Chromophore Environment Provides Clue to "Kindling Fluorescent Protein" Riddle*

Dmitriy M. ChudakovDagger §, Alexei V. FeofanovDagger , Nikolay N. MudrikDagger §, Sergey LukyanovDagger §, and Konstantin A. LukyanovDagger §

From the Dagger  Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Science and § Evrogen, Joint Stock Company, Miklukho-Maklaya 16/10, 117997 Moscow, Russia

Received for publication, November 25, 2002, and in revised form, December 19, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

asCP, the unique green fluorescent protein-like nonfluorescent chromoprotein from the sea anemone Anemonia sulcata, becomes fluorescent ("kindles") upon green light irradiation, with maximum emission at 595 nm. The kindled protein then relaxes to a nonfluorescent state or can be "quenched" instantly by blue light irradiation. In this work, we used asCP mutants to investigate the mechanism underlying kindling. Using site-directed mutagenesis we showed that amino acids spatially surrounding Tyr66 in the chromophore are crucial for kindling. We propose a model of the kindling mechanism, in which the key event is chromophore turning or cis-trans isomerization. Using site-directed mutagenesis we also managed to transfer the kindling property to the two other coral chromoproteins. Remarkably, most kindling mutants were capable of both reversible and irreversible kindling. Also, we obtained novel variants that kindled upon blue light irradiation. The diversity of photoactivated fluorescent proteins that can be developed by site-directed mutagenesis is promising for biotechnological needs.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Recently a GFP-like1 chromoprotein from the sea anemone Anemonia sulcata was discovered (1). This protein, named asCP, absorbs light effectively, with a maximum at 568 nm, and causes purple coloration of anemone tentacle tips. Initially nonfluorescent, asCP becomes fluorescent (kindles) in response to intense green light irradiation, with an excitation maximum at 575 nm and an emission maximum at 595 nm. The protein then relaxes back to its initial nonfluorescent state, or it can be quenched instantly by short blue light irradiation. Both kindling in green light and quenching in blue light are reversible processes for the wild-type protein. The nature of these striking changes remains unclear; here we propose an explanation based on the wild-type and mutant asCP properties.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Cloning, Expression, and Mutagenesis-- For heterologous expression of proteins, full-length coding regions were cloned into the pQE30 vector (Qiagen). Proteins fused to an N-terminal 6xHis tag were expressed in Escherichia coli and purified using the Talon metal affinity resin (Clontech). Site-directed mutagenesis was performed by overlap extension PCR with primers containing the appropriate target substitutions (2).

Screening-- Screening of E. coli colonies expressing mutant proteins was performed using a Nikon Optiphot fluorescent microscope and an Olympus US SZX12 fluorescent stereo microscope. Photographs were made using an Olympus DP50 camera.

Spectroscopy-- Absorption spectra were recorded on a Beckman DU520 UV/VIS spectrophotometer. A Varian Cary eclipse fluorescence spectrophotometer was used to measure excitation-emission spectra and as a light source to determine the action spectrum for the quenching of asCP-A148G mutant. The following external light sources were used: 458 nm (Ar-ion laser line), 430-490 nm (fluorescent microscope filter), 460-490 nm (fluorescent microscope filter), 514 nm (Ar-ion laser line), 532 nm (Nd laser line), 543 nm (HeNe laser line), and 546 (±14 nm, fluorescent microscope filter) as the light sources for estimating asCP kindling and quenching optima; 430-490 nm (fluorescent microscope filter) and 460-490 nm (fluorescent microscope filter) as the quenching light sources for asCP and its mutants and as the kindling light sources for hcCP-N165A,G mutants; 532 nm (Nd laser line) and 546 (±14) nm (fluorescent microscope filter) as the kindling light sources for the asCP, its mutants and for those cgCP and hcCP mutants that kindle in green light.

    RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

asCP Kindling and Relaxation-- asCP kindles in response to green light and quenches in blue light. As the kindling light intensity is relatively high, it was difficult to measure the optimum kindling wavelength precisely. We roughly estimated kindling and quenching wavelength optima using different light sources. asCP irradiation at 532, 543, or 546 nm caused bright kindling (hereinafter referred to as kindling green light). However, the 514 nm light caused only low level kindling, but it quenched previously kindled asCP considerably, indicating a probable intersection with a blue quenching light. The blue light irradiation at 458, 430-490, or 460-490 nm caused no kindling and quenched previously kindled asCP. It is likely that the kindling optimum corresponds to the absorption peak at 568 nm (Fig. 1A). (Extinction coefficient = ~120,000 M-1 cm-1 according to our refined data. Previous studies showed a lower extinction coefficient, probably due to the incomplete protein maturation (1)). The action spectrum for the quenching was determined precisely using an asCP mutant (see "asCP-A148G Mutant Study").


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 1.   Changes in excitation and absorption spectra during kindling. A, normalized asCP absorption spectra before kindling (solid line) and fluorescence excitation spectra after kindling in green light (dotted line). B, difference between kindled and initial asCP-A148G mutant protein absorption spectra. The main absorption peak at 568 nm falls, but absorption growth is observed with a peak at 445 nm. C, relative decrease of the fluorescent brightness for the asCP-A148G mutant after quenching using different wavelengths of light irradiation. Fluorescent emission brightness at 610 nm was measured using 570 nm excitation light.

The intensity and duration of the green light illumination needed for kindling depend on several parameters: the protein sample thickness and transparency, protein concentration, and wavelength used. In the protein sample, kindling is observed when kindling speed (which depends both on irradiation power and duration) is higher than the kindled asCP relaxation speed. For example, irradiation of asCP expressing E. coli colony in fluorescent microscope through 10× objective (TRITC filter set, 100-W lamp) causes quick kindling, which reaches its maximum in ~10-20 s. Irradiation through a 5× objective causes very slow kindling, which reaches its maximum in about 10 min. A quarter of 5× objective intensity causes no visible kindling.

We also measured the relaxation kinetics of the kindled asCP at room temperature, which was shown to have a 7-s half-life. However, kindled asCP stayed fluorescent for a much longer period of time, for more than 24 h, upon immediate cooling with liquid nitrogen and subsequent placement at -70 °C.

Chromophore Environment in GFP-like Proteins-- Currently, the x-ray structures for two GFP-like proteins are known, i.e. GFP itself and coral DsRed protein (3-6). These data can be used as three-dimensional models and are suitable for the analysis of asCP and other GFP-like fluorescent proteins (FPs) and nonfluorescent chromoproteins (CPs). According to the x-ray structure, Tyr66 of the GFP chromophore is oriented to Thr203 and His148, forming a hydrogen bond with the latter (3, 4). Our previous studies also showed the importance of the amino acids at positions 148, 203, and, interestingly, 165 for the fluorescent properties of coral GFP-like proteins (1, 7, 8) (to facilitate comparisons, we have denoted all amino acids according to their alignment with GFP).

Remarkably, position 148 is clearly different between coral FPs and CPs cloned to date. The known coral FPs (about 20) contain Ser148 (Table I). Similar to GFP, the DsRed crystal structure shows that the chromophore is stabilized through position 148 (5, 6). It is highly probable that in most coral FPs the conserved Ser148 is engaged in chromophore stabilization in the fluorescent state. In CPs (except asCP), position 148 is Cis or Asn (Table I), whereas asCP contains the uncommon Ala148. At the same time, asCP is an exceptional protein, capable of being in both the nonfluorescent or "chromo" state and the fluorescent state. (The chromo state indicates that the protein has a high extinction coefficient but a low quantum yield, whereas in the fluorescent state the protein is characterized by a high quantum yield.) Noticeably, a single X148S mutation makes coral CPs (including asCP) fluorescent, with the excitation/emission characteristics corresponding to their absorption peaks (1, 8). It is likely that in coral CP X148S mutants, the chromophore state is similar to that in FPs.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Amino acids in positions 148, 165, and 203 in Aequorea victoria GFP and Anthozoa chromoproteins and fluoroproteins
Data on fluoroproteins were derived from Labas et al. (14).

Like Ala148, Ser165 in asCP is also exceptional. Typically this position is occupied by Val or Ile in FPs or by Asn in CPs (Table I). Position 203 does not differ so definitely between coral FPs and CPs, although most FPs contain His203.

Properties of asCP Mutants-- We examined the kindling properties of asCP with mutations at amino acid position 148, in combination with wild-type Ser165 and mutated S165V. His203 was also mutated. Table II lists the properties of the mutant proteins. Random mutagenesis of Ala148 in asCP gave mutants with Gly, Ser, Cys, Asn and Thr148, which folded in E. coli at 37 °C. All of these mutants were intensely colored except for the poorly folded A148T. Mutants A148C and A148N lost the kindling property and stayed nonfluorescent, the A148G kept the wild-type protein properties in general (see detailed study below), and mutants A148S and A148T became fluorescent and kept the kindling property (although A148T lost the quenching property). It is reasonable to assume that changes to essential position 148 affected asCP fluorescent and kindling properties because this position is important for FPs chromophore stabilization. However, interestingly, our results demonstrated that changes in position 148 caused no significant alteration in protein coloration.

                              
View this table:
[in this window]
[in a new window]
 
Table II
asCP mutant properties

It is noteworthy that the asCP-S165V mutant was fluorescent and completely lost all its kindling/quenching properties. Moreover, this mutation made asCP fluorescent independently of position 148. Thus, asCP mutant S165V with mutation at position 148 (A148G, A148S, A148C, or A148N) were fluorescent and incapable of quenching or additional kindling (A148T/S165V mutant folding was poor). At the same time, all S165V mutants were relatively weakly colored compared with wild-type asCP and A148X mutants.

Mutation of His203 also influenced asCP kindling. The asCP-A148S/H203S mutant was fluorescent with an emission maximum at 590 nm and, in contrast to the A148S mutant, was incapable of kindling or quenching.

Model of the Kindling Mechanism-- Two plausible explanations can be proposed for a protein switching from a chromo to fluorescent form: either a change in chromophore charge or a change in chromophore environment. Neutral and anionic forms of the GFP chromophore have clearly different excitation spectra, peaking at 396 and 476 nm, respectively, but have close emission maxima at 508 and 503 nm (9). A simultaneous decrease of the 396 nm and increase of the 476 nm excitation peaks was observed in the course of irreversible GFP photoactivation (10, 11). However, the excitation maximum wavelength of the kindled asCP was very close to its absorption maximum before kindling (Fig. 1A). This argues for the same chromophore charge of both initial nonfluorescent and kindled fluorescent asCP, suggesting that some other changes put the chromophore into a new state. The influence of temperature on kindled asCP relaxation time (see above) indicates that protein conformation changes are involved.

On the basis of our findings, we propose a model whereby kindling is related to cis-trans isomerization (or turning) of the excited chromophore from a chromo to a fluorescent state. As Ser148, characteristic of coral FPs, makes asCP fluorescent, we presume that the asCP chromophore fluorescent (kindled) state is similar to the fluorescent state of coral FPs and GFP. Therefore, if kindling is tied with the chromophore cis-trans isomerization, asCP Tyr66 should contact Ser165 before kindling. (The latter follows from the DsRed crystal map. In DsRed, Ile165 spatially blocks this conformation (Fig. 2A).) Therefore, we suggest that the exceptional wild-type Ser165 in asCP stabilizes the chromophore in the chromo state. However, the excited chromophore has a chance to isomerize to the fluorescent state, close to that of GFP and DsRed (Fig. 2, B and C). Notably, the S165V change makes asCP fluorescent. This mutant loses all of the kindling/quenching properties of the wild-type protein, suggesting that the chromo state is blocked by Val165.


View larger version (49K):
[in this window]
[in a new window]
 
Fig. 2.   DsRed chromophore and asCP kindling model. A, schematic stereo outline of the DsRed chromophore and selected neighboring residues in a "space-filled" representation. Carbon atoms are shown in gray, nitrogen atoms in blue, and oxygen atoms in red. This image was generated using RasMol 2.6 software based on PDB file 1G7K (6). B and C, model of the asCP chromophore cis-trans isomerization, which leads to asCP kindling. Schematic outline of the chromophore (known for DsRed) and selected neighboring residues in chromo (B, before kindling) and fluorescent (C, after kindling) asCP states. Carbon atoms are shown in gray, nitrogen atoms in blue, and oxygen atoms in red. Images were generated using RasMol 2.6 software. Computer modeling for asCP was performed using Swiss-PdbViewer and HyperChem 5.01 software based on DsRed crystal structure.

As in GFP Thr203 is known to take part in chromophore stabilization, and asCP contains His203, which is present in most coral FPs, it is possible that His203 takes part in the temporary asCP chromophore stabilization in the kindled state. The proposed model correlates well with asCP mutants properties (see Table II).

For example, previously mentioned asCP-A148S mutant is fluorescent and is capable of additional kindling in green light and quenching in blue light. Within several minutes after kindling or quenching irradiation is stopped, the protein is restored to its initial fluorescent brightness. Within the proposed model, these properties of the A148S mutant may be explained as such; Ser148 functions to stabilize the chromophore in the fluorescent state as it does in coral FPs. Nevertheless, some portion of the chromophore rests stabilized by Ser165 in the chromo state, making the protein capable of additional kindling. In turn, the fluorescent chromophore fraction may be quenched, as the chromo state is not prohibited.

Mutants asCP-A148C and A148N (both variants are typical for coral CPs) lose kindling ability and stay nonfluorescent, although intensely colored, with an absorption maximum close to wild-type asCP. The A148C or A148N mutation probably makes the fluorescent state noncompetitive, so that no fluorescence or kindling is observed. However, mutants asCP-A148C/S165V and asCP-A148N/S165V were fluorescent and incapable of kindling or quenching, indicating that all chromophores were in the fluorescent state, which was noncompetitive in the presence of Ser165.

Remarkably, cis-trans chromophore photoisomerization was proposed earlier to explain GFP switching from the fluorescent to the long-lived "dark" state. Quantum chemical calculations showed a high probability of excited GFP chromophore cis-trans isomerization (12). Extending our model, we may also surmise that two chromophore conformations are common for the chromo, fluorescent, and kindling GFP-like proteins. We have summarized this hypothesis in Table III, taking the initial GFP chromophore state as cis and the dark state as trans.

                              
View this table:
[in this window]
[in a new window]
 
Table III
Possible chromophore conformations in GFP-like proteins

AsCP-A148G Mutant Study-- The A148G mutant behaved similar to wild-type asCP, with very low initial fluorescence and an ability to kindle in green light and quench in blue light. However, the kindled state half-life of the A148G mutant was 50 s, which is severalfold longer than the wild-type protein. We used the A148G mutant, the closest to wild-type asCP, as a model because its prolonged kindled state half-life allowed us to measure and compare the absorption spectra before and after reversible kindling.

We found that absorption at 568 nm fell considerably in the course of A148G kindling (Fig. 3A), and simultaneous absorption growth at 445 nm was observed (Fig. 1B). (We observed the same effect on the wild-type protein, but the fast relaxation hampered accurate measurement.) The fluorescence excitation maximum of kindled A148G and asCP was 575 nm, making it evident that the kindled chromophore still absorbs at this wavelength. Therefore, we suggest that there are at least two equilibrium forms of the kindled chromophore. The first is fluorescent, with the excitation peaked at 575 nm, whereas the second absorbs at 445 nm and is nonfluorescent. It is likely that Tyr66 is protonated in this second state, as a close absorption maximum (430 nm) was registered for acid-denatured asCP (13). It is also very likely that this equilibrium state is responsible for kindled protein quenching, which happens upon blue light irradiation. We determined the action spectrum for the quenching using asCP-A148G mutant by measuring the decrease of its weak initial fluorescence upon different wavelength light irradiation (Fig. 1C). The quenching wavelength optimum was shown to be close to the absorption peak, which appears after asCP-A148G kindling.


View larger version (31K):
[in this window]
[in a new window]
 
Fig. 3.   Imaging of kindling proteins in E. coli colonies. A, chromo-fluorescent switching of asCP-A148G mutant. The round-shaped part of the E. coli colony expressing the A148G mutant was kindled with intense green light for 1 min using a fluorescent microscope (TRITC filter set, 100-W lamp, 10× magnification). This part fluoresces brightly, but absorption of the irradiated region is low. After several minutes, the kindled protein relaxed to the nonfluorescent state while its absorption recovered. B, dual red fluorescent labeling using two kindling proteins. E. coli colonies expressing hcCP-N165A and asCP-A148G mutants were grown on the same Petri dish. Colonies were irradiated by blue light (460-490 nm) for several seconds using a fluorescent microscope. Immediately after blue light irradiation, colonies expressing hcCP mutant were kindled and fluoresced brightly. After 2 min of green light irradiation (TRITC filter set), the hcCP mutant relaxed to the nonfluorescent state, whereas asCP mutant was kindled and fluoresced brightly. This switching could be repeated many times.

Transfer of the Kindling Property to Nonkindling Chromoproteins-- To confirm the crucial role of positions 165, 148, and 203 in kindling and to obtain novel kindling variants, we made several point mutations in two other coral chromoproteins, hcCP (from Heteractis crispa (7)) and cgCP (from Condilactis gigantea (7)). As a result, we managed to transfer the kindling property to both proteins and also identified a novel type of kindling upon blue light irradiation.

Wild-type cgCP contains Cys148, Asn165, and Leu203. A single C148S mutation made this protein fluorescent with an emission maximum at 620 nm, also making it capable of additional kindling in green light. The cgCP-C148S plus N165S mutant was weakly fluorescent and could be brightly kindled in green light, similar to asCP.

For hcCP we started with a mutant containing a number of folding substitutions (A5S, T39A, L181H, P208L, K211E), because wild-type protein folding is very poor in E. coli at 37 °C. We made several changes of Cys148, Asn165, and Ile203. Among the mutants obtained, hcCP-C148S/N165S/I203H and hcCP-C148A/N165G/I203H clearly showed the kindling property. Both of these proteins were weakly fluorescent and could be brightly kindled in green light.

The only mutant protein obtained on the basis of hcCP and cgCP that demonstrated quenching under blue light was the cgCP and hcCP hybrid protein: cgCP-C148S before Ser165 and hcCP after Ser165. This mutant protein was fluorescent and could be additionally kindled in green light and considerably quenched in blue light, similar to the asCP-A148S mutant.

Novel Type of Kindling-- A novel type of kindling was found in two hcCP mutants containing a single change at position 165, N165A and N165G (with no additional random mutations). These nonfluorescent proteins kindled quickly upon blue light irradiation. In the kindled form they were fluorescent with an emission peak at 620 nm and excitation at 590 nm. The hcCP-N165A mutant fluorescence was observed for about 1 min after kindling, whereas the N165G mutant was fluorescent for 10 s.

In contrast to other kindling variants, the kindling wavelength of these mutants was different than the fluorescent excitation wavelength, giving advantages for biotechnology applications, as there is no background kindling in the course of object tracking. Also, dual red fluorescent labeling becomes possible (Fig. 3B).

Irreversible Kindling-- Surprisingly, for most mutant kindling proteins (but not for wild-type asCP) the irradiation with kindling light of greater intensity caused irreversible kindling. For example, irreversibly kindled A148G mutant gave stable red fluorescence with an intensity up to 40 times brighter than the unkindled protein, reaching about 50% of reversibly kindled protein brightness. The fluorescence of the irreversibly kindled A148G mutant protein did not fade for at least a year after kindling. Further studies are needed to understand the nature of this irreversible effect.

Concluding Comments-- The kindling effect was relatively easy to transfer to nonkindling, nonfluorescent, GFP-like Anthozoa chromoproteins. This was achieved by performing amino acid substitutions at positions 148, 165, and 203 around chromophore Tyr66, confirming the key role of these positions in kindling.

asCP Ala148 and Ser165 positions mutagenesis gave proteins with essentially modified or blocked kindling and quenching. The properties of these mutants enabled us to propose a model that implies that asCP kindling is linked to chromophore cis-trans isomerization. Although Ser165 takes part in chromophore stabilization in the initial chromo state, His203 takes part in kindled chromophore temporary stabilization in the fluorescent (kindled) state, which is similar to the fluorescent chromophore state in GFP and DsRed.

    FOOTNOTES

* This work was supported by grants from the Russian Foundation for Basic Research (01-04-49037) and the Russian Foundation for Support of Domestic Science (to S. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Institute of Bioorganic Chemistry, Miklukho-Maklaya 16/10, 117997 Moscow, Russia. Tel.: 7-095-429-80-20; E-mail: kluk@ibch.ru.

Published, JBC Papers in Press, December 19, 2002, DOI 10.1074/jbc.M211988200

    ABBREVIATIONS

The abbreviations used are: GFP, green fluorescent protein; FP, fluorescent protein; CP, chromoprotein; TRITC, tetramethylrhodamine isothiocyanate.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

1. Lukyanov, K. A., Fradkov, A. F., Gurskaya, N. G., Matz, M. V., Labas, Y. A., Savitsky, A. P., Markelov, M. L., Zaraisky, A. G., Zhao, X., Fang, Y., Tan, W., and Lukyanov, S. A. (2000) J. Biol. Chem. 275, 25879-25882[Abstract/Free Full Text]
2. Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K., and Pease, L. R. (1989) Gene 77, 51-59[CrossRef][Medline] [Order article via Infotrieve]
3. Yang, F., Moss, L. G., and Phillips, G. N. (1996) Nat. Biotechnol. 14, 1246-1251[Medline] [Order article via Infotrieve]
4. Ormö, M., Cubitt, A. B., Kallio, K., Gross, L. A., Tsien, R. Y., and Remington, S. J. (1996) Science 273, 1392-1395[Abstract]
5. Wall, M. A., Socolich, M., and Ranganathan, R. (2000) Nat. Struct. Biol. 7, 1133-1138[CrossRef][Medline] [Order article via Infotrieve]
6. Yarbrough, D., Wachter, R. M., Kallio, K., Matz, M. V., and Remington, S. J. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 462-467[Abstract/Free Full Text]
7. Gurskaya, N. G., Fradkov, A. F., Terskikh, A., Matz, M. V., Labas, Y. A., Martynov, V. I., Yanushevich, Y. G., Lukyanov, K. A., and Lukyanov, S. A. (2001) FEBS Lett. 19, 16-20
8. Bulina, M., Chudakov, D., Mudrik, N., and Lukyanov, K. (2002) BMC Biochem. 3, 7[CrossRef][Medline] [Order article via Infotrieve]
9. Heim, R., Prasher, D. C., and Tsien, R. Y. (1994) Proc. Natl. Acad. Sci. U. S. A. 20, 12501-12504
10. Yokoe, H., and Meyer, T. (1996) Nat. Biotechnol 14, 1252-1256[Medline] [Order article via Infotrieve]
11. Patterson, G. H., and Lippincott-Schwartz, J. A. (2002) Science 13, 1873-1877
12. Weber, W., Helms, V., McCammon, J. A., and Langhoff, P. W. (1999) Proc. Natl. Acad. Sci. U. S. A. 25, 6177-6182[CrossRef]
13. Martynov, V. I., Savitsky, A. P., Martynova, N. Y., Savitsky, P. A., Lukyanov, K. A., and Lukyanov, S. A. (2001) J. Biol. Chem. 276, 21012-21016[Abstract/Free Full Text]
14. Labas, Y. A., Gurskaya, N. G., Yanushevich, Y. G., Fradkov, A. F., Lukyanov, K. A., Lukyanov, S. A., and Matz, M. V. (2002) Proc. Natl. Acad. Sci. U. S. A. 2, 4256-4261[CrossRef]


Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.