of 23
S
1
Supporting Information
Incorporation of aliphatic proline residues into recombinantly
-
produced insulin
Stephanie L. Breunig
1
, Janine C. Quijano
2
, Cecile Donohue
2
, Amy Henrickson,
3
Borries Demeler,
3,4
Hsun Teresa Ku
2
,5
, and David A. Tirrell
1
*
1
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California
91125, United States.
2
Department of Translational Research and Cellular Therapeutics, Diabetes and Metabolism Research
Institute,
Beckman Institute
City of Hope
, Duarte, California 91010, United States.
3
University of Lethbridge, Dep
artment
of Chemistry and Biochemistry, Lethbridge, Alberta
T1K 3M4
,
Canada
.
4
University of Montana, Department of Chemistry and Biochemistry, Missoula, MT 59801, U
nited States.
5
Irell & Manella Graduate School of Biological Science
, City of Hope, Duarte, California 91010, United
States.
*
C
orresponding author. Email: tirrell@caltech.edu
Table of Contents
Materials and methods
................................
................................
................................
..................
S
2
Chemicals
................................
................................
................................
................................
.
S
2
Enzymes
................................
................................
................................
................................
...
S
2
Strains and
plasmids
................................
................................
................................
................
S3
Primers
................................
................................
................................
................................
.....
S
3
Nucleotide and amino acid sequences
................................
................................
....................
S
3
Screening ncPro incorporation
................................
................................
................................
S5
Proinsulin expression
................................
................................
................................
...............
S
5
Proinsulin refolding
................................
................................
................................
..................
S
6
Insulin maturation and
purification
................................
................................
.........................
S7
MALDI
-
TOF MS
................................
................................
................................
.........................
S
7
Circular dichroism spectroscopy
................................
................................
..............................
S
7
Reduction of
blood glucose in diabetic mice
................................
................................
...........
S
8
Fibrillation
................................
................................
................................
................................
S
8
Transmission electron microscopy
................................
................................
..........................
S9
ANS fluorescence
................................
................................
................................
.....................
S
9
Analytica
l
ultracentrifugation
................................
................................
................................
..
S
9
Models of ins
-
4
R
-
Me and ins
-
4
S
-
Me hexamers
................................
................................
.......
S
9
Mass spectrometry characterization of dissolved insulin fibrils
................................
...........
S10
Calculations of proline and proline analog conformation
................................
.....................
S
10
Table S1: Incorporation of non
-
canonical proline residues
................................
.........................
S
1
1
Table S
2
:
Mass spectrometry characterization of insulin variants
................................
..............
S
1
2
S
2
Table S
3
:
Expression conditions and insulin yields
................................
................................
......
S
1
3
Table S
4
:
Summary of insulin
variant
characterization
................................
...............................
S
1
4
Table S5: Sedimentation coefficients of insulin and ins
-
4
S
-
Me
................................
...................
S
1
5
Figure S1: SDS
-
PAGE after ncPro incorporation
................................
................................
..........
S
1
6
Figure S2:
Purity of insulin samples assessed by SDS
-
PAGE
................................
........................
S
1
7
Figure S
3
:
Changes in CD
signal after dilution are not due to protein denaturation
..................
S
1
8
Figure S4: Equilibrium CD spectra after dilution
................................
................................
..........
S
1
9
Figure S
5
: Models of
ins
-
4
R
-
Me and ins
-
4
S
-
Me hexamers
................................
..........................
S
20
Figure S6:
Deconvoluted mass spectra of insulin and ins
-
4ene fibrils
................................
........
S
2
1
Figure S
7
:
Conformations of proline, 4
-
methyleneproline, and 3,4
-
dehydroproline
.................
S
2
2
References
................................
................................
................................
................................
....
S
2
3
Materials and
m
ethods
Chemicals:
All chemicals were purchased from MilliporeSigma
unless otherwise indicated. 4
-
methyleneproline (4ene) was purchased as the N
-
boc protected version from Acros Organics, and
deprotected with trifluoroacetic acid (TFA) in dichloromethane. 4ene was extracted with H
2
O and
lyophilized; complete deprotection and >95% purity w
ere
verified by
1
H NMR. All other proline
analogs were used as received: 4
R
-
methylproline (4
R
-
Me) was purchased from Advanced
Chemblocks as the hydrochloride salt;
1
H NMR analysis indicated the presence of 5
-
10% of the
4
S
-
methylproline dia
stereomer. 2
-
M
ethylproline (2
-
Me) was purchased from Advanced
ChemBlocks as the hydrochloride salt. 4
S
-
M
ethylproline (4
S
-
Me) was purchased from AstaTech
as the hydrochloride salt. L
-
cis
-
pyrrolidine
-
2,4
-
dicarboxylic acid (4
S
-
COOH) was purchased from
Boc Sciences. L
-
trans
-
pyrrolidine
-
2,4
-
dicarboxylic acid (4
R
-
COOH) was purchased from Tocris
Biosciences. L
-
pyroglutamic acid (5
-
oxo) was purchased from Aldrich. 4,4
-
dimethylproline (44
-
diMe) was purchased from J&W Pharmlab. Piperazine
-
2
-
carboxylic acid (Pip
-
Az) w
as purchased
from Ark Pharm as the dihydrochloride salt. (2
S
,5
S
)
-
5
-
hydroxypiperidine
-
2
-
carboxylic acid (Pip
-
OH) was purchased from Ark Pharm. 4
S
-
aminoproline (4
S
-
NH
2
) was purchased from Toronto
Research Chemicals as the dihydrochloride salt. 3
R
-
hydroxyproline (3
R
-
OH) was purchased from
Combi
-
Blocks. 3
S
-
hydroxyproline (3
S
-
OH) was purchased from Ark Pharm. 4
-
O
xoproline (4
-
oxo)
was purchased as the hydrobromide salt from MilliporeSigma.
Enzymes:
Restriction enzymes, kinases, and ligases were purchased from New England Biolabs. Trypsin was
purchased from MilliporeSigma. Carboxypeptidase B was purchased from Worthington
Biochemical. Glu
-
C peptidase was purchased from Promega.
S
3
Strains and plasmids:
The proline
-
auxotrophic
E. coli
strain CAG18515 was obtained from the Coli Genetic Stock Center
(CGSC) at Yale University. Strain DH10B was used for standard cloning operations;
electrocompetent CAG18515 were transformed with purified plasmid products.
The plasmid pQE80_H27R
-
PI_proS contains an IPTG
-
inducible proinsulin gene and the
E. coli
prolyl
-
tRNA synthetase gene controlled by its endogenous promoter. Proinsulin is translationally
fused to an N
-
terminal leader peptide (H27R) that increases expression yields,
1
and a 10x
-
his tag
to facilitate proinsulin enrichment after refolding. The gene for H27R
-
PI was ordered as a g
-
Block
gene fragment from Integrated DNA Technologies (IDT) after codon optimization of the N
-
terminal leader peptide. A restriction enzyme cloni
ng approach (XhoI and BamHI restriction
enzyme cut sites) was used to replace the hexahistidine
-
tagged proinsulin gene in the plasmid
pQE80PI
-
proS, which was described previously.
2
Correct installation of the gene of interest was
verified by Sanger sequencing.
A blunt
-
end ligation approach was used to install the C443G and M157Q mutations. The
proS
-
containing plasmid was amplified with primers AL01004_fwd & AL01004_rev (C443G), or
AL01005_fwd & AL01005_rev2 (M157Q). The linear PCR product was phosphorylated (T4 PNK)
and circularized (T4 DNA ligase). Correct installation of the point mutation was ver
ified by Sanger
sequencing.
Primers:
DNA oligos were purchased from Integrated DNA Te
chnologies (IDT).
Nucleotides responsible for
installing the M157Q and C443G mutations are
underlined
.
AL01004_fwd:
ATACCGTAGC
C
ACCCATCGTCAGG
AL01004_rev:
CGGGGTAACGCGTGTGGT
AL01005_fwd:
AGCATCTTTC
TG
CAGGAATTCGC
AL01005_rev2:
TACTCTTTCCATACTTCTCAGGAATCC
Nucleotide and amino acid sequences:
H27R
-
PI: The g
-
Block gene fragment was purchased from IDT. The coding sequence is in
UPPERCASE, XhoI and BamHI cut sites are
underlined
.
gccctttcgtcttcac
ctcgag
aaatcataaaaaatttatttgctttgtgagcggataacaattataatagattcaattgtgagcggataac
aatttcacacagaattcattaaagaggagaaattaactATGACAATGATCACTAATTCACCCGAGATTTCCCACCATCA
TCATCATCATCACCACCACCATCAGTTGATCTCGGAGGCCCGTTTTGTGAACCAGCACCTGTGCGGTAGC
CACCTGGTGGAAGCTCTGTACCTGGTTTGCGGTGAGC
GTGGTTTCTTCTACACGCCAAAGACCCGCCGT
GAAGCTGAAGATCTGCAGGTGGGCCAGGTAGAACTGGGCGGTGGTCCGGGTGCCGGCTCTCTGCAACC
GCTGGCACTGGAAGGTTCCCTGCAAGCGCGTGGTATCGTAGAGCAGTGCTGTACTTCTATCTGCTCCCT
GTACCAGCTGGAGAACTACTGTAATTAA
ggatcc
gcatgcgagc
S
4
The sequence of the H27R leader peptide is
underlined
, proinsulin is in
bold
. The A
-
chain and B
-
chain in mature insulin are colored
red
and
blue
, respectively.
MTMITNSPEISHHHHHHHHHHQLISEAR
FVNQHLCGSHLVEALYLVCGERGFFYTPKT
RREAEDLQVGQVE
LGGGPGAGSLQPLALEGSLQAR
GIVEQCCTSICSLYQLENYCN
proS
: the endogenous
proS
promoter is in
bold
, the coding sequence is
underlined
.
ATTCACGC
CCTTCTCTTTTGACATTTCTTTTGCACTGGTAAACTAAATCAC
TTTTTTTTGTCCCAGGCTCGC
CTTGAGCCTGTTCTACCTTCCAACTGGAACCGTAACAAC
ATGCGTACTAGCCAATACCTGCTCTCCACTCT
CAAGGAGACACCTGCCGACGCCGAGGTGATCAGCCATCAGCTGATGCTGCGCGCCGGGATGATCCGCA
AGCTGGCCTCCGGGTTATATACCTGGCTGCCGACCGGCGTGCGCGTTCTGAAAAAAGTCGAAAACATCG
TGCGTGAAGAGATGAACAACGCCGGTGCGATCGAGGTGTCGATGCCGGTGGTTCAGCCAGCCGATTTG
TGGCAAGAGAGTGGTCGTT
GGGAACAGTACGGTCCGGAACTGCTGCGTTTTGTTGACCGTGGCGAGCG
TCCGTTCGTACTCGGCCCAACTCATGAAGAAGTTATCACTGACCTGATTCGTAACGAGCTTAGCTCTTAC
AAACAGCTGCCGCTGAACTTCTATCA
GATCCAGACCAAGTTCCGCGACGAAGTGCGTCCGCGTTTCGGC
GTCATGCGTTCCCGCGAATTCCTGATGAAAGATGCTTACTCTTTCCATACTTCTCAGGAATCCCTGCAGGA
AACCTACGATGCAATGTATGCGGCCTACAGCAAAATCTTCAGCCGCATGGGGCTGGATTTCCGCGCCGT
ACAAGCCGACACCGGTTCTATCGGCGGCAGCGCCTCTCACGAATTCCAGGTGCTGGCGCAGAGCGGTG
AAGAC
GATGTGGTCTTCTCCGACACCTCTGACTATGCAGCGAACATTGAACTGGCAGAAGCTATCGCGC
CGAAAGAACCGCGCGCTGCTGCTACCCAGGAAATGACGCTGGTTGATACGCCGAACGCGAAAACCATC
GCGGAACTGGTTGAACAGTTCAATCTGCCGATTGAGAAAACGGTTAAGACTCTGCTGGTTAAAGCGGTT
GAAGGCAGCAGCTTCCCGCAGGTTGCGCTGCTGGTGCGCGGTGATCACGAGCTGA
ACGAAGTTAAAGC
AGAAAAACTGCCGCAGGTTGCAAGCCCGCTGACTTTCGCGACCGAAGAAGAAATTCGTGCCGTGGTTAA
AGCCGGTCCGGGTTCACTGGGTCCGGTAAACATGCCGATTCCGGTGGTGATTGACCGTACCGTTGCGGC
GATGAGTGATTTCGCTGCTGGTGCTAACATCGATGGTAAACACTACTTCGGCATCAACTGGGATCGCGA
TGTCGCTACCCCGGAAGTTGCAGATATCCGTAACGT
GGTGGCTGGCGATCCAAGCCCGGATGGCCAGG
GTAGGCTGCTGATCAAACGTGGTATCGAAGTTGGTCACATCTTCCAGCTGGGTACCAAGTACTCCGAAG
CACTGAAAGCCTCCGTACAGGGTGAAGATGGCCGTAACCAAATCCTGACGATGGGTTGCTACGGTATCG
GGGTAACGCGTGTGGTAGCTGCGGCGATTGAGCAGAACTACGACGAACGAGGCATCGTATGGCCTGAC
GCTATCGCGCCGTTCCAG
GTGGCGATTCTGCCGATGAACATGCACAAATCCTTCCGCGTACAAGAGCTT
GCTGAGAAACTGTACAGCGAACTGCGTGCACAAGGTATCGAAGTGCTGCTGGATGACCGCAAAGAGCG
TCCGGGCGTGATGTTTGCTGATATGGAACTGATCGGTATTCCGCACACTATTGTGCTGGGCGACCGTAA
CCTCGACAACGACGATATCGAATATAAATATCGTCGCAACGGCGAGAAACAGTTAATTAAGACTGGTG
A
CATCGTCGAATATCTGGTGAAACAGATTAAAGGCTGA
MRTSQYLLSTLKETPADAEVISHQLMLRAGMIRKLASGLYTWLPTGVRVLKKVENIVREEMNNAGAIEVSMP
VVQPADLWQESGRWEQYGPELLRFVDRGERPFVLGPTHEEVITDLIRNELSSYKQLPLNFYQIQTKFRDEVRP
RFGVMRSREFLMKDAYSFHTSQESLQETYDAMYAAYSKIFSRMGLDFRAVQADTGSIGGSASHEFQVLAQS
GEDDVVFSDTSDYAANIELAEAIAPKEPRAAATQEMTLVD
TPNAKTIAELVEQFNLPIEKTVKTLLVKAVEGSS
FPQVALLVRGDHELNEVKAEKLPQVASPLTFATEEEIRAVVKAGPGSLGPVNMPIPVVIDRTVAAMSDFAAG
ANIDGKHYFGINWDRDVATPEVADIRNVVAGDPSPDGQGRLLIKRGIEVGHIFQLGTKYSEALKASVQGEDG
RNQILTMGCYGIGVTRVVAAAIEQNYDERGIVWPDAIAPFQVAILPMNMHKSFRVQELAEKLYSELRAQGIE
VLLDDR
KERPGVMFADMELIGIPHTIVLGDRNLDNDDIEYKYRRNGEKQLIKTGDIVEYLVKQIKG*
S
5
Screening ncPro incorporation
:
A single colony of
E. coli
strain CAG18515
/
pQE80_H27R
-
PI_proS was used to inoculate a culture
of Luria Bertani (LB) medium supplemented with ampicillin. The culture was grown overnight at
37°C until stationary phase was reached, then diluted 1:100 into 100 mL of 1x M9 medium,
supplemented with all
twenty
canonical
amino acids. The medium composition of M9 is as
follows: 8.5 mM NaCl, 18.7 mM NH
4
Cl, 22 mM KH
2
PO
4
, 47.8 mM Na
2
HPO
4
, 0.1 mM CaCl
2
, 1 mM
MgSO
4
, 3 mg L
-
1
FeSO
4
, 1 μg L
-
1
trace metals [Cu
2+
, Mn
2+
, Zn
2+
, MoO
4
2
-
], 35 mg L
-
1
thiamine HCl,
10 mg
L
-
1
biotin, 20 mM D
-
glucose, 100 mg L
-
1
ampicillin, 50 mg L
-
1
of each L
-
amino acid.
The culture was grown at 37°C until it reached OD ~0.8, after which it was subjected to a medium
shift: cells were pelleted via centrifugation (5
,000
g, 5 min, 4°C) and washed twice with 10 mL
ice
-
cold 0.9% NaCl. Washed cells were resuspended in 80 mL of 1.25x M9
Pro, a 1.25x
concentrated form of M9 that omits proline. The culture was split into 4 mL aliquots, and
incubated for 30 min at 37°C to deplet
e residual proline. A 1 mL solution containing 2.5 mM ncPro
and 1.5 M NaCl was added (0.5 mM ncPro and 0.3
M NaCl working concentrations). After 30 min
of incubation at 37°C to allow for ncPro uptake, proinsulin expression was induced by the
addition of 1 mM IPTG. Cultures were incubated for 2.5 h at 37°C, after which cells were
harvested via centrifugation an
d stored at
-
80°C until further processing.
Cell pellets were thawed and lysed with B
-
PER Complete (Thermo Fisher Scientific) for 1 h at
room temperature with shaking, then centrifuged (20
,000
g, 10 min) and the supernatant
discarded. The pellet (containing insoluble proinsulin) was washed once with Triton wash buffer
(2 M urea, 20 mM Tris, 1% Triton X
-
100, pH 8.0), and twice with ddH
2
O. The pellet was
resuspended in solubilization buffer (8 M urea, 300 mM NaCl, 50 mM NaH
2
PO
4
, pH 8.0), and
proinsulin was allowed to dissolve for 1 h at room temperatur
e with shaking. Samples were
centrifuged, and the supernatant removed for analysis by SDS
-
PAGE and MALDI
-
TOF (described
in the section entitled “MALDI
-
TOF MS” below)
.
Proinsulin expression
:
A single colony of
E. coli
strain CAG18515 harboring plasmid pEQ80_H27R
-
PI_proS (or the
corresponding plasmid with a point mutation in the
proS
gene; see Table S3)
was used to
inoculate 70 mL of LB medium containing ampicillin, and the culture was grown overnight at 37°C
to stationary phase. The overnight culture was used to inoculate 5 L (as 4x1.25 L cultures) of 1x
Andrew’s Magical Medium (AMM),
3
a defined medium containing all 20 proteinogenic amino
acids, in 2.8 L Fernbach flasks. The composition of AMM was the following: 3.60 g L
-
1
glucose, 3.5
g L
-
1
KH
2
PO
4
, 6.56 g L
-
1
K
2
HPO
4
3H
2
O, 3.5 g L
-
1
(NH
4
)
2
HPO
4
, 8.37 g L
-
1
MOPS, 0.72 g L
-
1
tricine, 2.92
g L
-
1
NaCl, 0.51 g L
-
1
NH
4
Cl, 0.26 g L
-
1
MgCl
2
7H
2
O, 50 mg L
-
1
K
2
SO
4
, 0.246 mg L
-
1
MgSO
4
7H
2
O,
12.3 mg L
-
1
CaCl
2
,
2H
2
O, 2.8 mg L
-
1
FeSO
4
7H
2
O, 0.5 mg L
-
1
thiamine, 24 μg L
-
1
boric acid, 1 μg L
-
1
trace metals (Cu
2+
, Mn
2+
, Zn
2+
, MoO
4
2
-
), and 50 mg L
-
1
each amino acid.
When growth reached mid
-
exponential phase (OD
600
~0.8), the culture was subjected to a
medium shift: cells were pelleted via centrifugation (5
,000
g, 5 min, 4°C) and washed twice with
100 mL ice
-
cold 0.9% NaCl. Washed cells were resuspended in 1 L of 1.25x AMM
-
Pro, a 1.25x
concentrated form of AMM that omits proline. Cells were incubated for 30 min at 37°C to deplete
S
6
residual proline, after which 250 mL of a solution containing 2.5
-
5.0 mM ncPro (see Table 4.S3)
and 2.5 M NaCl was added (0.5
-
1.0 mM ncPro and 0.5 M NaCl working concentrations). After 30
min of incubation at 37°C to allow for ncPro uptake, proinsulin expr
ession was induced by the
addition of
isopropylthio
-
b
-
galactosidase (IPTG, 1 mM)
. Cultures were incubated overnight at
37°C, after which cells were harvested via centrifugation and stored at
-
80°C until further
processing.
Proline
-
containing proinsulin was expressed using strain CAG18515 harboring plasmid pQE80
-
H27R
-
PI_proS in 7.5 L (as 6 x 1.25 L cultures) of Terrific Broth (TB). IPTG
(1 mM)
was added at
mid
-
log phase (OD
600
~0.8) to induce proinsulin expression. Cultures were incubated at 37°C for
3 h, after which cells were harvested via centrifugation and stored at
-
80°C until further
processing
.
Proinsulin refolding
:
Cell pellets were warmed from
-
80°C to room temperature and resuspended in 5 mL IB buffer
(50 mM tris, 100 mM NaCl, 1 mM EDTA, pH 8.0) per gram cell pellet.
L
ysozyme
(
1 mg L
-
1
)
and
phenylmethylsulfonyl fluoride (PMSF, 1 mM)
were added, and the slurry
was placed
on ice for 30
min before cells were lysed via sonication. The lysate was centrifuged (14
,000
g, 30 min, 4°C) and
the soluble fraction was discarded. The pellet was washed twice with IB buffer + 1% Triton X
-
100,
once with IB buffer, and once with wate
r; this final step required centrifugation for 45 min. The
washed inclusion body pellet was resuspended in a minimal amount of water, and the mass of
proinsulin in the inclusion body pellet was estimated by SDS
-
PAGE.
In preparation for proinsulin refolding, the inclusion body was resuspended in 3 M urea and 10
mM cysteine in water, such that the proinsulin concentration was 1 mg proinsulin per L total
slurry. To dissolve proinsulin, the pH was adjusted to 12 and sample
stirred for 1 h at room
temperature. At this stage, ncPro incorporation was assessed by MALDI
-
TOF,
as
described in the
section entitled “MALDI
-
TOF MS” below. The solubilized proinsulin solution was diluted ten
-
fold
into refold
ing
buffer (10 mM CAPS, pH 10
.6) that had been pre
-
cooled to 4°C. The pH of the
refold
ing
solution was adjusted to 10.7 and the sample stored at 4°C; care was taken to ensure
that the solution pH remained between 10.6 and 10.8 throughout the refolding process.
Proinsulin refolding progress was monitored by reverse
-
phase HPLC, and usually reach
ed
completion within 50 h.
Proinsulin was enriched from the refold
ing
solution after adjusting the pH to 8.0 and incubating
the sample overnight with Ni
-
NTA resin and 10 mM imidazole. The resin was washed with wash
buffer (25 mM imidazole in PBS, pH 8.0), and proinsulin was eluted with elution buffer (250 mM
imidazole in PB
S, pH 8.0). Fractions containing proinsulin were combined and extensively
dialyzed against 10 mM sodium phosphate, pH 8.0
.
S
7
Insulin maturation and purification:
Refolded proinsulin was warmed to 37°C and digested with trypsin (20 U mL
-
1
) and
carboxypeptidase
-
B (10 U mL
-
1
) at 37°C for 90 min to remove the N
-
terminal tag and C
-
chain.
Digestion was halted by adjusting the pH to ~3 with 6 N HCl.
Insulins were immediately purified after proteolysis by reverse
-
phase HPLC on a C
4
column
(Penomenex Jupiter 5 μm particle size, 300 Å pore size, 250x10 mm) using 0.1% TFA in water
(solvent A) and 0.1% TFA in acetonitrile (solvent B) as mobile phases. A gradient of 25
-
32% solvent
B was applied over 65 min, and fractions containing insul
in were collected. Samples for purity
analysis were removed at this stage; the remaining portion of the fraction was lyophilized. Each
insulin fraction was analyzed by analyt
ical reverse
-
phase HPLC, MALDI
-
TOF MS (Figure 2e
-
h), and
SDS
-
PAGE (Figure S2) to verify sample quality and ensure ≥95% purity for all downstream
analyses. Lyophilized insulin powders were stored at
-
20°C until further use
.
MALDI
-
TOF MS:
To assess levels of incorporation of ncPros into the corresponding proinsulins, samples were
digested with Glu
-
C, which
yields
a peptide fragment containing ProB28 (
50
RGFFYT
P
KTRRE). A 20
μL aliquot of proinsulin was subjected to cysteine reduction (5 mM DTT, 55°C for 20 min) and
alkylation (15 mM iodoacet
a
mide,
room temperature
for 15 min in the dark), prior to 10
-
fold
dilution into 100 mM NH
4
HCO
3
, pH 8.0 (100 μL final volume). Digestion was started with addition
of 0.6 μL Glu
-
C (0.5 μg μL
-
1
in ddH
2
0) at 37
°C for 2.5 h. The digestion reaction was quenched by
adding 10 μL of 5% TFA. Peptides were desalted using ZipTip C
18
columns (MilliporeSigma)
according to the manufacturer’s protocol. Desalted peptides (in 50% acetonitrile
, ACN;
0.1% TFA)
were diluted 3:1 into the matrix solution (α
-
cyanohydroxycinnamic acid in 50% ACN, 0.1% TFA)
and analyzed by MALDI
-
TOF MS. Analog incorporation was calculated by comparing the area
under the curve (AUC) of the ncPro form of the peptide (m/z = 157
2 for 4
R
-
Me and 4
S
-
Me, and
1570 for 4ene) with the AUC of the canonical proline peptide (m/z = 1558).
HPLC
-
purified insulins were analyzed as full
-
length, mature proteins. Aliquots directly from HPLC
purification (~30% ACN, 0.1% TFA) were mixed 1:1 with matrix solution (sinapic acid in 30% ACN,
0.1% TFA) before analysis by MALDI
-
TOF MS
.
Circular dichroism spectroscopy
:
Equilibrium measurements:
The circular dichroism spectra of insulin samples (60 μM in 100 mM
sodium phosphate, pH 8.0) were measured at 25°C in 1 mm quartz cuvettes on an Aviv Model
430 Circular Dichroism Spectrophotometer using a step size of 0.5 nm and averaging time of 1 s.
A re
ference buffer spectrum was subtracted from each sample spectrum.
Kinetic measurements:
Insulin samples in 100 mM sodium phosphate buffer pH 8.0 were dialyzed
overnight against 28.6 mM tris buffer, pH 8.0 (Slide
-
A
-
Lyzer dialysis cassettes, 3.5 kDa MWCO,
ThermoFisher). Insulins were formulated as follow
s
: 600 μM insulin, 250 μM ZnCl
2
, 25 mM m
-
cresol, 25 mM tris buffer, pH 8. To a stirred buffer solution containing 2.98 mL of 25 mM tris, pH
8.0 in a 10 mm quartz cuvette was injected 20 μL of the insulin formulation (150
-
fold dilution).
S
8
Ellipticity was monitored at 222 nm over 120 s (1 s kinetic interval, 0.5 s time constant, 10 nm
bandwidth) at 25°C. A typical run led to a rapid drop in CD signal as mixing occurred (~5 s), then
a gradual rise to an equilibrium ellipticity representative
of an insulin monomer. Data preceding
the timepoint with the greatest negative ellipticity (representing the mixing time) were omitted
from further analysis. Runs were discarded if the maximum change in mean residue ellipticity
from equilibrium did not exc
eed 750 deg cm
2
dmol
-
1
, which indicated poor mixing. The remaining
data were fit to a mono
-
exponential function using Scipy (Python). The data presented here are
from at least two separate insulin HPLC fractions, measured on two different days.
For quality control, an equilibrium spectrum for each protein was obtained after dilution as
described above; all spectra
approached
that of the insulin monomer
4
(
Figure S4
). The CD
spectrum of human insulin under pre
-
dilution formulation conditions was obtained using a 0.1
mm quartz cuvette. In each case, a blank spectrum containing all buffers and ligands was
subtracted from the sample spectrum
.
Reduction of blood glucose in diabetic mice
:
Adult (8 week old) male C57BL/6J mice were ordered from Jackson Laboratory (Bar Harbor, ME)
.
Mice were maintained under
specific pathogen
-
free conditions, and experiments were
conducted according to procedures approved by the Institutional Animal Care and Use
Committee (IACUC) at the City of Hope. Adult (8
-
12 week old) male mice were injected
intraperitoneally (
6
5 mg kg
-
1
day
-
1
for 3
consecutive
days) with freshly prepared streptozotocin
(STZ) in 50 mM citrate buffer, pH 4.5 to induce diabetes.
Insulin
-
dependent d
iabetes was
confirmed 3 weeks after the last dose of STZ by detection of high glucose levels (200
-
600 mg
dL
-
1
) as measured by a glucomonitor (Freestyle, Abbott Diabetes Care, Alameda, CA) in blood
sampled from the lateral tail vein. Insulin analogs were diluted to 100 μg mL
-
1
in formulation
buffer (1.6 mg mL
-
1
m
-
cresol, 0.65 mg mL
-
1
phenol, 3.8 mg mL
-
1
sodium
phosphate pH 7.4, 16 mg
mL
-
1
glycerol, 0.8 μg mL
-
1
ZnCl
2
).
Insulin analogs were injected (35 μg kg
-
1
) subcutaneously at the
scruff of diabetic 12
-
13 week old C57BL/6J mice.
B
lood glucose was measured at 0, 10, 20, 30,
40, 50, 60, 80, 100, 120, and 150 min
.
Fibrillation
:
Insulin samples (60 μM in 100 mM sodium phosphate, pH 8.0) were centrifuged at 22,000 g for
1 h at 4°C, prior to the addition of 1 μM thioflavin T (ThT). Each insulin (200 μL) was added to a
96
-
well, black, clear bottom plate (Greiner Bio
-
One) and sealed.
Samples were shaken
continuously at 960 rpm on a Varioskan multimode plate reader at 37°C, and fluorescence
readings were recorded every 15 min (444 nm excitation, 485 nm emission). Fibrillation runs
were performed on at least two separate HPLC fractions,
each in triplicate or quadruplicate, on
at least two different days. The growth phase of each fibrillation replicate was fit to a linear
function, and fibrillation lag times were reported as the x
-
intercept of this fit. Fibril samples were
stored at 4°C u
ntil analysis by TEM
and mass spectrometry
.
S
9
Transmission electron microscopy
:
Insulin fibrils were centrifuged (5
,000
g, 1 min), then washed twice and resuspended in ddH
2
O.
Fibrils were stained with 2% uranyl acetate on a 300
-
mesh formvar/carbon coated copper grid
(Electron Microscopy Sciences) and imaged on a Tecnai T12 LaB6 120 eV transmission electron
microscope
.
ANS fluorescence
:
I
nsulin
s
(
1 μM
)
were
mixed with 5 μM ANS in 100 mM phosphate buffer, pH 8.0. Fluorescence
emission spectra were measured in 1 cm quartz cuvettes at ambient temperature using a PTI
QuantaMaster fluorescence spectrofluorometer. A 350 nm excitation wavelength and scan rate
of 2
nm s
-
1
were used. Measurements for each insulin were performed in triplicate from three
separate HPLC fractions. Spectra were smoothed before plotting and determining the emission
maxima
.
Analytical ultracentrifugation
:
Insulins were dialyzed against 2
8.6
mM tris buffer, pH 8.0, and formulated at 300 μM insulin, 12.5
mM
m
-
cresol, and 125 μM ZnCl
2
. Ligand
-
free insulins were formulated from the same dialysis
sample. The insulin samples were then diluted 75
-
fold into 25 mM tris buffer, pH 8.0 (4 μM
insulin,
167 μM
m
-
cresol, 1.7 μM ZnCl
2
), conditions identical to those after dilution in the CD
dissociation kinetics experiments.
Diluted insulins were incubated at room temperature for at
least one hour after dilution
prior to analysis
.
Velocity sedimentation experiments were performed at the Canadian Center for Hydrodynamics
at the University of Lethbridge.
300 μM insulin s
amples
were measured by interference optics,
due to the high
absorbance
from the
protein and
m
-
cresol
; they were measured in 3 mm titanium
centerpieces from Nanolytics, fitted in a standard Beckman Coulter cell housing using a 3mm
spacer above and below the centerpiece
.
D
iluted
samples
(4 μM insulin)
were measured using
absorbance optics
at 225 nm
in standard Beckman Co
ulter 1.2 epon
-
charocol centerpieces
. All
samples were measured at 50,000 RPM and 20°C in standard Beckman Coulter cell housings
fitted with a 1.2 cm epon
-
charcoal centerpiece and sapphire windows
. All data were analyzed
with UltraScan III version 4.0 release 6606.
5
Velocity data were initially fitted with the two
-
dimensional spectrum analysis
6
to determine meniscus position and time
-
and radially
-
invariant
noise. Subsequent noise
-
corrected data were analyzed by the
enhanced van Holde
-
Weischet
analysis
7
to generate diffusion
-
corrected
integral
sedimentation coefficient distributions
.
Models of ins
-
4
R
-
Me and ins
-
4
S
-
Me hexamers
:
Crystal structures of the T
6
(PDB: 1MSO) and R
6
(1EV6) insulin hexamers were downloaded from
the Protein Data Bank and visualized with Pymol. The hydrogen atoms at the C
g
position of
ProB28 were replaced with methyl groups; no additional energy minimization was used
.
S
10
Mass spectrometry characterization of dissolved insulin fibrils
:
Samples containing insulin fibrils were
centrifuged (5
,000
g, 1 min, 4°C), washed twice with
ddH
2
O and dissolved in dimethylsulfoxide (DMSO). Dissolved fibrils were reduced (5 μM DTT,
55°C, 20 min), then diluted ten
-
fold into
MS loading buffer (2% ACN, 0.2%
formaic acid,
FA
,
in
water)
. 8 μL of this
sample
was injected onto a
Thermo EASY
-
Spray column (ES902, C18, 2um,
100A, 75 um x 25 cm)
equipped
with an Acclaim PepMap trapping column (C18, 3um, 100A, 75
um x 2 cm)
, and
analyzed
using
a Thermo Orbitrap Eclipse Tribrid Mass Spectrometer coupled
wi
th a Thermo Easy nLC
-
1200. The resulting raw files
were
deconvoluted using MASH Explorer
.
8
Calculations of proline and proline analog conformation
:
The equilibrium geometry conformations of the N
-
methyl, O
-
methyl ester protected versions of
proline, 4
-
methyleneproline, and 3,4
-
dehydroproline in water were calculated using Spartan
Student (Wavefunction) at the B3LYP/G
-
31+G** level of theory. Pseudorota
tion parameters
were calculated from the dihedral angles about the pyrrolidine ring, as previously reported.
9
S
11
Table S
1
. Incorporation of non
-
canonical proline residues
into recombinant proinsulin
.
ncPro
Expected
m/z
Observed m/z
Expected ∆ m/z
#
Observed ∆ m/z
‡#
Incorporation
efficiency
Proline
1557.90
1557.19 ± 0.02
2
-
Me
1571.91
n.d.
*
14.01
3
R
-
OH
1573.88
1573.184 ± 0.006
15.98
15.5 ± 0.7
0.672 ± 0.004
3
S
-
OH
1573.88
1573.184 ± 0.008
15.98
15.990 ± 0.005
0.537 ± 0.002
4
S
-
NH
2
1572.90
1572.191 ± 0.001
15.00
14.990 ± 0.006
0.17 ± 0.03
4
R
-
COOH
1601.89
n.d.
43.99
4
S
-
COOH
1601.89
n.d.
43.99
4
-
oxo
1571.87
1571.182 ± 0.009
13.97
13.974 ± 0.009
0.15 ± 0.01
4
-
ene
1569.89
1569.218 ± 0.008
11.99
11.971 ± 0.001
0.926 ±
0.002
4
R
-
Me
1571.91
1571.241 ± 0.006
14.01
13.90 ± 0.02
0.853 ± 0.004
4
S
-
Me
1571.91
1571.26 ± 0.01
14.01
14.017 ± 0.004
0.775 ± 0.007
44
-
diMe
1585.93
n.d.
28.03
5
-
oxo
1571.87
n.d.
13.97
Pip
-
OH
1587.91
n.d.
30.01
Pip
-
Az
1572.90
n.d.
15.00
Photo
-
p
ro
%
528.6
1
528.61
8.66
8.67
0.373
Average ± standard deviation of two technical replicates
#
Mass shift compared to the proline
-
containing peptide present in the spectrum
*
n.d., not detected
%
Digested photo
-
proline peptide was
analyzed by LC
-
ESI
-
MS, due to diazirine photolysis during MALDI
-
TOF analysis. We quantified the [M+3H]
+3
ion for the proline and ncPro
-
containing peptides. We also note
the presence (27%; m/z = 519.3) of a
n ion corresponding to
replacement of
proline
by
3,4
-
dehydroproline
(dhp)
.
The incorporation efficiency for photo
-
pro reported here is with respect to the proline and
dhp
ions
:
photo
-
pro incorporation efficiency =
!"#
!
"
#$#
%
!&#
!"#
!&#
$
!"#
'
"
!
$
!"#
!
"
#$#
%
!&#
S
12
Table S
2
. Mass spectrometry characterization of insulin
variants
.
Digested peptide
Mature insulin
Protein
Expected
m/z
Observed m/z
Incorporation
efficiency
Expected
m/z
Observed m/z
Insulin
1557.78
1557.43 ± 0.06
5808.6
580
7.7
± 0.
4
Ins
-
4
R
-
Me
1571.86
1571.83 ± 0.03
0.901 ± 0.010
5822.6
5822.
2
± 0.
3
Ins
-
4
S
-
Me
1571.86
1571.70 ± 0.03
0.
896 ±
0.0
02
5822.6
5822.34 ± 0.01
Ins
-
4ene
1569.84
1569.56 ± 0.02
0.
9
417
±
0.0
0
05
5820.6
582
1.2
± 0.
3
Average ± standard deviation of two technical
replicates
S
13
Table S
3.
Expression conditions and i
nsulin yields.
Protein
ProRS variant
[ncPro] (mM)
[NaCl] (M)
Approx. proinsulin
yield (mg L
-
1
)
Approx. mature
insulin yield (mg L
-
1
)
Insulin
*
wt
35
8.0
Ins
-
4
R
-
Me
C443G
0.5
0.5
29
3.9
Ins
-
4
S
-
Me
wt
0.5
0.5
53
7.9
Ins
-
4ene
M157Q
1.0
0.5
23
1.5
Yields determined by measuring absorbance (280 nm) after
proinsulin refolding and
Ni
-
NTA enrichment
.
*
Expressed in terrific broth (TB)
S
14
Table S
4
. Summary of insulin
variant
characterization.
Protein
Ellipticity ratio
(208/222 nm)
Fibrillation lag time (h)
Hexamer dissociation
t
1/2
(s)
*
ANS emission
maximum (nm)
Insulin
1.24 ± 0.03
16.6 ± 4.1
18.4 ± 2.8
470 ± 5
Ins
-
4
R
-
Me
1.37 ± 0.05
15.0 ± 3.5
9.8 ± 1.8
465 ± 3
Ins
-
4
S
-
Me
1.19 ± 0.06
12.5 ± 2.5
9.9 ± 2.5
466 ± 6
Ine
-
4ene
1.32 ± 0.05
8.2 ± 4.0
17.0 ± 2.3
450 ± 11
60 μM insulin, 100 mM phosphate, pH 8.0
*
150
-
fold dilution of 600 μM insulin, 250 μM ZnCl
2
, 25 mM
m
-
cresol, 25 mM tris, pH 8.0
S
15
Table S
5
.
Sedimentation coefficients of insulin and ins
-
4
S
-
Me.
Protein
Formulation
*
Dilution
Ligand
-
free
%
Insulin
3.3
S
1.1
S
2.4
S
Ins
-
4
S
-
Me
3.4
S
1.1
S
3.1
S
*
300 μM insulin,
125
μM ZnCl
2
,
12.5
mM
m
-
cresol, 25 mM tris, pH 8.0
4
μM insulin,
1.7
μM ZnCl
2
,
167
μ
M
m
-
cresol
,
25 mM tris, pH 8.0
%
300 μM insulin, 25 mM tris, pH 8.0
S
16
Figure
S
1
. SDS
-
PAGE
analysis of proinsulin expression in media supplemented with
non
-
canonical proline
analog
s
.
Proinsulin (12.7 kDa) was expressed after a medium shift to ncPro
-
containing medium. The inclusion body fraction was isolated, solubilized, and analyzed by SDS
-
PAGE.
S
17
Figure S2. Purity
of insulin samples assessed by SDS
-
PAGE.
HPLC
-
purified insulin (a), Ins
-
4
R
-
Me
(b), Ins
-
4
S
-
Me (c), and Ins
-
4ene
(d)
were analyzed by SDS
-
PAGE to validate purity; shown are
representative lanes corresponding to individual HPLC fractions
.
S
18
Figure S
3
.
Changes in CD signal after dilution are not due to protein denaturation
.
At 60 μM,
insulin is expected to exist as a dimer at pH 8,
as a
monomer in 20% ethanol, and
in
denatured
form
in 8 M guanidinium chloride. These spectra are overlaid with equilibrium spectra collected
before and after dilution for kinetic CD measurements.
Spectra below 210
-
215
nm were omitted
for some samples due to high levels of buffer absorbance at these wavelengths.
S
19
Figure S
4
.
Equilibrium CD spectra after dilution
.
Equilibrium far
-
UV spectra of
insulin
(a),
Ins
-
4
R
-
Me
(b),
Ins
-
4
S
-
Me
(c), and
Ins
-
4ene
(d)
after 150
-
fold dilution from the hexamer formulation.
Conditions after dilution from the hexamer formulation are as follows: 4 μM insulin variant, 167
μM
m
-
cresol, 1.67 μM ZnCl
2
, 25 mM tris buffer, pH 8.0.
S
20
Figure S
5
.
Models of ins
-
4R
-
Me and ins
-
4S
-
Me hexamers
. 4
S
-
Me and 4
R
-
Me were modeled in
the structures of the R
6
(
a
) and T
6
(
b
) insulin hexamers (PDB ID: 1EV3 & 1MSO, respectively).
Atoms near to each methyl substituent are indicated; distance measurements are in Å.