Subscriber access provided by UNIV OF NEW ENGLAND ARMIDALE
Communication
Sequential Protein Expression and Capsid Assembly In Cell: Towards the Study of Multi-Protein Viral Capsids Using Solid-State NMR Techniques Sebastien Alphonse, Boris Itin, Reza Khayat, and Ranajeet Ghose Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00003 • Publication Date (Web): 21 Feb 2018 Downloaded from http://pubs.acs.org on February 23, 2018
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Biochemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 9 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biochemistry
Sequential Protein Expression and Capsid Assembly In Cell: Towards the Study of Multi-Protein Viral Capsids Using SolidState NMR Techniques Sébastien Alphonse†, Boris Itin§, Reza Khayat†,‡ and Ranajeet Ghose*,†,‡,¶ †
Department of Chemistry and Biochemistry, The City College of New York, 160 Convent Avenue, New York, NY 10031 (USA), ‡PhD Programs in Biochemistry and Chemistry, The Graduate Center of CUNY, New York, NY 10016 (USA), ¶PhD Program in Physics, The Graduate Center of CUNY, New York, NY 10016 (USA), §New York Structural Biology Center, 89 Convent Avenue, New York, New York, 10027 (USA)
Supporting Information Placeholder
ABSTRACT: While solid-state NMR (ssNMR) has emerged as a powerful technique to study viral capsids, current studies are limited to capsids formed from single proteins or single polyproteins. The ability to selectively label individual protein components within multi-protein viral capsids and the resulting spectral simplification will facilitate the extension of ssNMR techniques to complex viruses. In vitro capsid assembly by combining individually purified, labeled and unlabeled components in NMR quantities is not a viable option for most viruses. To overcome this barrier, we present a method that utilizes sequential protein expression and in cell assembly of component-specifically labeled viral capsids in amounts suitable for NMR studies. We apply this approach to purify capsids of bacteriophage φ6 isotopically-labeled on only one of its four constituent protein components, the NTPase P4. Using P4-labeled φ6 capsids and the sensitivity enhancement provided by dynamic nuclear polarization (DNP) we illustrate the utility of this method to enable ssNMR studies on complex viruses.
In recent years, magic angle spinning (MAS) solidstate NMR (ssNMR) spectroscopy1 has been successfully utilized to probe the structure2-5 and dynamics6-8 of viral capsids and to characterize the genomes of intact viruses9. The emergence of dynamic nuclear polarization (DNP)10 and 1H-detection techniques11 for signal enhancement have greatly benefited these investigations. However, all these ssNMR studies have focused on virus capsids that are assemblies of single proteins or single polyproteins. New approaches, both biochemical and
spectroscopic, are needed to extend these techniques to complex multi-protein viral capsids. These species are expected to produce spectra with significant resonance overlap when all components are NMR-labeled using identical labeling schemes. The ability to NMR-label individual protein components within these multiprotein capsids would result in substantial spectral simplification and enable the efficient extension of ssNMR approaches to multi-component viruses. Here, we demonstrate the feasibility of generating a multi-protein viral capsid selectively NMR-labeled on a single protein component using the inner capsid of bacteriophage φ6 as our model system. φ6 consists of two protein capsids surrounded by a lipid coat12. The inner capsid, also known as the polymerase complex, encloses the three-segmented doublestranded RNA (dsRNA) genome and is assembled from four distinct protein components (Figure S1): 120 copies of P1, the major capsid protein are arranged as asymmetric dimers on an icosahedral T=1 lattice; 72 copies of the NTPase P4 assemble as hexamers on the 5-fold axis; ~10-12 copies of the RNA-directed RNA polymerase P2 are located on the 3-fold axes, ~60 copies of the accessory protein P7 are most likely located near the 3-fold axis in close proximity to P2. Poranen et. al demonstrated that the φ6 procapsid (PC, the term procapsid denotes the inner capsid without its genomic contents) can be assembled in vitro by mixing purified P1, P2, P4 and P7 proteins. PC assembly was shown to proceed through a well-defined pathway requiring the presence of P4 hexamers to stabilize assembly intermediates; PC formation was not possible in the absence of P413. Given the criti-
ACS Paragon Plus Environment
Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
cal role of P4 in PC formation, we selected it for NMRlabeling in the context of the multi-component φ6 PC.
Figure 1. (a) 15N CP-MAS spectra of P4PC containing UN-labeled P4 recorded with (red) or without (blue) microwave irradiation. (b) A 2D NCO spectrum of P4PC containing 15N-labeled P4 using the directly-connected natural abundance 13C. The bottom right inset shows an aliased Arg Nη/Cζ correlation in expansion. Spectra were recorded at 121 K using a 3.2 mm HCN-DNP probe at 600 MHz (395 GHz) using a ωr = 12 kHz. For the 2D NCO experiment, non-uniform sampling was used (50%). Reconstructed data were processed using Gaussian apodization in both dimensions (LB=-10 Hz, GM=0.1).
15
At first, we attempted to assemble φ6 PCs in vitro using the methodology described by Poranen et al.13. Each protein component (P1, P2, P4 and P7) was individually expressed in E. coli and purified using standard approaches. Assembly was then initiated by mixing P1, P2, P4 and P7 in proportions expected in the φ6 PC in presence of 6% PEG3350. The assembled PCs were then purified using a 10-30% sucrose gradient. Our hope was that success in this simple, direct approach would enable straightforward component-specific NMR labeling. However, in our hands, the assembly efficiency was found to be about 25%, significantly lower than the 50% reported by Poranen et al.13. We attribute this difference to the far higher initial concentrations of the component proteins, especially P1, necessitated by the requirement for large quantities of assembled PCs needed for NMR studies compared to the relatively small amounts needed for biochemical/enzymatic assays. Additionally, the tendency of isolated P1 to aggregate, even at low-tomoderate concentrations, made impractical its purifica-
tion and subsequent use for in vitro assembly of PCs in sufficient quantities for NMR studies. To circumvent these complications with in vitro PC assembly, and taking inspiration from the LEGO-NMR strategy of Mund et al.14 we attempted to assemble φ6 PCs specifically NMR-labeled on only one of its four protein components within E. coli cells i.e. using in vivo assembly. The four proteins that comprise the φ6 PC were cloned into two separate plasmids, allowing their expression in appropriate sequence for in cell PC assembly. First, the gene for the P4 protein, chosen here for isotopic enrichment, was cloned into an IPTG-inducible T7 expression vector (pRSF1b) compatible with glucose-based media for flexible, cost-effective 13Cenrichment. Genes corresponding to the three remaining proteins (P1, P2 and P7) were cloned into a glucoseincompatible araBD expression vector (pBAD) that can be induced by arabinose. These two constructs were cotransformed into an E. coli cell line containing a tunable lysY system which allows expression of T7 lysozyme, a natural competitor of T7 RNA polymerase. Transformed cells were then grown in an isotope enriched M9medium and induced with IPTG to express uniformly 15 N- or 13C,15N-labeled P4. After several hours of induction, depending on the temperature, protein expression was quenched by inducing T7 lysozyme. The cells were then harvested, washed and re-suspended in an unlabeled glycerol-based medium. Expression of unlabeled P1, P2 and P7 proteins was then induced by addition of arabinose in the presence of casamino acids to avoid partial proteolysis of isotope-labeled P4. Labeled P4, unlabeled P1, P2 and P7 could then self-assemble within the E. coli cells to form φ6 PC particles. These particles were subsequently purified using a minor modification of a previously described protocol15, 16. Analysis of the φ6 PCs obtained using this “sequential expression and in-cell assembly” approach using SDS-PAGE (Figure S2) confirmed the presence of the four constituent proteins in appropriate amounts. The integrity of the purified PC particles was tested further using negativestaining transmission electron microscopy (TEM). The overall morphologies of the PC particles were in line with that described by Sun et al.17 (Figure S3), displaying two sub-populations that differed in their occupancies of the P4 binding sites. This underscores a previous observation that although P4 is critical for the assembly and the stability of functional PCs13, 17 not all of the 12 potential P4-binding sites are necessarily or permanently occupied. In order to test the incorporation of labeled P4 within the assembled φ6 PCs we relied on dynamic nuclear polarization (DNP) enhanced ssNMR measurements at 600 MHz (using a 395 GHz gyrotron) under cryogenic conditions (120 K). All NMR samples were supplemented with DNP “juice” containing the paramagnetic biradical AMUpol18. For the initial experiments, we utilized PCs containing uniformly 15N-labeled P4. The level of DNP
ACS Paragon Plus Environment
Page 2 of 9
Page 3 of 9 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biochemistry
enhancement (ε) was measured using simple 1H/15N cross-polarization (CP) experiment recorded with or without hyperpolarization induced by microwave irradiation. The ε value of 41 obtained (Figure 1a) was in line with enhancements seen in comparable systems e.g. assemblies of CA-SP1 polyprotein from HIV-1 virus that displayed ~2-64-fold enhancement depending on experimental conditions10; 35-60-fold enhancement were seen for the intact Pf1 bacteriophage19. This sample could also be used to perform a 2D DNP-NCO experiment20 utilizing the directly connected 13C at natural abundance (Figure 1b).
leading to reduced signal intensity in NMR spectra. We estimate that in about 90% of the particles in our preparation most of the 12 P4 sites on the PC are unoccupied (P4 occupancy in at least a single site is required for a stable PC). It has been shown by Sun et al.17 that incubation of the purified PCs with an excess of separately purified P4 followed by an additional round of PC purification enhances their P4 content. Since NMR-labeled P4 can be expressed in significant amounts in isolation (Figure S4), this procedure adapted for the present case would increase the amount of P4PC leading to increased signal in the NMR spectra.
To further investigate the properties of the φ6 PCs obtained using our approach, we utilized PCs constituted from U-13C,15N-labeled P4 and compared them to purified isolated U-13C,15N-labeled P4 hexamers (Figure S4) under similar conditions. A 60-fold enhancement was observed for isolated P4 hexamers under DNP conditions (using 1H/13C CP) compared to a 33-fold enhancement for the in-particle P4 (referred to as P4PC) (Figure 2a). This 2-fold difference in enhancement is likely the result of decreased accessibility of the biradical to P4PC compared to isolated P4 hexamers. A similar explanation has been proposed before10. A comparison of the 13C-13C DNP-DARR spectrum of isolated P4 hexamers with that of P4PC showed similar overall patterns of peaks, largely coincident with that calculated using SHIFTX221 and the structure of P422 (Figure S5). However, the spectrum of isolated P4 displayed broad lines (Figure 2b), while that of P4PC displayed significantly narrower lines for isolated resonances. The characteristic linewidths for well-resolved peaks in the spectrum of P4PC were estimated to be in the 0.4-0.8 ppm range. Two possible reasons for these differences can be conceived – (1) the high local symmetry of the P4 hexamer units when assembled on the PC compared to individual hexamers that likely retain substantial conformational heterogeneity; (2) greater exposure to the biradical in the case of isolated P4 (also attributed to the larger ε value, as stated above) leading to a higher degree of paramagnetic relaxation enhancement. It is notable, however, the 13C-13C DNP-DARR spectra of both species showed sparse correlations involving methyl groups (Figure S6). It has been shown that methyl dynamics at cryogenic temperatures interfere with 1H decoupling and reduce CP efficiency leading to attenuated intensity of methyl resonances. These deleterious effects can be overcome to a large extent by 2Hlabeling23. As noted previously, the EM images of the assembled φ6 PCs indicated variable occupancies of the P4 binding sites (Figure S3). In our case, based on the symmetry properties of the φ6 capsid and the expected copy numbers of the component proteins, a PC sample would consist of ~17% labeled material if all 12 potential binding sites were occupied by NMR-labeled P4 hexamers. This is clearly not true in the present case, as noted above,
Figure 2. (a) 13C CP-MAS spectra of isolated U-15N,13Clabeled hexameric P4 (top) and P4PC containing U-15N,13Clabeled P4 recorded with (red) or without (blue) microwave irradiation. (b) Expansions of selected regions of 13C13 C DNP-DARR spectra of isolated U-15N,13C-labeled hexameric P4 (green) and P4PC containing U-15N,13C-labeled P4 (magenta) illustrating specific correlations: Gly Cα− C’ (top left), Tyr Cε1/2−Cζ (top right), Cα− C’ for several residue types (Ile, Pro, Ser, Thr and Val; bottom left), Arg Cδ−Cγ, Leu Cβ−Cγ, Ile Cβ−Cγ1, Lys Cε−Cδ (bottom right). All spectra were recorded at 120 K using a 1.9 mm HCN-DNP probe at 600 MHz (395 GHz) using a ωr = 23 kHz and spin-diffusion time of 8 ms. Data were processed
ACS Paragon Plus Environment
Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
using squared cosine-bell apodization in both dimensions. Also shown for reference as orange ovals are resonances expected based on chemical shifts calculated from the structure of P4 using SHIFTX2.
The approach presented is easily generalizable to the other components of the φ6 PC by replacing the gene for P4 (chosen here for labeling) on the pRSF1b system by genes corresponding to either the P2 or P7 proteins. Labeling of P1 should also be straightforward despite the fact that its high local concentration, without the presence P4, triggers aggregation into forms incompatible with stable PC formation. In a modified approach, we would express P2, P4 and P7 under the dependency of the araBD promoter in a glycerol based medium, followed by switching to a glucose-based medium for expression and labeling of P1. Glucose, being a strong antagonist of the araBD promoter, should suppress the expression of P2, P4 and P7. To summarize, using sequential protein expression and in cell assembly of φ6 PCs we have demonstrated our ability to produce multi-component viral capsids NMRlabeled in component-specific fashion. The quality of our initial DNP-enhanced ssNMR spectra suggest the viability of NMR measurements on these systems. However, further optimization of sample conditions, isotopelabeling schemes and indeed, experimental parameters, are needed to enable detailed analyses. These efforts are currently underway in the laboratory.
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
(10)
ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website.
(11)
Detailed experimental procedures and supplementary data, including Table S1 and Figures S1−S6 (PDF). (12)
AUTHOR INFORMATION Corresponding Author
(13)
*
[email protected] Notes
(14)
The authors declare no competing financial interests.
ACKNOWLEDGMENT
(15)
The authors thank Prof. Paul Gottlieb and Dr. Alexandra Alimova for the kind gift of the pLM638 plasmid and advice on purification of φ6 PCs. This work is supported by a grant from the NSF (MCB 1412007). Instrumentation at the NYSBC is supported by grants from the Simons Foundation (349237), NYSTAR and the NIH (GM103310, CO6RR015495, S10RR029249).
(16)
(17)
REFERENCES (1)
Quinn, C. M., Lu, M., Suiter, C. L., Hou, G., Zhang, H., and Polenova, T. (2015) Magic angle spinning NMR of viruses, Prog. NMR Spectr. 86-87C, 21-40.
(18)
Page 4 of 9 Han, Y., Ahn, J., Concel, J., Byeon, I.-J. L., Gronenborn, A. M., Yang, J., and Polenova, T. (2010) Solid-state NMR studies of HIV-1 capsid protein assemblies, J. Am. Chem. Soc. 132, 1976-1987. Goldbourt, A., Gross, B. J., Day, L. A., and McDermott, A. E. (2007) Filamentous phage studied by magic-angle spinning NMR: resonance assignment and secondary structure of the coat protein in Pf1, J. Am. Chem. Soc. 129, 2338-2344. Chen, B., and Tycko, R. (2010) Structural and dynamical characterization of tubular HIV-1 capsid protein assemblies by solid state nuclear magnetic resonance and electron microscopy, Protein Sci. 19, 716-730. Morag, O., Sgourakis, N. G., Baker, D., and Goldbourt, A. (2015) The NMR-Rosetta capsid model of M13 bacteriophage reveals a quadrupled hydrophobic packing epitope, Proc. Natl. Acad. Sci. USA 112, 971-976. Byeon, I. J., Hou, G., Han, Y., Suiter, C. L., Ahn, J., Jung, J., Byeon, C. H., Gronenborn, A. M., and Polenova, T. (2012) Motions on the millisecond time scale and multiple conformations of HIV-1 capsid protein: implications for structural polymorphism of CA assemblies, J. Am. Chem. Soc. 134, 6455-6466. Wang, M., Quinn, C. M., Perilla, J. R., Zhang, H., Shirra, R., Jr., Hou, G., Byeon, I. J., Suiter, C. L., Ablan, S., Urano, E., Nitz, T. J., Aiken, C., Freed, E. O., Zhang, P., Schulten, K., Gronenborn, A. M., and Polenova, T. (2017) Quenching protein dynamics interferes with HIV capsid maturation, Nature Commun. 8, 1779. Lorieau, J. L., Day, L. A., and McDermott, A. E. (2008) Conformational dynamics of an intact virus: order parameters for the coat protein of Pf1 bacteriophage, Proc. Natl. Acad. Sci. USA 105, 10366-10371. Sergeyev, I. V., Day, L. A., Goldbourt, A., and McDermott, A. E. (2011) Chemical shifts for the unusual DNA structure in Pf1 bacteriophage from dynamic-nuclear-polarization-enhanced solid-state NMR spectroscopy, J. Am. Chem. Soc. 133, 2020820217. Gupta, R., Lu, M., Hou, G., Caporini, M. A., Rosay, M., Maas, W., Struppe, J., Suiter, C., Ahn, J., Byeon, I. J., Franks, W. T., Orwick-Rydmark, M., Bertarello, A., Oschkinat, H., Lesage, A., Pintacuda, G., Gronenborn, A. M., and Polenova, T. (2016) Dynamic nuclear polarization enhanced MAS NMR spectroscopy for structural analysis of HIV-1 protein assemblies, J. Phys. Chem. B 120, 329-339. Barbet-Massin, E., Felletti, M., Schneider, R., Jehle, S., Communie, G., Martinez, N., Jensen, M. R., Ruigrok, R. W., Emsley, L., Lesage, A., Blackledge, M., and Pintacuda, G. (2014) Insights into the structure and dynamics of measles virus nucleocapsids by 1H-detected solid-state NMR, Biophys. J. 107, 941-946. Alphonse, S., and Ghose, R. (2017) Cystoviral RNA-directed RNA polymerases: Regulation of RNA synthesis on multiple time and length scales, Virus Res. 234, 135-152. Poranen, M. M., Paatero, A. O., Tuma, R., and Bamford, D. H. (2001) Self-assembly of a viral molecular machine from purified protein and RNA constituents, Mol. Cell 7, 845-854. Mund, M., Overbeck, J. H., Ullmann, J., and Sprangers, R. (2013) LEGO-NMR spectroscopy: a method to visualize individual subunits in large heteromeric complexes, Angew. Chem. Int. Ed. Engl. 52, 11401-11405. Frilander, M., and Bamford, D. H. (1995) In vitro packaging of the single-stranded RNA genomic precursors of the segmented double-stranded RNA bacteriophage φ6: the three segments modulate each other's packaging efficiency, J. Mol. Biol. 246, 418-428. Pirttimaa, M. J., Paatero, A. O., Frilander, M. J., and Bamford, D. H. (2002) Nonspecific nucleoside triphosphatase P4 of double-stranded RNA bacteriophage φ6 is required for singlestranded RNA packaging and transcription, J. Virol. 76, 1012210127. Sun, X., Pirttimaa, M. J., Bamford, D. H., and Poranen, M. M. (2013) Rescue of maturation off-pathway products in the assembly of pseudomonas phage φ6, J. Virol. 87, 1327913286. Sauvée, C., Rosay, M., Casano, G., Aussenac, F., Weber, R. T., Ouari, O., and Tordo, P. (2013) Highly efficient, water-soluble
ACS Paragon Plus Environment
Page 5 of 9 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biochemistry
(19)
(20)
(21)
(22)
polarizing agents for dynamic nuclear polarization at high frequency, Angew. Chem. Int. Ed. Engl. 52, 10858-10861. Sergeyev, I. V., Itin, B., Rogawski, R., Day, L. A., and McDermott, A. E. (2017) Efficient assignment and NMR analysis of an intact virus using sequential side-chain correlations and DNP sensitization, Proc. Natl. Acad. Sci. USA 114, 5171-5176. Baldus, M., Petkova, A. T., Herzfeld, J., and Griffin, R. G. (1998) Cross polarization in the tilted frame: assignment and spectral simplification in heteronuclear spin systems, Molec. Phys. 95, 1197-1207. Han, B., Liu, Y., Ginzinger, S. W., and Wishart, D. S. (2011) SHIFTX2: significantly improved protein chemical shift prediction, J. Biomol. NMR 50, 43-57. El Omari, K., Meier, C., Kainov, D., Sutton, G., Grimes, J. M., Poranen, M. M., Bamford, D. H., Tuma, R., Stuart, D. I., and Mancini, E. J. (2013) Tracking in atomic detail the functional specializations in viral RecA helicases that occur during evolution, Nucleic Acids Res. 41, 9396-9410.
(23)
Ni, Q. Z., Markhasin, E., Can, T. V., Corzilius, B., Tan, K. O., Barnes, A. B., Daviso, E., Su, Y., Herzfeld, J., and Griffin, R. G. (2017) Peptide and protein dynamics and lowtemperature/DNP magic angle spinning NMR, J. Phys. Chem. B 121, 4997-5006.
ACS Paragon Plus Environment
Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Figure 1 177x201mm (300 x 300 DPI)
ACS Paragon Plus Environment
Page 6 of 9
Page 7 of 9 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biochemistry
Figure 2 256x419mm (300 x 300 DPI)
ACS Paragon Plus Environment
Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Table of Contents graphic 44x38mm (300 x 300 DPI)
ACS Paragon Plus Environment
Page 8 of 9
Page 9 of 9 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biochemistry
Possible Cover Image 280x364mm (300 x 300 DPI)
ACS Paragon Plus Environment
