was then washed three times in deionized water. This entire process was performed in a laminar flow to avoid contamination of the sample surface. The experiment was conducted at room temperature in tapping mode, in which the cantilever oscillates at its resonance frequency or very close to contact.
Results and discussion
Crotamine was isolated using a two-step chromatography procedure: size exclusion followed by ion exchange. The identity and homogeneity of the peptides were confirmed using mass spectrometry which detected three crotamine isoforms due to the low resolution of the ion exchange column used in the second purification step. The molecular mass of the most abundant isoform was 4883.2548 Da (data not shown).
Although fluorescence can be a very helpful indicator of molecular change, when it comes to changes in secondary structure, such measurements are difficult to interpret directly (α-helix and β-sheet). When the structure of native protein containing α-helical and β-sheet areas is denatured it undergoes an unfolding process. Often, the fraction of non-covalent bonds in α-helical, β-sheet and aperiodic conformations may be estimated using highly sensitive measurements, such as circular dichroism (CD) and infrared (IR) spectroscopy. In favorable cases, the spectral effects of conformational changes caused by physical and biological phenomena may be observed .
Pelton and McLean  elucidated that short helices can result in reduced bands in the spectra. Coronado et al.  illustrated that both α-helix structures in crotamine are formed by few residues, with a single turn in α2 and two turns in α1. The CD spectra of β-sheets display a negative band near 216 nm, a positive band between 195 and 200 nm, and a negative band near 175 nm. However, the position and magnitude of these bands is variable, resulting in less accurate predictions for β-structure than for α-helices using CD . Our data indicate that radiation induced the transition of a small portion of the random coil regions towards an α-helical conformation. By using a secondary structure prediction tool, we observed that a region containing three cysteines (D24-K38) is prone to forming α-helices . It is possible that this does not occur in the native peptide as these residues form disulfide bonds that stabilize the molecule. Irradiation may cause disruption in these reticulation bonds, enabling the formation of a non-native helical domain.
We also performed CD spectroscopy with temperature changes in an attempt to assess modifications in the thermal stability of crotamine after exposure to radiation. Initially, the temperature was maintained at 20 °C (Fig. 2a) and subsequently increased by 10 °C every 30 min until a final temperature of 90 °C was reached (Fig. 2b to h). We observed that structural changes due to thermal denaturation lead to changes in CD spectra and it seems that complete denaturation occurred at 90 °C. After reaching the maximum temperature, we reduced the sample temperature to 20 °C (Fig. 2i) and took a new reading which showed only a partial recovery of structural elements. Irradiated crotamine displayed partial denaturation and it is apparent that this form of the toxin was more sensitive to temperature since at 70 °C only random conformation could be observed.
Differential scanning calorimetry (DSC) is extremely useful for characterizing temperature- induced conformational changes in proteins, especially with respect to the factors associated with protein stability . The thermodynamic analysis showed that irradiation modified molecule enthalpy. Endothermic transitions decreased while exothermic transitions increased.
Figure 3b shows an irregular spectrum with a first component indicating that the irradiated toxin protein melted at 22.23 °C, which reflects lower enthalpy in the irradiated sample. In other words, the energy required to denature the toxin exposed to radiation is much lower, showing that structural stability was significantly affected by the irradiation.
Infrared spectroscopy is a well-established experimental procedure used to analyze the secondary structure of polypeptides and proteins associated with the vibration of repeating structural units, especially the frequency at which amine bonds absorb infrared radiation. The use of the infrared spectroscopy technique to assess protein secondary structure has undergone a renaissance with the development of Fourier transform infrared spectroscopy.
Nine normal modes are allowed for the amide band of proteins in order of decreasing frequency: A, B, and I-VII. The amide band I (80 % C = O stretch, near 1650 cm−1) is the most commonly used to assign secondary structures to proteins .
In Fig. 5 the spectra of native crotamine are presented as a smooth curve with peaks along the amide I region, while well-defined peaks appear on the curve of the irradiated crotamine. It is evident that the regions most affected by radiation were the spectral regions corresponding to the α-helix (1650 and 1655 cm−1), since major differences were observed at these wave numbers. It was also observed that the curve of the irradiated crotamine shifted approximately 20 cm−1 to the left, suggesting lower stability in this protein, corroborating the results of the DSC. Given the effects of radiation associated with the ionization of the molecule and its ability to modify the protein structure, these phenomena may be associated with a strong interaction between protein products of lower molecular mass and may demonstrate the formation of peptides, amino acids or even protein aggregates [17, 41]. The presence of these molecular neighbors may modify the interaction of the peptide groups, changing the total energy of the molecule.
The quality of data from atomic force microscopy (AFM) used for imaging and manipulating biomolecules has improved due to advances in equipment, sample preparation and image acquisition conditions. This is the only instrument that can image samples in aqueous solution at the subnanometer scale .
Based on our findings, we conclude that crotamine suffered conformational changes after being exposed to 2 kGy gamma radiation doses from a cobalt-60 source.
The fluorescence analysis allowed us to observe small changes in the tertiary structure of the protein, shown by quenching of the tryptophan fluorophores which are partially exposed in the native molecule.
The results of circular dichroism and infrared spectroscopy showed that there were changes to the secondary structure of the toxin. Such changes in the secondary structure of proteins may be related to an attenuation or abolishment in its biological activities, such as decreased myotoxic activity. This fact has been demonstrated by previous studies [14–17].
Subjecting the samples to changes in temperature was also used as a mean of comparing the thermal stability of the native and irradiated toxin. Circular dichroism and differential scanning calorimetry showed that irradiation induces energy (enthalpy) changes in the peptide, making it more susceptible to thermal denaturation. AFM reinforces these observations, showed by the formation of “soluble aggregates” in the irradiated sample.
Taken as a whole, our data indicate that the irradiation of crotamine in solution leads to significant and apparently homogeneous conformational changes, with alterations of structural elements and a loss of enthalpy, resulting in reduced structural stability. The use of crotamine to investigate the impact of gamma ray exposure on peptides provides important insights into the potential of radiation as a tool for attenuating toxins and contributing to the design of safer antigens for vaccine and antiserum production.
Fourier transformed infrared
Atomic force microscopy
Differential scanning calorimetry
The authors would like to thank the Coordination for the Improvement of Higher Education Personnel (CAPES) for their financial support, Dr. Maria Cecília Salvadori of the Institute of Physics at the University of São Paulo (USP) and Dr. Denise Maria Zezell of the Center for Lasers and Applications at the Nuclear and Energy Research Institute (IPEN – CNEN/SP). Special thanks are also extended to the Center for the Study of Venoms and Venomous Animals (CEVAP) of UNESP. RSFJ is a CNPq DTI fellow researcher (310395/2014-3).
- Calmette A. Les animaux venimeux et la sérotherapie antivenimeuse. In: Les Venins. Paris: Masson et Cie; 1907.Google Scholar
- Rosenfeld G. Ação neurotóxica de venenos ofídicos (Crotalus e Micrurus) no sistema nervoso central. Interpretação clínica. Ciênc Cult. 1971;23 Suppl:S1–45.Google Scholar
- Magalhães RA, Ribeiro MMF, Rezende NA, Amaral CFS. Rabdomióilise secundária a acidente ofídico crotálico (Crotalus durissus terrificus). Rev Inst Med Trop Sao Paulo. 1986;28(4):228–33.PubMedGoogle Scholar
- Souza FA, Spencer PJ, Rogero JR, Nascimento N, Dal Pai-Silva M, Gallacci M. 60Co gamma irradiation prevents Bothrops jararacussu venom neurotoxicity and myotoxicity in isolated mouse neuromuscular junction. Toxicon. 2002;40(8):1101–6.View ArticlePubMedGoogle Scholar
- Oussedik-Oumehdi H, Laraba-Djebari F. Irradiated Cerastes cerastes venom as a novel tool for immunotherapy. Immunopharmacol Immunotoxicol. 2008;30(1):37–52.PubMedGoogle Scholar
- Costa LM, Takeda AK, Barbosa SFC, Berra JAP, Adelina MGF, Soerensen B, et al. Estudo comparativo da resposta imune em cavalos ao veneno de Crotalus durisssus terrificus, in natura, tratado com formaldeído e submetido à ação térmica. Vac Soros. 1985;1:24–9.Google Scholar
- Guidolin R, Dias da Silva W, Higashi HG, Caricati CP, Lima MLSR, Morais JF. Hiperimunização de cavalos soroprodutores com venenos botrópico e crotálico tratados por glutaraldeído. Mem Inst Butantan. 1989;51:85–90.Google Scholar
- Moroz C, Goldblum N, De Vries A. Preparation of Vipera palestinae antineurotoxin using carboxymethyl-cellulose-bound neurotoxin as antigen. Nature. 1963;200:228–33.View ArticleGoogle Scholar
- Okonogi T, Hattori Z, Ogiso A, Mitsui S. Detoxification by persimmon tannin of snake venoms and bacterial toxins. Toxicon. 1979;17(5):524–7.View ArticlePubMedGoogle Scholar
- Relyveld EH, Girard O, Cheyroux M, Asso J, De Rudder J. Nouveau procede d’inactivation pour la preparation de vaccins. Dev Biol Stand. 1974;33:236–348.Google Scholar
- Guidolin R, Higashi HG, Nishikawa AK, Yamagushi IK, Lima MLSR, Morais JF, et al. Venenos botrópicos pré-tratados com inibidores ativos para os sítios enzimáticos em proteases e com substâncias quelantes preservam seu poder imunogênico. Mem Inst Butantan. 1989;51(3):107–15.Google Scholar
- Heneine IF, Heneine LGD, Daniel JP, Nascimento MCS, Rocha OA. Properties of protein toxins and venoms modified by controlled iodination. Anais Acad Ciênc Est São Paulo. 1988;57(2):55–66.Google Scholar
- Grasset E. Anavenoms and their use in the preparation of antivenoms sera. Trans R Soc Trop Med Hyg. 1945;38:463–88.PubMedGoogle Scholar
- Murata Y, Nishikawa AK, Nascimento N, Higashi HG, Dias da Silva W, Rogero JR. Gamma irradiation reduces the toxic activities of Crotalus durissus terrificus venom but does not affect their immunogenic activities. Toxicon. 1992;28:617–28.Google Scholar
- Nascimento N, Seebart CS, Francis B, Rogero JR, Kaiser II. Influence of ionizing radiation on crotoxin: biochemical and immunological aspects. Toxicon. 1996;34(1):123–31.View ArticlePubMedGoogle Scholar
- Clissa PB, Nascimento N, Rogero JR. Toxicity and immunogenicity of Crotalus durissus terrificus venom treated with different doses of gamma rays. Toxicon. 1999;37(8):1131–41.View ArticlePubMedGoogle Scholar
- Nascimento N, Spencer PJ, Andrade Junior HF, Guarnieri MC, Rogero JR. Effects of gamma radiation on snake venoms. Radiat Phys Chem. 1998;52(1–6):665–9.Google Scholar
- Ferreira Junior RS, Nascimento N, Martinez JC, Alves JB, Meira DA, Barraviera B. Immunization with native and cobalt 60-irradiated Crotalus durissus terrificus venom in swiss mice: assessment of the neutralizing potency of antisera. J Venom Anim Toxins incl Trop Dis. 2005;11(3):299–314 [http://www.scielo.br/scielo.php?pid=S1678-91992005000300008&script=sci_arttext]Google Scholar
- Ferreira Junior RS, Nascimento N, Couto R, Alves JB, Meira DA, Barraviera B. Laboratory evaluation of young ovines inoculated with natural or 60Co-irradiated Crotalus durissus terrificus venom during hyperimmunization process. J Venom Anim Toxins incl Trop Dis. 2006;12(4):620–31 [http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1678-91992006000400009].Google Scholar
- Caproni P, Baptista JA, de Almeida TL, Passos LAC, Nascimento N. Study of irradiated bothropstoxin-1 with 60Co gamma rays: immune system behavior. J Venom Anim Toxins incl Trop Dis. 2009;15(2):216–25 [http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1678-91992009000200005]Google Scholar
- Butler J, Hoey BM, Swallow AJ. Radiation chemistry. Annu Rep Prog Chem. 1987;83:129–75.Google Scholar
- Butler J, Land EJ, Swallow AJ. Chemical mechanisms of the effects of high energy radiation on biological systems. Radiat Phys Chem. 1984;24(3–4):273–82.Google Scholar
- Moreira EC, Nascimento N, Rosa GJ, Rogero JR, Vassilieff VS. Effects of gamma irradiation on the behavioral properties of crotoxin. Braz J Med Biol Res. 1997;30(2):245–9.PubMedGoogle Scholar
- Gallacci M, Nunes EC, Moreira EG, Nascimento N, Rogero JR, Vassilieff VS. Reduction of crotoxin-induced neuromuscular blockade by gamma radiation. Toxicon. 1998;36(6):941–5.View ArticlePubMedGoogle Scholar
- Casare MS, Spencer PJ, Campos LA, Nascimento N. Study of gamma-radiation effects on crotamine and crotoxin. J Radioanal Nucl Ch. 2006;269(3):571–7.Google Scholar
- Silva MC, Baptista JA, Spencer PJ, Nascimento N. Effects of 60Co radiation on the molecular structure of crotamine. Radiat Phys Chem. 2004;71(1):417–8.Google Scholar
- Netto DP, Chiacchio SB, Bicudo PL, Alfieri AA, Balarim MRS, Nascimento N. Hematological changes in sheep inoculated with natural and Cobalt60-irradiated Crotalus durissus terrificus venom (Laurenti, 1768). J Venom Anim Toxins incl Trop Dis. 2004;10(1):34–52 [http://www.scielo.br/scielo.php?pid=S1678-91992004000100004&script=sci_arttext]Google Scholar
- Fundação Nacional de Saúde. Manual de diagnóstico e tratamento de acidentes por animais peçonhentos. Brasília: Ministério da Saúde; 1998. p. 131.Google Scholar
- Laure CJ. Die Primar strutur des crotamins. Hope Seylers z Physiol Chem. 1975;356:213–5.Google Scholar
- Siqueira AM, Martins NF, de Lima ME, Diniz CR, Cartier A, Brown D, et al. A proposed 3D structure for crotamine based on homology building, molecular simulations and circular dichroism. J Mol Graph Model. 2002;20(5):389–98.PubMedGoogle Scholar
- Coronado MA, Gabdulkhakov A, Georgieva D, Sankaran B, Murakami MT, Arni RK, et al. Structure of the polypeptide crotamine from the Brazilian rattlesnake Crotalus durissus terrificus. Acta Crystallogr D Biol Crystallogr. 2013;69(Pt 10):1958–64.PubMed CentralPubMedGoogle Scholar
- Boni-Mitake M, Costa H, Spencer PJ, Vassilieff VS, Rogero JR. Effects of 60Co gamma radiation on crotamine. Braz J Med Biol Res. 2001;34(12):1531–8.PubMedGoogle Scholar
- Oliveira KC, Silva MN, Gonçalves KO, Spencer PJ, Nascimento N. DSC analysis of irradiated proteins from Crotalus durissus terrificus. Rev Bras Pesq Des. 2011;13:155–62.Google Scholar
- Naumann D. FT-Infrared and FT-Raman spectroscopy in biomedical research. Appl Spectrosc Rev. 2001;36(2–3):239–98.Google Scholar
- Naumann D. Infrared spectroscopy in microbiology. In: Meyers RA, editor. Encyclopedia of Analytical Chemistry. United Kingdom: John Wiley & Sons, Chichester; 2000. p. 102–31.Google Scholar
- Ferreira AAP, Yamanaka H. Microscopia de força atômica aplicada em imunoensaios. Quim Nova. 2006;29(1):137–42.Google Scholar
- Pelton JT, McLean LR. Spectroscopic methods for analysis of protein secondary structure. Anal Biochem. 2000;227(2):167–76.Google Scholar
- Chou PY, Fasman GD. Prediction of protein conformation. Biochemistry. 1974;13(2):222–45.View ArticlePubMedGoogle Scholar
- Sanchez-Ruiz JM. Differential scanning calorimetry of proteins. In: Biswas BB, Roy S, editors. Subcellular biochemistry. Proteins: structure, function and engineering. 24th ed. New York: Plenum Press; 1995.Google Scholar
- Beekes M, Lasch P, Naumann D. Analytical applications of Fourier transform-infrared (FT-IR) spectroscopy in microbiology and prion research. Vet Microbiol. 2007;123(4):305–19.PubMedGoogle Scholar
- Cui FZ, Lin YB, Zhang DM, Tian MB. Irradiation effects on secondary structure of protein induced by keV ions. Radiat Phys Chem. 2001;60(1–2):35–8.Google Scholar
- Engel A, Müller DJ. Observing single biomolecules at work with the atomic force microscope. Nat Struct Biol. 2000;7(9):715–8.PubMedGoogle Scholar
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.