Incorporation
of Aliphatic
Proline
Residues
into Recombinantly
Produced
Insulin
Stephanie
L. Breunig,
Janine
C. Quijano,
Cecile
Donohue,
Amy
Henrickson,
Borries
Demeler,
Hsun
Teresa
Ku, and David
A. Tirrell
*
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ACS Chem.
Biol.
2023,
18, 2574−2581
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ABSTRACT:
Analogs
of proline
can be used
to expand
the chemical
space
about
the residue
while
maintaining
its uniquely
restricted
conformational
space.
Here,
we demonstrate
the incorporation
of 4
R
-
methylproline,
4
S
-methylproline,
and
4-methyleneproline
into
re-
combinant
insulin
expressed
in
Escherichia
coli
. These
modified
proline
residues,
introduced
at position
B28,
change
the biophysical
properties
of insulin:
Incorporation
of 4-methyleneproline
at B28
accelerates
fibril
formation,
while
4-methylation
speeds
dissociation
from
the
pharmaceutically
formulated
hexamer.
This
work
expands
the scope
of
proline
analogs
amenable
to incorporation
into recombinant
proteins
and demonstrates
how
noncanonical
amino
acid mutagenesis
can be
used
to engineer
the therapeutically
relevant
properties
of protein
drugs.
■
INTRODUCTION
Proline
is unique
among
the canonical
amino
acids:
the cyclic
pyrrolidine
side
chain
restricts
the conformational
space
accessible
to the residue.
Replacing
proline
with
any of the
proteinogenic
amino
acids
through
standard
mutagenesis
approaches
necessarily
grants
greater
conformational
freedom.
Alternatively,
noncanonical
proline
(ncPro)
residues
expand
the
chemical
space
about
proline
while
maintaining
a
pyrrolidine
(or
pyrrolidine-like)
side
chain.
Because
the
conformational
preferences
of many
proline
analogs
are
known,
1,2
ncPro
residues
are useful
as probes
of conforma-
tional
effects
on protein
behavior.
Proline
analogs
have
enabled
investigators
to elucidate
the importance
of a key proline
cis
-
trans
isomerization
event
in 5HT
3
receptor
opening,
3
modify
the
properties
of elastin-like
proteins,
4
−
6
determine
the
molecular
origins
of collagen
stability,
1,7
and probe
the role
of
cis
-
trans
isomerization
in
β
2-microglobulin
fibrillation.
8,9
Although
Chatterjee
and co-workers
have
reported
progress
in
the development
of orthogonal
prolyl-tRNA
synthetase/tRNA
pairs,
10
to date,
only
residue-specific
(rather
than
site-specific)
incorporation
approaches
have
been
successful
in introducing
proline
analogs
into recombinant
proteins.
11
Using
E. coli
as an
expression
host,
these
approaches
have
allowed
efficient
incorporation
of various
3- and
4-functionalized
proline
residues
and
those
with
modified
ring
sizes
and
composi-
tions.
12
Insulin
(Figure
1a) is a 5.8 kDa peptide
hormone
normally
released
from
the pancreatic
β
cells
in response
to elevated
levels
of blood
glucose.
Its binding
to the insulin
receptor
induces
intracellular
responses
that
ultimately
lower
blood
glucose
concentrations.
13
Diabetes
mellitus
is a result
of
dysfunctional
insulin
signaling,
either
through
an impaired
ability
to produce
and secrete
insulin
(type
1) or through
insulin
resistance
(type
2). Subcutaneous
injection
of
exogenous
insulin
is a common
strategy
in diabetes
treatment,
especially
for individuals
with
type
1 diabetes.
The
recombinant
production
of insulin
14
has significantly
advanced
diabetes
treatment
by enabling
both
its production
at scale
and
the creation
of modified
insulins
with
desirable
properties
through
standard
mutagenesis
and
chemical
modification
approaches.
15
−
18
To mimic
the insulin-action
profile
of a healthy
pancreas,
two
broad
classes
of insulin
analogs
have
been
developed:
long-acting
(or basal)
insulins
and fast-acting
insulins
(FAIs).
19
Long-acting
variants
recapitulate
the lower
levels
of insulin
secretion
that
maintain
metabolism
in an anabolic
state.
Conversely,
FAIs
aim
to mimic
the transient
increases
in
insulin
concentration
stimulated
by elevated
blood
glucose
after
a meal.
Typical
insulin
replacement
therapy
relies
on a
combination
of regular
basal
insulin
treatments
and
FAI
injections
before
meals.
Received:
September
12, 2023
Revised:
October
24, 2023
Accepted:
October
27, 2023
Published:
November
14,
2023
Articles
pubs.acs.org/acschemicalbiology
© 2023
The Authors.
Published
by
American
Chemical
Society
2574
https://doi.org/10.1021/acschembio.3c00561
ACS Chem.
Biol.
2023,
18, 2574
−
2581
This article is licensed under CC-BY 4.0
Many
FDA-approved
insulin
variants
have
been
engineered
by altering
the amino
acid
sequence
in ways
that
cause
pronounced
pharmacokinetic
effects.
20
Notably,
insulin
aspart
17,21
(NovoLog,
marketed
by Novo
Nordisk)
and insulin
lispro
15,22
(Humalog,
Eli Lilly)
both
involve
changes
to
ProB28,
a key residue
15
near
the C-terminus
of the B-chain
(Figure
1a).
Insulin
aspart
is achieved
by the single
point
mutation
ProB28Asp,
while
insulin
lispro
contains
an inversion
of ProB28
and
LysB29;
both
changes
destabilize
insulin
oligomers.
Because
the rate-limiting
step for insulin
absorption
into the bloodstream
is dissociation
of oligomer
to monomer
23
(Figure
1b), these
changes
accelerate
insulin’s
onset
of action.
Insulins
are also
prone
to chemical
and physical
denatura-
tion,
24
−
26
processes
slowed
by the formation
of protective
oligomers.
As a result,
insulin
production
and
distribution
require
a cold
chain,
27
and maintaining
protein
stability
in
continuous
subcutaneous
insulin
infusion
pumps
is challeng-
ing.
28
Intrigued
by the role that ProB28
plays
in insulin
biophysics,
we recently
introduced
a series
of ncPro
residues
at position
B28 of human
insulin.
29,30
Because
mature
insulin
contains
one
proline,
a residue-specific
replacement
approach
results
in site-
specific
proline
replacement
without
the
need
for an
orthogonal
aminoacyl-tRNA
synthetase/tRNA
pair.
These
efforts,
which
focused
on proline
analogs
known
to incorporate
well
into
recombinant
proteins
in
E. coli
,
5
illustrated
how
proline
mutagenesis
of insulin
can
be used
to tune
its
biophysical
characteristics.
29,30
Here,
we demonstrate
the efficient
incorporation
of three
new
aliphatic
proline
residues
(4
R
-methylproline,
4
R
-Me;
4
S
-
methylproline,
4
S
-Me;
and 4-methyleneproline,
4ene;
Figure
1c) at position
B28 of recombinantly
produced
insulin;
these
insulin
variants
will be referred
to as ins-4
R
-Me,
ins-4
S
-Me,
and ins-4ene,
respectively.
We find
that
these
modifications
alter
insulin
behavior:
replacement
of ProB28
with
4ene
speeds
fibril
formation,
while
4-methylation
accelerates
hexamer
dissociation
without
affecting
stability
against
physical
denaturation.
This
work
expands
the range
of proline
analogs
that can be incorporated
into recombinant
proteins
in
E. coli
. It
also
demonstrates
how
small
molecular
changes
introduced
through
noncanonical
amino
acid mutagenesis
can be used
to
Figure
1.
Proline
mutagenesis
at position
B28 of human
insulin.
a. Crystal
structure
of insulin
(PDB
1MSO),
highlighting
ProB28
located
at the
dimer
interface.
b. Simplified
scheme
of insulin
dissociation
after
injection.
Insulin
exists
as a hexamer
in the R state
in the presence
of zinc and
phenolic
ligands
such
as in the pharmaceutical
formulation.
After
injection,
insulin
dissociates
into lower-order
oligomeric
species
that can diffuse
more
easily
across
the capillary
membrane,
enter
the bloodstream,
and bind
to the insulin
receptor.
c. The structure
of proline
and the aliphatic
proline
analogs
used
in this study.
Figure
2.
Mass
spectrometry
of insulin
variants.
a-d. Characterization
of proline
analog
incorporation.
The solubilized
inclusion
body
(containing
proinsulin)
after
expression
in medium
supplemented
with
proline
(a), 4R-Me
(b), 4S-Me
(c), or 4ene
(d) was digested
with
Glu-C
and analyzed
by MALDI-TOF
MS. The peptide
that contains
position
B28 of mature
insulin
is
50
RGFFYT
P
KTRRE
(expected
m
/
z
= 1557.8).
e-h. MALDI-
TOF
characterization
of mature
and purified
insulin
variants:
human
insulin
(e), ins-4
R
-Me
(f), ins-4
S
-Me
(g), and Ins-4ene
(h). The peaks
at
m
/
z
∼
6050
correspond
to adducts
of the sinapic
acid matrix.
ACS Chemical
Biology
pubs.acs.org/acschemicalbiology
Articles
https://doi.org/10.1021/acschembio.3c00561
ACS Chem.
Biol.
2023,
18, 2574
−
2581
2575
probe
and engineer
the therapeutically
relevant
properties
of
protein
drugs.
■
RESULTS
AND
DISCUSSION
Aliphatic
Proline
Residues
Are Accepted
by the
E. coli
Translational
Machinery.
To identify
an expanded
set of
ncPro
residues
accepted
by the
E. coli
translational
machinery,
we expressed
proinsulin
(a precursor
to insulin)
under
conditions
that
favor
ncPro
incorporation.
12
We monitored
ncPro
replacement
by proinsulin
expression
and
mass
spectrometry
and noted
a range
of incorporation
efficiencies
for the 15 commercially
available
proline
analogs
tested
(Figure
S1 and
Table
S1).
Notably,
the aliphatic
proline
residues
4
R
-Me,
4
S
-Me,
and
4ene
led to high
levels
of
proinsulin
expression
and
good
(
∼
90%)
incorporation
efficiencies
under
the optimized
conditions
(Figure
2a-d,
Table
S2, Table
S3).
We replaced
ProB28
of human
insulin
with
each
of these
three
aliphatic
proline
analogs
(Figure
2e-h)
and determined
the resulting
effects
on insulin
behavior.
Proline
Analogs
Do Not
Affect
Insulin
Secondary
Structure
or Bioactivity.
The
secondary
structure
of each
insulin
variant
was
assessed
by circular
dichroism
(CD)
spectroscopy
(Figure
3a-c;
Table
S4).
The
CD spectrum
of
insulin
is sensitive
to the state
of oligomerization
of the
protein:
for monomeric
insulins,
the
ratio
of negative
ellipticities
at 208 and 222 nm is increased
compared
to that
of the insulin
dimer.
31
The
far-UV
CD spectrum
for each
variant
closely
matched
that
of human
insulin
at 60
μ
M,
suggesting
that
proline
replacement
has
not
significantly
perturbed
the secondary
structure
or dimer
formation
under
these
conditions.
To validate
biological
activity
in vivo
,
insulins
were
formulated
with
zinc
and
phenolic
ligands
and
injected
subcutaneously
into
diabetic
mice;
blood
glucose
was
monitored
over
2.5 h. Rodent
models
can
assess
insulin
activity
but cannot
distinguish
differences
in time
to onset
of
action
for human
insulin
and fast-acting
analogs.
32
Because
the
C-terminus
of the B-chain
does
not interact
with
the insulin
receptor,
33,34
we did not expect
modification
of ProB28
to
affect
bioactivity.
As anticipated,
all of the insulin
variants
reduced
the blood
glucose
in diabetic
mice
(Figure
3d).
4-Methylation
of ProB28
Speeds
Hexamer
Dissocia-
tion.
Increased
negative
ellipticity
at 222
nm in the CD
spectrum
of insulin
is a signature
of oligomerization.
31,35
Adopting
a method
reported
by Gast
and co-workers,
36
we
measured
the rate
of dissociation
of insulin
variants
to the
monomer
state.
Insulins
were
formulated
under
conditions
that
mimic
the pharmaceutical
formulation
(600
μ
M insulin,
25
mM
m
-cresol,
250
μ
M
ZnCl
2
) and
favor
the R
6
hexamer
state.
37
Dissociation
was
monitored
by tracking
the mean
residue
ellipticity
at 222 nm over
time
after
150-fold
dilution
into
ligand-free
buffer
(Figure
4a). CD spectra
after
dilution
are distinct
from
that of chemically
denatured
insulin
(Figure
S3, Figure
S4),
confirming
that changes
in the CD signal
are
not a result
of denaturation.
While
ins-4ene
dissociated
at a
rate similar
to that of human
insulin
(t
1/2
= 17.0
±
2.3 and 18.4
±
2.8 s, respectively),
dissociation
of ins-4
R
-Me
(t
1/2
= 9.8
±
Figure
3.
Circular
dichroism
spectroscopy
and bioactivity
of insulin
variants.
a-c. Far-UV
circular
dichroism
spectra
of insulin
and insulin
variants
(60
μ
M in 100 mM phosphate
buffer,
pH 8.0).
The spectrum
of each
insulin
variant
is overlaid
with
that of human
insulin
(gray).
d. Insulins
were
injected
subcutaneously
into diabetic
mice,
and blood
glucose
was measured
over
time
after
injection.
Figure
4.
Hexamer
dissociation
kinetics
of insulin
variants.
a. Equilibrium
CD spectra
of insulin
before
and after
dilution.
To measure
the
dissociation
kinetics,
the decrease
in negative
ellipticity
at 222 nm was monitored
over
time
after
dilution.
b-e. Representative
dissociation
plots
for
insulin
(b), ins-4
R
-Me
(c), ins-4
S
-Me
(d), and ins-4ene
(e). Each
dilution
experiment
was fit to a monoexponential
function,
and the half-life
for
each
displayed
replicate
is indicated.
f. Summary
of dissociation
half-life
values.
ACS Chemical
Biology
pubs.acs.org/acschemicalbiology
Articles
https://doi.org/10.1021/acschembio.3c00561
ACS Chem.
Biol.
2023,
18, 2574
−
2581
2576