Rational Design of Self-assembling Artificial Proteins Utilizing a Micelle-Assisted Protein Labeling Technology (MAPLabTech): Testing the Scope

TLDR: In this article, the synthesis of self-assembling artificial proteins (SAPs) using micelle-assisted protein labeling technology (MAPLabTech) has been described by developing a chemical method for site-specific labeling of a surfaceexposed cysteine residue of globular proteins.

Abstract: Self-assembling artificial proteins (SAPs) have gained enormous interest in recent years due to their applications in different fields. Synthesis of well-defined monodisperse SAPs is accomplished predominantly through genetic methods. However, the last decade witnessed the use of few chemical technologies for that purpose. In particular, micelle-assisted protein labeling technology (MAPLabTech) has made huge progress in this area. The first generation MAPLabTech focused on site-specific labeling of the active-site residue of serine proteases to make SAPs. Further, this methodology was exploited for labeling of N-terminal residue of a globular protein to make functional SAPs. In this study, we describe the synthesis of novel SAPs by developing a chemical method for site-specific labeling of a surface-exposed cysteine residue of globular proteins. In addition, we disclose the synthesis of redox- and pH-sensitive SAPs and their systematic self-assembly and dis-assembly studies using complementary biophysical studies. Altogether these studies further expand the scope of MAPLabTech in different fields such as vaccine design, targeted drug delivery, diagnostic imaging, biomaterials, and tissue engineering.

Figures

Figure 2. Characterization and self-assemby data for Try-OEG-SS-C12-2T. a MALDI-TOF. data of reaction mixture. b. MALDI-TOF data of purified conjugate. c. DLS data of purified conjugate. d. SEC chromatogram of purified conjugate. e. SEC-MALS data of purified conjugate.

Table 1. Comparison of SEC data of N-terminus vs. thiol conjugate of BSA.

Figure 5. Characterization and pH-dependent disassembly studies. a. MALDI-TOF data of reaction mixture. b. MALDI-TOF data of purified conjugate. e. Dis-assembly studies on day 1 and day 2. d. Dis-assembly studies on day 5 and day 7.

Figure 4. Control experiments a. SEC chromatogram of native trypsin (black) DTT treated trypsin (red). b. Try-OEG (black) and DTT treated try-OEG (red). c. Try-OEG-C12-2T (black) and DTT treated Try-OEG-C12-2T (red) respectively.

Figure 1. a. MALDI-TOF data of Native BSA and reaction mixture. b. MALDI-TOF data of purified BSA conjugate c. SEC chromatogram of BSA-OEG-C18-1T (Thiol) vs BSA-OEG-C181T (N-term). d. BSA (PDB accession: 3V03) structure showing thiol residue (yellow) with respect to N-terminus location (green). Negatively charged residues like Asp and Glu are colored red. Positively charged residues like Arg and Lys are colored blue.

Figure 3. Dis-assembly data of Try-OEG-SS-C12-2T complex a. Dis-assembly studies at varying concentrations of DTT. b. Time-dependent disassembly studies c. MALDI-TOF spectra of purified (black) and cleaved conjugate (red).

Full text

Rational Design of Self-assembling Artificial Proteins Utilizing a Micelle-
Assisted Protein Labeling Technology (MAPLabTech): Testing the Scope
Mullapudi Mohan Reddy
1
, Pavankumar Bhandari
1
and Britto S Sandanaraj*
1, 2
Department of Chemistry
1
and Department of Biology
2
Indian Institute of Science Education and Research – Pune
Email: sandanaraj.britto@iiserpune.ac.in
Abstract
Self-assembling artificial proteins (SAPs) have gained enormous interest in recent years
due to their applications in different fields. Synthesis of well-defined monodisperse SAPs is
accomplished predominantly through genetic methods. However, the last decade witnessed the
use of few chemical technologies for that purpose. In particular, micelle-assisted protein labeling
technology (MAPLabTech) has made huge progress in this area. The first generation
MAPLabTech focused on site-specific labeling of the active-site residue of serine proteases to
make SAPs. Further, this methodology was exploited for labeling of N-terminal residue of a
globular protein to make functional SAPs. In this study, we describe the synthesis of novel SAPs
by developing a chemical method for site-specific labeling of a surface-exposed cysteine residue
of globular proteins. In addition, we disclose the synthesis of redox- and pH-sensitive SAPs and
their systematic self-assembly and dis-assembly studies using complementary biophysical
studies. Altogether these studies further expand the scope of MAPLabTech in different fields
such as vaccine design, targeted drug delivery, diagnostic imaging, biomaterials, and tissue
engineering.
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted August 2, 2021. ; https://doi.org/10.1101/2021.08.01.454616doi: bioRxiv preprint

Introduction
Self-assembling artificial proteins (SAPs) are an interesting class of biomacromolecules
that would spontaneously self-associate to forms diverse protein nanostructures
1–5
. These
proteins found applications in various fields such as vaccine design
6,7
, targeted drug delivery
8
, in
vivo imaging,
9,10
and tissue engineering.
11,12
Among the various technologies available to design
SAPs; computational protein design
13–16
has made tremendous progress in the last decade.
Similarly, rational protein design
17–19
and directed evolution technology
20
also yielded very
impressive results. All three technologies utilize a microbial host system to synthesize the target
proteins and are therefore restricted to naturally occurring 20 amino acids.
Ashutosh and coworkers introduced a new strategy to synthesize SAPs in which they
combined a genetic method with a post-translational modification strategy to synthesize hybrid
protein-lipid conjugate
21
. They have shown that these custom-designed proteins self-assemble to
form interesting protein nanostructures. Although this method provides opportunities to go
beyond the standard 20 amino acids, it is still restricted to a small number of building blocks. In
addition, incorporation of every new building block requires elaborate engineering of the host
system which restricts the broader utility of this method for various applications. Therefore, it is
important to develop a general methodology that can incorporate a wide range of chemical
entities onto the self-assembling proteins without compromising salient features of natural
proteins such as a single chemical entity and the presence of well-defined functional groups in
the 3d-space. Toward that goal, our group invented a new method called ‘Micelle-Assisted
Protein Labeling Technology (MAPLabTech)
22–26
. During the last several years, we have utilized
MAPLabTech for the design of different families of well-defined monodisperse globular SAPs
which include protein amphiphiles
22
, protein-dendron conjugates
23
, protein-peptides
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted August 2, 2021. ; https://doi.org/10.1101/2021.08.01.454616doi: bioRxiv preprint

conjugates
24
, photo-responsive protein amphiphiles
22
, multi-responsive protein-dendron
conjugates
25
, and redox-sensitive protein conjugates
26
. Although extremely powerful, the above
method was only applicable to proteins that belong to the serine protease family
22–26
. This is a
serious limitation and therefore restricts the use of this method for a variety of applications such
as vaccine design
6,7
and antibody-drug conjugates.
27
To improve the scope of the existing
method, we recently introduced a new method that combines N-terminal bioconjugation
methodology
28
along with MAPLabTech
29
. This method indeed increases the diversity of
proteins that can be used as scaffolds for the construction of SAPs. However, this method has
three major limitations; (i) First, a vast number of proteins undergoes post-modification of the N-
terminal group because of this reason, the above strategy would not work for those proteins (ii)
secondly, in some cases, the N-terminal amine group may not be solvent-exposed and therefore
not be available for the bioconjugation reaction (iii) finally, this method will not work if the
protein contains proline in the second position
28
.
Site-specific labeling of a cysteine residue of a globular has been widely used for the
synthesis of water-soluble non-self-assembling protein-polymer conjugates which include
therapeutic proteins, antibody-drug conjugates. The ability to introduce cysteine moiety onto a
protein at a predetermined position makes this methodology extremely powerful for a variety of
applications. Although there are few reports on the synthesis of SAPs using chemical methods
30–
35
, they have major limitations such as (i) they are not monodispersed (ii) bioconjugation reaction
is mostly carried out in water/organic solvent mixture (iii) a viable and scalable method was not
reported for purification. Therefore, it is important to develop a chemical methodology that
addresses the above limitations. Towards that goal, herein, we report a chemical methodology for
site-specific labeling of a cysteine residue of globular protein. The newly synthesized semi-
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted August 2, 2021. ; https://doi.org/10.1101/2021.08.01.454616doi: bioRxiv preprint

synthetic was purified and characterized through various analytical techniques. Detailed studies
show the designed protein self-assembled to form monodisperse protein nanoparticles. In
addition, we also report the design, synthesis self-assembly, and disassembly studies of redox-
and pH-responsive artificial proteins.
Results
Synthesis of Cysteine-Reactive amphiphilic activity-based probe
The probe contains three structural parts (i) maleimide targeting group (ii) precisely defined
hydrophilic oligoethylene glycol and, (iii) hydrophobic tail (Scheme 1). The choice of maleimide
group is based on its selectivity towards cysteine residue in the presence of other potential
functional groups such as primary amine, alcohol, etc. The choice of the chain length of
oligoethylene glycol and hydrophobic tail length/branching was based on our extensive previous
experience. Accordingly, we synthesized a maleimide probe (MA-OEG-C18-1T) through multi-
step organic synthesis (Scheme 1). In brief, hydrophilic alkyne (1) and hydrophobic azide (2)
were allowed to react in the presence of 1M sodium ascorbate and 1M CuSO
4
for 16 hrs at room
temperature to get compound 3. The amphiphilic tosylate (3) was treated with sodium azide to
get compound 4. The azide residue was subjected to reduction using triphenylphosphine to get
compound 5 followed by treatment with N-(methoxycarbonyl) maleimide to get the final
compound 6 (MA-OEG-C18-1T).
Bioconjugation Reaction and Self-assembly Studies
Bovine serum albumin (BSA) is chosen as a target protein following reasons (i) it is a globular
proteins having a medium size and (ii) it contains one cysteine residue located on the outer
surface of the protein. Triton X-100 was used to solubilize MA-OEG-C18-1T and the
bioconjugation was carried out at room temperature in a 100% aqueous medium for 12 hours.
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted August 2, 2021. ; https://doi.org/10.1101/2021.08.01.454616doi: bioRxiv preprint

The progress of the reaction was monitored using MALDI-TOF MS (Figure 1a). After 12 hours,
we observed 70% conversion. After that reaction was over, the mixture was purified (Figure 1b)
by ion-exchange chromatography (IEX) followed by size-exclusion chromatography (SEC). The
self-assembling ability of the designed BSA conjugate was tested by analytical SEC (Figure 1c).
As expected, the artificial BSA conjugate self-assemble to make protein nanoparticles of bigger
size as evident from the elution profile seen in the SEC chromatogram. The relative molecule
weight of protein nanoparticles was obtained from the standard calibration curve (Table 1). The
obtained data suggest that the protein nanoparticles contain about 9-10 subunits of individual
proteins. It is interesting to note that this conjugate self-assembled to form a slightly bigger
complex than that of its corresponding N-terminus conjugate of the same spacer and tail group
(Figure 1c, 1d and Table 1).
Molecular Design and Synthesis of redox-sensitive amphiphilic activity-based probe
Compound 7 was obtained by hydrolysis of compound 8 in sodium hydroxide. The obtained
compound was allowed to react with 2, 2’-disulfanediylbis (ethan-1-ol) in presence of EDC and
DMAP in DCM to afford compound 9. This was followed by the activation using N N’-DSC, in
the presence of triethyl amine to yield compound 10. The activated ester 10 was then reacted
with amine (11) in the presence of Et
3
N and DMF to obtain compound 12. Then, the resultant
diphosphonate ester 12 was heated with lithium bromide in DMF to get monophosphonate ester
13 which finally on fluorination using DAST in DCM afforded flurophosphonate 14 (FP-OEG-
SS-C12-2T).
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted August 2, 2021. ; https://doi.org/10.1101/2021.08.01.454616doi: bioRxiv preprint

Frequently asked questions

Q1. What have the authors contributed in "Rational design of self-assembling artificial proteins utilizing a micelle-assisted protein labeling technology (maplabtech): testing the scope" ?

In this study, the authors describe the synthesis of novel SAPs by developing a chemical method for site-specific labeling of a surface-exposed cysteine residue of globular proteins. ( which was not certified by peer review ) is the author/funder. Altogether these studies further expand the scope of MAPLabTech in different fields such as vaccine design, targeted drug delivery, diagnostic imaging, biomaterials, and tissue engineering. 

Q2. What is the synthesis of the protein nanoparticles?

The authors hypothesis that the selective cleave of “disulfide functionality” would convert self-assembling protein nanoparticles into non-selfassembling monomeric proteins. 

Q3. What are the applications of self-assembling artificial proteins?

These proteins found applications in various fields such as vaccine design6,7, targeted drug delivery8, in vivo imaging,9,10 and tissue engineering. 

Q4. What was the reaction used to get the final compound?

The azide residue was subjected to reduction using triphenylphosphine to get compound 5 followed by treatment with N-(methoxycarbonyl) maleimide to get the final compound 6 (MA-OEG-C18-1T). 

Q5. What are the advantages of self-assembling artificial proteins?

Self-assembling artificial proteins (SAPs) are an interesting class of biomacromoleculesthat would spontaneously self-associate to forms diverse protein nanostructures1–5. 

Q6. What is the impressive feature of the SEC-MALS data?

One of the most impressive features of the SEC-MALS data is that it gives information about the polydispersity of the protein nanoparticles. 

Q7. What was the main purpose of the study?

After systematic self-assembly studies, the authors focused their attention on disassembly studies of protein nanoparticles Try-OEG-SS-C12-2T. 

Q8. What are the limitations of the method?

Although this method provides opportunities to go beyond the standard 20 amino acids, it is still restricted to a small number of building blocks. 

Q9. What are the main limitations of the new method?

Although there are few reports on the synthesis of SAPs using chemical methods30– 35, they have major limitations such as (i) they are not monodispersed (ii) bioconjugation reaction is mostly carried out in water/organic solvent mixture (iii) a viable and scalable method was not reported for purification. 

Q10. What is the effect of the presence of imine functionality on the synthesis of a?

After careful analysis, the authors figure out the presence of “imine functionality” next to the carbonyl group decreases the susceptibility of imine group hydrolysis at acidic pH. 

Q11. What is the molecular weight of the protein nanoparticles?

To get information on the molecular weight of the protein nanoparticles, the authors have carried out sizeexclusion chromatography coupled with multi-angle light scattering (SEC-MALS) studies. 

Q12. What is the scope of MAPLab 2.0?

MAPLab 2.0 was developed for labeling of N-terminal residue of any given protein which increased the scope of this technology substantially29. 

Q13. What is the structure of the protein TyrOEG-C12-2T?

the authors made another self-assembling artificial protein TyrOEG-C12-2T, this protein structure exactly mimics the structure of Tyr-OEG-SS-C12-2T, except the control protein, does not contain an engineered disulfide bond. 

Q14. How many equivalents of DTT are enough to achieve complete disassembly?

The disassembly studies in presence of different concentrations of DTT revealed that 10 equivalents of DTT are enough to achieve complete disassembly. 

Q15. What is the main limitation of MAPLabTech 2.0?

MAPLabTech 2.0 does not apply proteins that are subjected to N-terminal post-translational modification, and most importantly the N-terminal amino acid should be solvent-exposed. 

Q16. What is the kinetics of the disassembly of protein nanoparticle?

the authors tested the stability of the monomeric native protein in the presence of DTT, as expected, due to proteolysis the native protein was converted into small peptides as evident from the SEC results (Figure 4a).